Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors.

PubMed

Brevik, Asgeir; Karlsen, Anette; Azqueta, Amaya; Tirado, Anna Estaban; Blomhoff, Rune; Collins, Andrew

2011-01-01

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant-rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (-39%, p < 0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (-38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.

Bisphenol A Promotes Cell Survival Following Oxidative DNA Damage in Mouse Fibroblasts

PubMed Central

Gassman, Natalie R.; Coskun, Erdem; Stefanick, Donna F.; Horton, Julie K.; Jaruga, Pawel; Dizdaroglu, Miral; Wilson, Samuel H.

2015-01-01

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway. PMID:25693136

Recent Advances in the Structural Mechanisms of DNA Glycosylases

PubMed Central

Brooks, Sonja C.; Adhikary, Suraj; Rubinson, Emily H.; Eichman, Brandt F.

2012-01-01

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28 years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities. PMID:23076011

Alkylpurine glycosylase D employs DNA sculpting as a strategy to extrude and excise damaged bases.

PubMed

Kossmann, Bradley; Ivanov, Ivaylo

2014-07-01

Alkylpurine glycosylase D (AlkD) exhibits a unique base excision strategy. Instead of interacting directly with the lesion, the enzyme engages the non-lesion DNA strand. AlkD induces flipping of the alkylated and opposing base accompanied by DNA stack compression. Since this strategy leaves the alkylated base solvent exposed, the means to achieve enzymatic cleavage had remained unclear. We determined a minimum energy path for flipping out a 3-methyl adenine by AlkD and computed a potential of mean force along this path to delineate the energetics of base extrusion. We show that AlkD acts as a scaffold to stabilize three distinct DNA conformations, including the final extruded state. These states are almost equivalent in free energy and separated by low barriers. Thus, AlkD acts by sculpting the global DNA conformation to achieve lesion expulsion from DNA. N-glycosidic bond scission is then facilitated by a backbone phosphate group proximal to the alkylated base.

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

PubMed Central

Çağlayan, Melike; Horton, Julie K.; Dai, Da-Peng; Stefanick, Donna F.; Wilson, Samuel H.

2017-01-01

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides. PMID:28067232

A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD

PubMed Central

Rubinson, Emily H.; Metz, Audrey H.; O'Quin, Jami; Eichman, Brandt F.

2013-01-01

Summary DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases. PMID:18585735

Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

PubMed Central

Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

Expression of DNA repair genes in burned skin exposed to low-level red laser.

PubMed

Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

2014-11-01

Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

Effect of Amalaki rasayana on DNA damage and repair in randomized aged human individuals.

PubMed

Vishwanatha, Udupi; Guruprasad, Kanive P; Gopinath, Puthiya M; Acharya, Raviraj V; Prasanna, Bokkasa V; Nayak, Jayakrishna; Ganesh, Rajeshwari; Rao, Jayalaxmi; Shree, Rashmi; Anchan, Suchitra; Raghu, Kothanahalli S; Joshi, Manjunath B; Paladhi, Puspendu; Varier, Panniampilly M; Muraleedharan, Kollath; Muraleedharan, Thrikovil S; Satyamoorthy, Kapaettu

2016-09-15

Preparations from Phyllanthus emblica called Amalaki rasayana is used in the Indian traditional medicinal system of Ayurveda for healthy living in elderly. The biological effects and its mechanisms are not fully understood. Since the diminishing DNA repair is the hallmark of ageing, we tested the influence of Amalaki rasayana on recognized DNA repair activities in healthy aged individuals. Amalaki rasayana was prepared fresh and healthy aged randomized human volunteers were administrated with either rasayana or placebo for 45 days strictly as per the traditional text. The DNA repair was analyzed in peripheral blood mononuclear cells before and after rasayana administration and after 45 days post-rasayana treatment regimen. UVC-induced DNA strand break repair (DSBR) based on extent of DNA unwinding by fluorometric analysis, nucleotide excision repair (NER) by flow cytometry and constitutive base excision repair (BER) by gap filling method were analyzed. Amalaki rasayana administration stably maintained/enhanced the DSBR in aged individuals. There were no adverse side effects. Further, subjects with different body mass index showed differential DNA strand break repair capacity. No change in unscheduled DNA synthesis during NER and BER was observed between the groups. Intake of Amalaki rasayana by aged individuals showed stable maintenance of DNA strand break repair without toxic effects. However, there was no change in nucleotide and base excision repair activities. Results warrant further studies on the effects of Amalaki rasayana on DSBR activities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

PubMed Central

Hollis, Thomas; Ichikawa, Yoshitaka; Ellenberger, Tom

2000-01-01

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1–azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1–azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner. PMID:10675345

Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

PubMed

He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

2013-11-01

Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guliaev, Anton B.; Singer, B.; Hang, Bo

2004-05-05

Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA thanmore » that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.« less

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Strand displacement synthesis by yeast DNA polymerase ε

PubMed Central

Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-01-01

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.

PubMed

Wang, Li-Juan; Ren, Ming; Zhang, Qianyi; Tang, Bo; Zhang, Chun-Yang

2017-04-18

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

PubMed

Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

2014-04-01

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.

PubMed

Kuznetsov, N A; Kiryutin, A S; Kuznetsova, A A; Panov, M S; Barsukova, M O; Yurkovskaya, A V; Fedorova, O S

2017-04-01

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T m , and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T m (F/T) < T m (εA/T) < T m (Hx/T) < T m (A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Repair of Clustered Damage and DNA Polymerase Iota.

PubMed

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

DNA repair in mammalian mitochondria: Much more than we thought?

PubMed

Liu, Pingfang; Demple, Bruce

2010-06-01

For many years, the repair of most damage in mitochondrial DNA (mtDNA) was thought limited to short-patch base excision repair (SP-BER), which replaces a single nucleotide by the sequential action of DNA glycosylases, an apurinic/apyrimidinic (AP) endonuclease, the mitochondrial DNA polymerase gamma, an abasic lyase activity, and mitochondrial DNA ligase. However, the likely array of lesions inflicted on mtDNA by oxygen radicals and the possibility of replication errors and disruptions indicated that such a restricted repair repertoire would be inadequate. Recent studies have considerably expanded our knowledge of mtDNA repair to include long-patch base excision repair (LP-BER), mismatch repair, and homologous recombination and nonhomologous end-joining. In addition, elimination of mutagenic 8-oxodeoxyguanosine triphosphate (8-oxodGTP) helps prevent cell death due to the accumulation of this oxidation product in mtDNA. Although it was suspected for many years that irreparably damaged mtDNA might be targeted for degradation, only recently was clear evidence provided for this hypothesis. Therefore, multiple DNA repair pathways and controlled degradation of mtDNA function together to maintain the integrity of mitochondrial genome.

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

PubMed

Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

2016-06-21

In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer

PubMed Central

Limpose, Kristin L; Trego, Kelly S; Li, Zhentian; Leung, Sara W; Sarker, Altaf H; Shah, Jason A; Ramalingam, Suresh S; Werner, Erica M; Dynan, William S; Cooper, Priscilla K; Corbett, Anita H; Doetsch, Paul W

2018-01-01

Abstract Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer. PMID:29522130

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair*

PubMed Central

King, Brenee S.; Cooper, Karen L.; Liu, Ke Jian; Hudson, Laurie G.

2012-01-01

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts. PMID:23038248

Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone.

PubMed

DeMott, Michael S; Beyret, Ergin; Wong, Donny; Bales, Brian C; Hwang, Jae-Taeg; Greenberg, Marc M; Demple, Bruce

2002-03-08

Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1' carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase beta is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase beta with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "short-patch" base excision repair but instead function as suicide substrates in the formation of protein-DNA cross-links that may require alternative modes of repair.

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

PubMed Central

Eichman, Brandt F.; O’Rourke, Eyleen J.; Radicella, J.Pablo; Ellenberger, Tom

2003-01-01

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix–hairpin–helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases. PMID:14517230

Strand displacement synthesis by yeast DNA polymerase ε.

PubMed

Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-09-30

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair.

PubMed

Khoronenkova, Svetlana V; Dianov, Grigory L

2013-02-01

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

PubMed

Guzder, S N; Sung, P; Prakash, L; Prakash, S

1998-11-20

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

Oxidative DNA damage background estimated by a system model of base excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Rational Inhibitors of DNA Base Excision Repair (BER) Enzymes: New Tools for Elucidating the Role of the BER in Cancer Chemotherapy

DTIC Science & Technology

2005-05-01

Revised Manuscript Received March 3, 2005 ABSTRACT: Base flipping is a highly conserved strategy used by enzymes to gain catalytic access to DNA bases that...73.13. Jencks, W. P. (1985) A primer for the bema hapothle-an between uracil and other normal DNA bases at this inter- empirical-approach to the

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase β

PubMed Central

Mendez, Frances; Kozin, Elliott; Bases, Robert

2003-01-01

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase β, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase β was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase β repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase β also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood. PMID:14627201

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

PubMed

Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

2010-06-04

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

NASA Astrophysics Data System (ADS)

Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

2016-07-01

Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

DNA Damage Induced by Alkylating Agents and Repair Pathways

PubMed Central

Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

2010-01-01

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways.

PubMed

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T; Gasparutto, Didier; Geacintov, Nicholas E; Saparbaev, Murat

2015-06-05

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways*

PubMed Central

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T.; Gasparutto, Didier; Geacintov, Nicholas E.; Saparbaev, Murat

2015-01-01

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. PMID:25903131

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

PubMed Central

Roos, Wynand Paul; Krumm, Andrea

2016-01-01

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

The Influence of Hepatitis C Virus Therapy on the DNA Base Excision Repair System of Peripheral Blood Mononuclear Cells.

PubMed

Czarny, Piotr; Merecz-Sadowska, Anna; Majchrzak, Kinga; Jabłkowski, Maciej; Szemraj, Janusz; Śliwiński, Tomasz; Karwowski, Bolesław

2017-07-01

Hepatitis C virus (HCV) can infect extrahepatic tissues, including lymphocytes, creating reservoir of the virus. Moreover, HCV proteins can interact with DNA damage response proteins of infected cells. In this article we investigated the influence of the virus infection and a new ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin (OBV/PTV/r ± DSV ± RBV) anti-HCV therapy on the PBMCs (peripheral blood mononuclear cells, mainly lymphocytes) DNA base excision repair (BER) system. BER protein activity was analyzed in the nuclear and mitochondrial extracts (NE and ME) of PBMC isolated from patients before and after therapy, and from subjects without HCV, using modeled double-strand DNA, with 2'-deoxyuridine substitution as the DNA damage. The NE and ME obtained from patients before therapy demonstrated lower efficacy of 2'-deoxyuridine removal and DNA repair polymerization than those of the control group or patients after therapy. Moreover, the extracts from the patients after therapy had similar activity to those from the control group. However, the efficacy of apurinic/apyrimidinic site excision in NE did not differ between the studied groups. We postulate that infection of lymphocytes by the HCV can lead to a decrease in the activity of BER enzymes. However, the use of novel therapy results in the improvement of glycosylase activity as well as the regeneration of endonuclease and other crucial repair enzymes.

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

PubMed Central

Boiteux, Serge; Jinks-Robertson, Sue

2013-01-01

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice

USDA-ARS?s Scientific Manuscript database

Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

PubMed

Cannan, Wendy J; Tsang, Betty P; Wallace, Susan S; Pederson, David S

2014-07-18

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

PubMed Central

Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

2014-01-01

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

PubMed

Yamasaki, Hiroshi; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Sato, Marcello Otake; Ito, Akira

2006-01-01

Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology and the molecular detection of taeniasis using copro-DNA were discussed.

Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

PubMed

Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

2014-04-01

Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy. Copyright © 2014. Published by Elsevier B.V.

Base excision repair in Archaea: back to the future in DNA repair.

PubMed

Grasso, Stefano; Tell, Gianluca

2014-09-01

Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection

PubMed Central

Romano, Christine A.; Sontz, Pamela A.; Barton, Jacqueline K.

2011-01-01

Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30, Y55, Y75 and Y82 help mediate charge transport between DNA and the [4Fe-4S] cluster. Based on circular dichroism studies to measure protein stability, mutations at residues W178 and Y185 are found to destabilize the protein; these residues may function to protect the [4Fe-4S] cluster. Atomic force microscopy studies furthermore reveal a correlation in the ability of mutants to carry out protein/DNA CT and their ability to relocalize onto DNA strands containing a single base mismatch; EndoIII mutants that are defective in carrying out DNA/protein CT do not redistribute onto mismatch-containing strands, consistent with our model. These results demonstrate a link between the ability of the repair protein to carry out DNA CT and its ability to relocalize near lesions, thus pointing to DNA CT as a key first step in the detection of base damage in the genome. PMID:21651304

Direct and indirect roles of RECQL4 in modulating base excision repair capacity

PubMed Central

Schurman, Shepherd H.; Hedayati, Mohammad; Wang, ZhengMing; Singh, Dharmendra K.; Speina, Elzbieta; Zhang, Yongqing; Becker, Kevin; Macris, Margaret; Sung, Patrick; Wilson, David M.; Croteau, Deborah L.; Bohr, Vilhelm A.

2009-01-01

RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund–Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H2O2-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H2O2-induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H2O2, RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase β, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity. PMID:19567405

DNA repair targeted therapy: the past or future of cancer treatment?

PubMed Central

Gavande, Navnath S.; VanderVere-Carozza, Pamela S.; Hinshaw, Hilary D.; Jalal, Shadia I.; Sears, Catherine R.; Pawelczak, Katherine S.; Turchi, John J.

2016-01-01

The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy. PMID:26896565

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

PubMed

Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

2017-01-01

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Base Excision Repair of Oxidative DNA Damage

PubMed Central

David, Sheila S.; O’Shea, Valerie L.; Kundu, Sucharita

2010-01-01

Base excision repair plays an important role in preventing mutations associated with the common product of oxidative damage, 8-oxoguanine. Recent structural studies have shown that 8-oxoguanine glycosylases use an intricate series of steps to efficiently search and locate 8-oxoguanine lesions within the multitude of undamaged bases. The importance of prevention of mutations associated with 8-oxoguanine has also been illustrated by direct connections between defects in the BER glycosylase MUTYH and colorectal cancer. In addition, the properties of other guanine oxidation products and the BER glycosylases that remove them are being uncovered. This work is providing surprising and intriguing new insights into the process of base excision repair. PMID:17581577

Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

DOE PAGES

Hang, Bo

2010-01-01

DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N -nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O 6 -alkylguaninemore » DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects.« less

POLYMORPHISMS IN THE DNA BASE EXCISION REPAIR GENES APEX1 AND XRCC1 AND LUNG CANCER RISK IN XUAN WEI, CHINA

EPA Science Inventory

The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...

Identification of a residue critical for the excision of 3′-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family

PubMed Central

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Yang, Wei; González-Pacanowska, Dolores; Vidal, Antonio E.

2009-01-01

DNA single-strand breaks containing 3′-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3′-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H2O2). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H2O2. Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3′-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3′-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3′-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage. PMID:19181704

Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water.

PubMed

Andrew, Angeline S; Karagas, Margaret R; Hamilton, Joshua W

2003-04-10

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic. Copyright 2003 Wiley-Liss, Inc.

Mutants of the base excision repair glycosylase, endonuclease III: DNA charge transport as a first step in lesion detection.

PubMed

Romano, Christine A; Sontz, Pamela A; Barton, Jacqueline K

2011-07-12

Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30, Y55, Y75, and Y82 help mediate charge transport between DNA and the [4Fe-4S] cluster. On the basis of circular dichroism studies to measure protein stability, mutations at residues W178 and Y185 are found to destabilize the protein; these residues may function to protect the [4Fe-4S] cluster. Atomic force microscopy studies furthermore reveal a correlation in the ability of mutants to carry out protein/DNA CT and their ability to relocalize onto DNA strands containing a single base mismatch; EndoIII mutants that are defective in carrying out DNA/protein CT do not redistribute onto mismatch-containing strands, consistent with our model. These results demonstrate a link between the ability of the repair protein to carry out DNA CT and its ability to relocalize near lesions, thus pointing to DNA CT as a key first step in the detection of base damage in the genome.

Day and night variations in the repair of ionizing-radiation-induced DNA damage in mouse splenocytes.

PubMed

Palombo, Philipp; Moreno-Villanueva, Maria; Mangerich, Aswin

2015-04-01

In mammals, biological rhythms synchronize physiological and behavioral processes to the 24-h light-dark (LD) cycle. At the molecular level, self-sustaining processes, such as oscillations of transcription-translation feedback loops, control the circadian clock, which in turn regulates a wide variety of cellular processes, including gene expression and cell cycle progression. Furthermore, previous studies reported circadian oscillations in the repair capacity of DNA lesions specifically repaired by nucleotide excision repair (NER). However, it is so far only poorly understood if DNA repair pathways other than NER are under circadian control, in particular base excision and DNA strand break repair. In the present study, we analyzed potential day and night variations in the repair of DNA lesions induced by ionizing radiation (i.e., mainly oxidative damage and DNA strand breaks) in living mouse splenocytes using a modified protocol of the automated FADU assay. Our results reveal that splenocytes isolated from mice during the light phase (ZT06) displayed higher DNA repair activity than those of the dark phase (ZT18). As analyzed by highly sensitive and accurate qPCR arrays, these alterations were accompanied by significant differences in expression profiles of genes involved in the circadian clock and DNA repair. Notably, the majority of the DNA repair genes were expressed at higher levels during the light phase (ZT06). This included genes of all major DNA repair pathways with the strongest differences observed for genes of base excision and DNA double strand break repair. In conclusion, here we provide novel evidence that mouse splenocytes exhibit significant differences in the repair of IR-induced DNA damage during the LD cycle, both on a functional and on a gene expression level. It will be interesting to test if these findings could be exploited for therapeutic purposes, e.g. time-of-the-day-specific application of DNA-damaging treatments used against blood malignancies. Copyright © 2015 Elsevier B.V. All rights reserved.

Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.

PubMed

Li, Cuiping; Wang, Hailin

2015-08-07

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10(-19)mol in mass and 2.9pM in concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

Low power lasers on genomic stability.

PubMed

Trajano, Larissa Alexsandra da Silva Neto; Sergio, Luiz Philippe da Silva; Stumbo, Ana Carolina; Mencalha, Andre Luiz; Fonseca, Adenilson de Souza da

2018-03-01

Exposure of cells to genotoxic agents causes modifications in DNA, resulting to alterations in the genome. To reduce genomic instability, cells have DNA damage responses in which DNA repair proteins remove these lesions. Excessive free radicals cause DNA damages, repaired by base excision repair and nucleotide excision repair pathways. When non-oxidative lesions occur, genomic stability is maintained through checkpoints in which the cell cycle stops and DNA repair occurs. Telomere shortening is related to the development of various diseases, such as cancer. Low power lasers are used for treatment of a number of diseases, but they are also suggested to cause DNA damages at sub-lethal levels and alter transcript levels from DNA repair genes. This review focuses on genomic and telomere stabilization modulation as possible targets to improve therapeutic protocols based on low power lasers. Several studies have been carried out to evaluate the laser-induced effects on genome and telomere stabilization suggesting that exposure to these lasers modulates DNA repair mechanisms, telomere maintenance and genomic stabilization. Although the mechanisms are not well understood yet, low power lasers could be effective against DNA harmful agents by induction of DNA repair mechanisms and modulation of telomere maintenance and genomic stability. Copyright © 2018 Elsevier B.V. All rights reserved.

Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

PubMed

Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

2015-09-01

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

The 2015 Nobel Prize in Chemistry The Discovery of Essential Mechanisms that Repair DNA Damage.

PubMed

Lindahl, Tomas; Modrich, Paul; Sancar, Aziz

2016-01-01

The Royal Swedish Academy awarded the Nobel Prize in Chemistry for 2015 to Tomas Lindahl, Paul Modrich and Aziz Sancar for their discoveries in fundamental mechanisms of DNA repair. This pioneering research described three different essential pathways that correct DNA damage, safeguard the integrity of the genetic code to ensure its accurate replication through generations, and allow proper cell division. Working independently of each other, Tomas Lindahl, Paul Modrich and Aziz Sancar delineated the mechanisms of base excision repair, mismatch repair and nucleotide excision repair, respectively. These breakthroughs challenged and dismissed the early view that the DNA molecule was very stable, paving the way for the discovery of human hereditary diseases associated with distinct DNA repair deficiencies and a susceptibility to cancer. It also brought a deeper understanding of cancer as well as neurodegenerative or neurological diseases, and let to novel strategies to treat cancer.

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

PubMed

Nuñez, Nicole N; Majumdar, Chandrima; Lay, Kori T; David, Sheila S

2018-01-01

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S] 2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease. © 2018 Elsevier Inc. All rights reserved.

The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

PubMed

Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

2013-07-10

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottlieb, J.; Muzyczka, N.

1988-06-01

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat andmore » in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.« less

Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S.

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis andmore » mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.« less

Contribution of DNA unwrapping from histone octamers to the repair of oxidatively damaged DNA in nucleosomes

PubMed Central

Maher, Robyn L.; Prasad, Amalthiya; Rizvanova, Olga; Wallace, Susan S.; Pederson, David S.

2013-01-01

Reactive oxygen species generate ~20,000 oxidative lesions in the DNA of every cell, every day. Most of these lesions are located within nucleosomes, which package DNA in chromatin and impede base excision repair (BER). We demonstrated previously that periodic, spontaneous partial unwrapping of DNA from the underlying histone octamer enables BER enzymes to bind to oxidative lesions that would otherwise be sterically inaccessible. In the present study, we asked if these periodic DNA unwrapping events are frequent enough to account for the estimated rates of BER in vivo. We measured rates of excision of oxidative lesions from sites in nucleosomes that are accessible only during unwrapping episodes. Using reaction conditions appropriate for presteady-state kinetic analyses, we derived lesion exposure rates for both 601 and 5S rDNA-based nucleosomes. Although DNA unwrapping-mediated exposure of a lesion ~16 NT from the nucleosome edge occurred ~7–8 times per minute, exposure rates fell dramatically for lesions located 10 or more NT further in from the nucleosome edge. The rates likely are too low to account for observed rates of BER in cells. Thus, chromatin remodeling, either BER-specific or that associated with transcription, replication, or other DNA repair processes, probably contributes to efficient BER in vivo. PMID:24051050

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

PubMed Central

Svilar, David; Goellner, Eva M.; Almeida, Karen H.

2011-01-01

Abstract Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage–induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated β-lyase activity; (b) monofunctional; and (c) bifunctional with an associated β,δ-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease. Antioxid. Redox Signal. 14, 2491–2507. PMID:20649466

Nucleotide excision repair deficient mouse models and neurological disease

PubMed Central

Niedernhofer, Laura J.

2008-01-01

Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER deficiency. PMID:18272436

DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY

PubMed Central

Sawant, Akshada; Floyd, Ashley M.; Dangeti, Mohan; Lei, Wen; Sobol, Robert W.; Patrick, Steve M.

2017-01-01

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. PMID:28110804

Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

PubMed Central

Gratias, Ariane; Lepère, Gersende; Garnier, Olivier; Rosa, Sarah; Duharcourt, Sandra; Malinsky, Sophie; Meyer, Eric; Bétermier, Mireille

2008-01-01

Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants. PMID:18420657

Influence of oxidized purine processing on strand directionality of mismatch repair.

PubMed

Repmann, Simone; Olivera-Harris, Maite; Jiricny, Josef

2015-04-17

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

PubMed Central

Edwards, Sarah K.; Ono, Toshikazu; Wang, Shenliang; Jiang, Wei; Franzini, Raphael M.; Jung, Jong Wha; Chan, Ke Min; Kool, Eric T.

2015-01-01

The repair of oxidative damage to DNA is essential to avoidance of mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a chief source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report on the design and properties of fluorogenic probes that directly report on OGG1 (and bacterial homologue Fpg) activity in real time as the oxidized base is excised. The probes are short modified DNA oligomers containing fluorescent DNA bases and are designed to utilize the damaged DNA base itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probe designs containing these fluorophores, and we found an optimized probe OGR1 that yields a 60-fold light-up signal in vitro with OGG1 and Fpg, and can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes may be useful in quantifying enzyme activity and performing competitive inhibition assays. PMID:26073452

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice.

PubMed

Vartanian, Vladimir; Minko, Irina G; Chawanthayatham, Supawadee; Egner, Patricia A; Lin, Ying-Chih; Earley, Lauriel F; Makar, Rosemary; Eng, Jennifer R; Camp, Matthew T; Li, Liang; Stone, Michael P; Lasarev, Michael R; Groopman, John D; Croy, Robert G; Essigmann, John M; McCullough, Amanda K; Lloyd, R Stephen

2017-04-18

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB 1 ) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB 1 -induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB 1 -deoxyguanosine adduct (AFB 1 -Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB 1 show significant increases in the levels of AFB 1 -Fapy-dG in Neil1 -/- vs. wild-type liver DNA. Further, Neil1 -/- mice are highly susceptible to AFB 1 -induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1 -/- The magnitude of this effect in Neil1 -/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB 1 -associated HCCs.

DNA Polymerase λ Inactivation by Oxidized Abasic Sites&

PubMed Central

Stevens, Adam J.; Guan, Lirui; Bebenek, Katarzyna; Kunkel, Thomas A.; Greenberg, Marc M.

2013-01-01

Base excision repair plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ is believed to play a backup role to DNA polymerase β in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA damaging agents, including several antitumor antibiotics, the C4′-oxidized abasic site following Ape1 incision (pC4-AP) and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single turnover kinetic experiments show that Pol λ excises DOB almost 13-times more slowly than a 5′-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β at the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β and the difference is attributable to a higher KI (33 ± 7 nM). Inactivation of Pol λ’s lyase activity by DOB also prevents the enzyme from carrying out polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. Protein modification involves reaction with released but-2-ene-1,4-dial. When acting on pC4-AP, Pol λ achieves approximately 4 turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA damaging agents that produce them. PMID:23330920

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

PubMed

Guo, Xiaoge; Jinks-Robertson, Sue

2013-12-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations. Copyright © 2013 Elsevier B.V. All rights reserved.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

PubMed Central

Guo, Xiaoge; Jinks-Robertson, Sue

2013-01-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral introduction of UV damage can potentially compromise genetic interpretations. PMID:24120148

Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

2002-03-01

Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appearsmore » to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.« less

Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schormann, Norbert; Zhukovskaya, Natalia; Bedwell, Gregory

We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymaticmore » function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.« less

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

PubMed

Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

2011-01-01

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

Ada response – a strategy for repair of alkylated DNA in bacteria

PubMed Central

Mielecki, Damian; Grzesiuk, Elżbieta

2014-01-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

PubMed Central

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

Background Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Methods Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. Results No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. Conclusion BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria. PMID:27598300

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

PubMed

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria.

Infrequent transposition of Ac in lettuce, Lactuca sativa.

PubMed

Yang, C H; Ellis, J G; Michelmore, R W

1993-08-01

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R1 plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2' promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

PubMed

Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings.

PubMed Central

Quaite, F. E.; Takayanagi, S.; Ruffini, J.; Sutherland, J. C.; Sutherland, B. M.

1994-01-01

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage. PMID:12244228

Uracil-DNA Glycosylase in Base Excision Repair and Adaptive Immunity

PubMed Central

Doseth, Berit; Visnes, Torkild; Wallenius, Anders; Ericsson, Ida; Sarno, Antonio; Pettersen, Henrik Sahlin; Flatberg, Arnar; Catterall, Tara; Slupphaug, Geir; Krokan, Hans E.; Kavli, Bodil

2011-01-01

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung+/+ and Ung−/− backcrossed mice. Interestingly, human cells displayed ∼15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ∼8-fold higher in mouse cells, constituting ∼50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung−/− mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity. PMID:21454529

The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen-Induced DNA-DNA Cross-Links in a Four-Stranded DNA Structure.

PubMed

Martin, Peter R; Couvé, Sophie; Zutterling, Caroline; Albelazi, Mustafa S; Groisman, Regina; Matkarimov, Bakhyt T; Parsons, Jason L; Elder, Rhoderick H; Saparbaev, Murat K

2017-12-12

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

Differential role of base excision repair proteins in mediating cisplatin cytotoxicity.

PubMed

Sawant, Akshada; Floyd, Ashley M; Dangeti, Mohan; Lei, Wen; Sobol, Robert W; Patrick, Steve M

2017-03-01

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA Repair in Drosophila: Mutagens, Models, and Missing Genes

PubMed Central

Sekelsky, Jeff

2017-01-01

The numerous processes that damage DNA are counterbalanced by a complex network of repair pathways that, collectively, can mend diverse types of damage. Insights into these pathways have come from studies in many different organisms, including Drosophila melanogaster. Indeed, the first ideas about chromosome and gene repair grew out of Drosophila research on the properties of mutations produced by ionizing radiation and mustard gas. Numerous methods have been developed to take advantage of Drosophila genetic tools to elucidate repair processes in whole animals, organs, tissues, and cells. These studies have led to the discovery of key DNA repair pathways, including synthesis-dependent strand annealing, and DNA polymerase theta-mediated end joining. Drosophila appear to utilize other major repair pathways as well, such as base excision repair, nucleotide excision repair, mismatch repair, and interstrand crosslink repair. In a surprising number of cases, however, DNA repair genes whose products play important roles in these pathways in other organisms are missing from the Drosophila genome, raising interesting questions for continued investigations. PMID:28154196

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences*

PubMed Central

Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

2015-01-01

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. PMID:26338705

Meiotic DNA Metabolism in Wild-Type and Excision-Deficient Yeast following Uv Exposure

PubMed Central

Resnick, Michael A.; Stasiewicz, Stanley; Game, John C.

1983-01-01

The effects of UV irradiation on DNA metabolism during meiosis have been examined in wild-type (RAD+) and mitotically defined excision-defective (rad1-1) strains of Saccharomyces cerevisiae that exhibit high levels of sporulation. The rad1-1 gene product is not required for normal meiosis: DNA synthesis, RNA synthesis, size of parental and newly synthesized DNA and sporulation are comparable in RAD+ and rad1-1 strains. Cells were UV irradiated at the beginning of meiosis, and the fate of UV-induced pyrimidine dimers as well as changes in DNA and DNA synthesis were followed during meiosis. Excision repair of pyrimidine dimers can occur during meiosis and the RAD1 gene product is required; alternate excision pathways do not exist. Although the rate of elongation is decreased, the presence of pyrimidine dimers during meiosis in the rad1-1 strain does not block meiotic DNA synthesis suggesting a bypass mechanism. The final size of DNA is about five times the distance between pyrimidine dimers after exposure to 4 J/m2. Since pyrimidine dimers induced in parental strands of rad1-1 prior to premeiotic DNA synthesis do not become associated with newly synthesized DNA, the mechanism for replicational bypass does not appear to involve a recombinational process. The absence of such association indicates that normal meiotic recombination is also suppressed by UV-induced damage in DNA; this result at the molecular level is supported by observations at the genetic level. PMID:6352404

Crystal Structures of Two Archaeal 8-Oxoguanine DNA Glycosylases Provide Structural Insight into Guanine/8-Oxoguanine Distinction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faucher, Frédérick; Duclos, Stéphanie; Bandaru, Viswanath

Among the four DNA bases, guanine is particularly vulnerable to oxidative damage and the most common oxidative product, 7,8-dihydro-8-oxoguanine (8-oxoG), is the most prevalent lesion observed in DNA molecules. Fortunately, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Ogg enzymes are divided into three separate families, namely, Ogg1, Ogg2, and archaeal GO glycosylase (AGOG). To date, structures of members of both Ogg1 and AGOG families are known but no structural information is available for members of Ogg2. Here we describe the first crystal structures of two archaeal Ogg2: Methanocaldococcus janischii Oggmore » and Sulfolobus solfataricus Ogg. A structural comparison with OGG1 and AGOG suggested that the C-terminal lysine of Ogg2 may play a key role in discriminating between guanine and 8-oxoG. This prediction was substantiated by measuring the glycosylase/lyase activity of a C-terminal deletion mutant of MjaOgg.« less

Integration of single-molecule detection with magnetic separation for multiplexed detection of DNA glycosylases.

PubMed

Li, Chen-Chen; Zhang, Yan; Tang, Bo; Zhang, Chun-Yang

2018-06-05

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

Ada response - a strategy for repair of alkylated DNA in bacteria.

PubMed

Mielecki, Damian; Grzesiuk, Elżbieta

2014-06-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

PubMed Central

Yang, Di; Fletcher, Sally C.; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J.

2017-01-01

Abstract Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. PMID:28973444

DNA Repair Deficiency in Neurodegeneration

PubMed Central

Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A.; Stevnsner, Tinna

2011-01-01

Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby causing Huntington’s disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair: homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage may be linked with the age-associated neurodegenerative disorders Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Mutation in the WRN protein leads to the premature aging disease Werner syndrome, a disorder that features neurodegeneration. In this article we review the evidence linking deficiencies in the DNA repair pathways with neurodegeneration. PMID:21550379

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faucher, Frédérick; Robey-Bond, Susan M.; Wallace, Susan S.

DNA is subject to a multitude of oxidative damages generated by oxidizing agents from metabolism and exogenous sources and by ionizing radiation. Guanine is particularly vulnerable to oxidation, and the most common oxidative product 8-oxoguanine (8-oxoG) is the most prevalent lesion observed in DNA molecules. 8-OxoG can form a normal Watson-Crick pair with cytosine (8-oxoG:C), but it can also form a stable Hoogsteen pair with adenine (8-oxoG:A), leading to a G:C {yields} T:A transversion after replication. Fortunately, 8-oxoG is recognized and excised by either of two DNA glycosylases of the base excision repair pathway: formamidopyrimidine-DNA glycosylase and 8-oxoguanine DNA glycosylasemore » (Ogg). While Clostridium acetobutylicum Ogg (CacOgg) DNA glycosylase can specifically recognize and remove 8-oxoG, it displays little preference for the base opposite the lesion, which is unusual for a member of the Ogg1 family. This work describes the crystal structures of CacOgg in its apo form and in complex with 8-oxo-2'-deoxyguanosine. A structural comparison between the apo form and the liganded form of the enzyme reveals a structural reorganization of the C-terminal domain upon binding of 8-oxoG, similar to that reported for human OGG1. A structural comparison of CacOgg with human OGG1, in complex with 8-oxoG containing DNA, provides a structural rationale for the lack of opposite base specificity displayed by CacOgg.« less

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

PubMed Central

Shell, Steven M.; Hawkins, Edward K.; Tsai, Miaw-Sheue; Hlaing, Aye Su; Rizzo, Carmelo J.; Chazin, Walter J.

2013-01-01

The xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results support an indirect read-out model for sensing the presence of lesions by human XPC and suggest XPC may act as a general sensor of damaged DNA capable of recognizing DNA containing lesions not repaired by NER. PMID:24051049

Alkyltransferase-like proteins: brokers dealing with alkylated DNA bases.

PubMed

Schärer, Orlando D

2012-07-13

A new pathway for the repair of DNA alkylation damage is described in this issue of Molecular Cell (Latypov et al., 2012). Alkyltransferase-like enzymes mark O(6)-alkylguanine lesions and, depending on adduct size, channel them into global genome or transcription-coupled nucleotide excision repair pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

A multistep damage recognition mechanism for global genomic nucleotide excision repair

PubMed Central

Sugasawa, Kaoru; Okamoto, Tomoko; Shimizu, Yuichiro; Masutani, Chikahide; Iwai, Shigenori; Hanaoka, Fumio

2001-01-01

A mammalian nucleotide excision repair (NER) factor, the XPC–HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC–HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC–HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC–HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC–HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC–HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER. PMID:11238373

A multistep damage recognition mechanism for global genomic nucleotide excision repair.

PubMed

Sugasawa, K; Okamoto, T; Shimizu, Y; Masutani, C; Iwai, S; Hanaoka, F

2001-03-01

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

Cytological evidence for DNA chain elongation after UV irradiation in the S phase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minka, D.F.; Nath, J.

1981-04-01

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G2, and the cells weremore » labeled with /sup 3/H-thymidine. Results showed significantly increased labeling during G2 of excision-deficient XP cells. Labeling was dependent on the time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.« less

Visualizing the Search for Radiation-damaged DNA Bases in Real Time.

PubMed

Lee, Andrea J; Wallace, Susan S

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Visualizing the search for radiation-damaged DNA bases in real time

NASA Astrophysics Data System (ADS)

Lee, Andrea J.; Wallace, Susan S.

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

PubMed

Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

2015-10-23

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate.

PubMed

Stanczyk, M; Sliwinski, T; Trelinska, J; Cuchra, M; Markiewicz, L; Dziki, L; Bieniek, A; Bielecka-Kowalska, A; Kowalski, M; Pastorczak, A; Szemraj, J; Mlynarski, W; Majsterek, I

2012-01-24

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL. © 2011 Elsevier B.V. All rights reserved.

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

PubMed Central

Parrilla-Doblas, Jara Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa

2017-01-01

ABSTRACT DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells. PMID:28277978

Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

PubMed Central

Rastogi, Rajesh P.; Richa; Kumar, Ashok; Tyagi, Madhu B.; Sinha, Rajeshwar P.

2010-01-01

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms. PMID:21209706

Nuclear organization of nucleotide excision repair is mediated by RING1B dependent H2A-ubiquitylation

PubMed Central

Chitale, Shalaka; Richly, Holger

2017-01-01

One of the major cellular DNA repair pathways is nucleotide excision repair (NER). It is the primary pathway for repair of various DNA lesions caused by exposure to ultraviolet (UV) light, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts. Although lesion-containing DNA associates with the nuclear matrix after UV irradiation it is still not understood how nuclear organization affects NER. Analyzing unscheduled DNA synthesis (UDS) indicates that NER preferentially occurs in specific nuclear areas, viz the nucleolus. Upon inducing localized damage, we observe migration of damaged DNA towards the nucleolus. Employing a LacR-based tethering system we demonstrate that H2A-ubiquitylation via the UV-RING1B complex localizes chromatin close to the nucleolus. We further show that the H2A-ubiquitin binding protein ZRF1 resides in the nucleolus, and that it anchors ubiquitylated chromatin along with XPC. Our data thus provide insight into the sub-nuclear organization of NER and reveal a novel role for histone H2A-ubiquitylation. PMID:28416769

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

NASA Technical Reports Server (NTRS)

Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

1999-01-01

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

Base Excision Repair Variants in Cancer

PubMed Central

Marsden, Carolyn G.; Dragon, Julie A.; Wallace, Susan S.; Sweasy, Joann B.

2018-01-01

Base excision repair (BER) is a key genome maintenance pathway that removes endogenously damaged DNA bases that arise in cells at very high levels on a daily basis. Failure to remove these damaged DNA bases leads to increased levels of mutagenesis and chromosomal instability, which have the potential to drive carcinogenesis. Next Generation sequencing efforts of the germline and tumors genomes of thousands of individuals has uncovered many rare mutations in BER genes. Given that BER is critical for genome maintenance, it is important to determine whether BER genomic variants have functional phenotypes. In this chapter we present our in silico methods for the identification and prioritization of BER variants for further study. We also provide detailed instructions and commentary on the initial cellular assays we employ to dissect potentially important phenotypes of human BER variants and highlight the strengths and weaknesses of our approaches. BER variants possessing interesting functional phenotypes can then be studied in more detail to provide important mechanistic insights regarding the role of aberrant BER in carcinogenesis. PMID:28645367

Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

PubMed Central

Shpakovski, G V; Berlin, Y A

1984-01-01

Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed. Images PMID:6091038

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms.

PubMed

Azevedo, Ana P; Silva, Susana N; De Lima, João P; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-06-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog ( E. coli ) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms

PubMed Central

Azevedo, Ana P.; Silva, Susana N.; De Lima, João P.; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-01-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility. PMID:28599464

A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

PubMed Central

Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

2011-01-01

DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products.

PubMed

Lee, Yujeong; Klauser, Paul C; Brandsen, Benjamin M; Zhou, Cong; Li, Xinyi; Silverman, Scott K

2017-01-11

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N 40 or N 100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O 2 -• or HOO • ) and hydrogen peroxide (H 2 O 2 ) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H 2 O 2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H 2 O 2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn 2+ and Mg 2+ , although including Mn 2+ increases the activity, and Mn 2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

PubMed

Bustamante, Paula; Covarrubias, Paulo C; Levicán, Gloria; Katz, Assaf; Tapia, Pablo; Holmes, David; Quatrini, Raquel; Orellana, Omar

2012-01-01

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation. Copyright © 2013 S. Karger AG, Basel.

Mutagenic cost of ribonucleotides in bacterial DNA

PubMed Central

Schroeder, Jeremy W.; Randall, Justin R.; Hirst, William G.; O’Donnell, Michael E.; Simmons, Lyle A.

2017-01-01

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genome-wide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand- and sequence-context–dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE. PMID:29078353

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

PubMed Central

Livingston, Alison L.; O’Shea, Valerie L.; Kim, Taewoo; Kool, Eric T.; David, Sheila S.

2009-01-01

Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication. PMID:18026095

A Protective Mechanism of Visible Red Light in Normal Human Dermal Fibroblasts: Enhancement of GADD45A-Mediated DNA Repair Activity.

PubMed

Kim, Yeo Jin; Kim, Hyoung-June; Kim, Hye Lim; Kim, Hyo Jeong; Kim, Hyun Soo; Lee, Tae Ryong; Shin, Dong Wook; Seo, Young Rok

2017-02-01

The phototherapeutic effects of visible red light on skin have been extensively investigated, but the underlying biological mechanisms remain poorly understood. We aimed to elucidate the protective mechanism of visible red light in terms of DNA repair of UV-induced oxidative damage in normal human dermal fibroblasts. The protective effect of visible red light on UV-induced DNA damage was identified by several assays in both two-dimensional and three-dimensional cell culture systems. With regard to the protective mechanism of visible red light, our data showed alterations in base excision repair mediated by growth arrest and DNA damage inducible, alpha (GADD45A). We also observed an enhancement of the physical activity of GADD45A and apurinic/apyrimidinic endonuclease 1 (APE1) by visible red light. Moreover, UV-induced DNA damages were diminished by visible red light in an APE1-dependent manner. On the basis of the decrease in GADD45A-APE1 interaction in the activating transcription factor-2 (ATF2)-knockdown system, we suggest a role for ATF2 modulation in GADD45A-mediated DNA repair upon visible red light exposure. Thus, the enhancement of GADD45A-mediated base excision repair modulated by ATF2 might be a potential protective mechanism of visible red light. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of topical application of sulfur mustard on mice skin and distant organs DNA repair enzyme signature.

PubMed

Sauvaigo, Sylvie; Sarrazy, Fanny; Batal, Mohamed; Caillat, Sylvain; Pitiot, Benoit; Mouret, Stéphane; Cléry-Barraud, Cécile; Boudry, Isabelle; Douki, Thierry

2016-01-22

Sulfur mustard (SM) is a chemical warfare agent that, upon topical application, damages skin and reaches internal organs through diffusion in blood. Two major toxic consequences of SM exposure are inflammation, associated with oxidative stress, and the formation of alkylated DNA bases. In the present study, we investigated the impact of exposure to SM on DNA repair, using two different functional DNA repair assays which provide information on several Base Excision Repair (BER) and Excision/Synthesis Repair (ESR) activities. BER activities were reduced in all organs as early as 4h after exposure, with the exception of the defense systems against 8-oxo-guanine and hypoxanthine which were stimulated. Interestingly, the resulting BER intermediates could activate inflammation signals, aggravating the inflammation triggered by SM exposure and leading to increased oxidative stress. ESR activities were found to be mostly inhibited in skin, brain and kidneys. In contrast, in the lung there was a general increase in ESR activities. In summary, exposure to SM leads to a significant decrease in DNA repair in most organs, concomitant with the formation of DNA damage. These synergistic genotoxic effects are likely to participate in the high toxicity of this alkylating agent. Lungs, possibly better equipped with repair enzymes to handle exogenous exposure, are the exception. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

PubMed

Poletto, Mattia; Yang, Di; Fletcher, Sally C; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J; Dianov, Grigory L

2017-09-29

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hide and seek: How do DNA glycosylases locate oxidatively damaged DNA bases amidst a sea of undamaged bases?

PubMed

Lee, Andrea J; Wallace, Susan S

2017-06-01

The first step of the base excision repair (BER) pathway responsible for removing oxidative DNA damage utilizes DNA glycosylases to find and remove the damaged DNA base. How glycosylases find the damaged base amidst a sea of undamaged bases has long been a question in the BER field. Single molecule total internal reflection fluorescence microscopy (SM TIRFM) experiments have allowed for an exciting look into this search mechanism and have found that DNA glycosylases scan along the DNA backbone in a bidirectional and random fashion. By comparing the search behavior of bacterial glycosylases from different structural families and with varying substrate specificities, it was found that glycosylases search for damage by periodically inserting a wedge residue into the DNA stack as they redundantly search tracks of DNA that are 450-600bp in length. These studies open up a wealth of possibilities for further study in real time of the interactions of DNA glycosylases and other BER enzymes with various DNA substrates. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

PubMed Central

Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

2016-01-01

DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

PubMed

Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar

2017-06-01

Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin? Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins.

PubMed

O'Rourke, Thomas W; Doudican, Nicole A; Mackereth, Melinda D; Doetsch, Paul W; Shadel, Gerald S

2002-06-01

The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. Copyright © 2016 Elsevier B.V. All rights reserved.

Polymerization by DNA polymerase eta is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells.

PubMed

Chijiwa, Shotaro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori; Kuraoka, Isao

2010-03-01

cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

PubMed Central

Friboulet, Luc; Postel-Vinay, Sophie; Sourisseau, Tony; Adam, Julien; Stoclin, Annabelle; Ponsonnailles, Florence; Dorvault, Nicolas; Commo, Frédéric; Saulnier, Patrick; Salome-Desmoulez, Sophie; Pottier, Géraldine; André, Fabrice; Kroemer, Guido; Soria, Jean Charles; Olaussen, Ken André

2013-01-01

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies. PMID:24036546

Mitochondrial DNA repair and damage tolerance.

PubMed

Stein, Alexis; Sia, Elaine A

2017-01-01

The accurate maintenance of mitochondrial DNA (mtDNA) is required in order for eukaryotic cells to assemble a functional electron transport chain. This independently-maintained genome relies on nuclear-encoded proteins that are imported into the mitochondria to carry out replication and repair processes. Decades of research has made clear that mitochondria employ robust and varied mtDNA repair and damage tolerance mechanisms in order to ensure the proper maintenance of the mitochondrial genome. This review focuses on our current understanding of mtDNA repair and damage tolerance pathways including base excision repair, mismatch repair, homologous recombination, non-homologous end joining, translesion synthesis and mtDNA degradation in both yeast and mammalian systems.

Premature aging and cancer in nucleotide excision repair-disorders

PubMed Central

Diderich, K.; Alanazi, M.; Hoeijmakers, J.H.J.

2014-01-01

During past decades the major impact of DNA damage on cancer as ‘disease of the genes’ has become abundantly apparent. In addition to cancer recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems not only protect against cancer but equally against too fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in man. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging. PMID:21680258

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

PubMed

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

PubMed Central

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E.; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated. PMID:23071448

Base Excision Repair Facilitates a Functional Relationship Between Guanine Oxidation and Histone Demethylation

PubMed Central

Li, Jianfeng; Braganza, Andrea

2013-01-01

Abstract Significance: Appropriately controlled epigenetic regulation is critical for the normal development and health of an organism. Misregulation of epigenetic control via deoxyribonucleic acid (DNA) methylation or histone methylation has been associated with cancer and chromosomal instability syndromes. Recent Advances: The main function of the proteins in the base excision repair (BER) pathway is to repair DNA single-strand breaks and deamination, oxidation, and alkylation-induced DNA base damage that may result from chemotherapy, environmental exposure, or byproducts of cellular metabolism. Recent studies have suggested that one or more BER proteins may also participate in epigenetic regulation to facilitate gene expression modulation via alteration of the state of DNA methylation or via a reaction coupled to histone modification. BER proteins have also been reported to play an essential role in pluripotent stem cell reprogramming. Critical Issues: One emerging function for BER in epigenetic regulation is the repair of base lesions induced by hydrogen peroxide as a byproduct of lysine-specific demethylase 1 (LSD1) enzymatic activity (LSD1/LSD2-coupled BER) for transcriptional regulation. Future Directions: To shed light on this novel role of BER, this review focuses on the repair of oxidative lesions in nuclear DNA that are induced during LSD1-mediated histone demethylation. Further, we highlight current studies suggesting a role for BER proteins in transcriptional regulation of gene expression via BER-coupled active DNA demethylation in mammalian cells. Such efforts to address the role of BER proteins in epigenetic regulation could broaden cancer therapeutic strategies to include epigenetic modifiers combined with BER inhibitors. Antioxid. Redox Signal. 18, 2429–2443. PMID:23311711

Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

PubMed Central

Duret, Laurent; Cohen, Jean; Jubin, Claire; Dessen, Philippe; Goût, Jean-François; Mousset, Sylvain; Aury, Jean-Marc; Jaillon, Olivier; Noël, Benjamin; Arnaiz, Olivier; Bétermier, Mireille; Wincker, Patrick; Meyer, Eric; Sperling, Linda

2008-01-01

Ciliates are the only unicellular eukaryotes known to separate germinal and somatic functions. Diploid but silent micronuclei transmit the genetic information to the next sexual generation. Polyploid macronuclei express the genetic information from a streamlined version of the genome but are replaced at each sexual generation. The macronuclear genome of Paramecium tetraurelia was recently sequenced by a shotgun approach, providing access to the gene repertoire. The 72-Mb assembly represents a consensus sequence for the somatic DNA, which is produced after sexual events by reproducible rearrangements of the zygotic genome involving elimination of repeated sequences, precise excision of unique-copy internal eliminated sequences (IES), and amplification of the cellular genes to high copy number. We report use of the shotgun sequencing data (>106 reads representing 13× coverage of a completely homozygous clone) to evaluate variability in the somatic DNA produced by these developmental genome rearrangements. Although DNA amplification appears uniform, both of the DNA elimination processes produce sequence heterogeneity. The variability that arises from IES excision allowed identification of hundreds of putative new IESs, compared to 42 that were previously known, and revealed cases of erroneous excision of segments of coding sequences. We demonstrate that IESs in coding regions are under selective pressure to introduce premature termination of translation in case of excision failure. PMID:18256234

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

PubMed Central

Liu, Geyi; Aronovich, Elena L.; Cui, Zongbin; Whitley, Chester B.; Hackett, Perry B.

2007-01-01

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase–transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. PMID:15133768

Impact of DNA repair on the dose-response of colorectal cancer formation induced by dietary carcinogens.

PubMed

Fahrer, Jörg; Kaina, Bernd

2017-08-01

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers, which is causally linked to dietary habits, notably the intake of processed and red meat. Processed and red meat contain dietary carcinogens, including heterocyclic aromatic amines (HCAs) and N-nitroso compounds (NOC). NOC are agents that induce various N-methylated DNA adducts and O 6 -methylguanine (O 6 -MeG), which are removed by base excision repair (BER) and O 6 -methylguanine-DNA methyltransferase (MGMT), respectively. HCAs such as the highly mutagenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) cause bulky DNA adducts, which are removed from DNA by nucleotide excision repair (NER). Both O 6 -MeG and HCA-induced DNA adducts are linked to the occurrence of KRAS and APC mutations in colorectal tumors of rodents and humans, thereby driving CRC initiation and progression. In this review, we focus on DNA repair pathways removing DNA lesions induced by NOC and HCA and assess their role in protecting against mutagenicity and carcinogenicity in the large intestine. We further discuss the impact of DNA repair on the dose-response relationship in colorectal carcinogenesis in view of recent studies, demonstrating the existence of 'no effect' point of departures (PoDs), i.e. thresholds for genotoxicity and carcinogenicity. The available data support the threshold concept for NOC with DNA repair being causally involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reversing DNA Methylation: Mechanisms, Genomics, and Biological Functions

PubMed Central

Wu, Hao; Zhang, Yi

2014-01-01

Methylation of cytosines in the mammalian genome represents a key epigenetic modification and is dynamically regulated during development. Compelling evidence now suggests that dynamic regulation of DNA methylation is mainly achieved through a cyclic enzymatic cascade comprised of cytosine methylation, iterative oxidation of methyl group by TET dioxygenases, and restoration of unmodified cytosines by either replication-dependent dilution or DNA glycosylase-initiated base excision repair. In this review, we discuss the mechanism and function of DNA demethylation in mammalian genomes, focusing particularly on how developmental modulation of the cytosine-modifying pathway is coupled to active reversal of DNA methylation in diverse biological processes. PMID:24439369

Isolation of a small molecule inhibitor of DNA base excision repair

PubMed Central

Madhusudan, Srinivasan; Smart, Fiona; Shrimpton, Paul; Parsons, Jason L.; Gardiner, Laurence; Houlbrook, Sue; Talbot, Denis C.; Hammonds, Timothy; Freemont, Paul A.; Sternberg, Michael J. E.; Dianov, Grigory L.; Hickson, Ian D.

2005-01-01

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3′-phosphodiesterase and 3′-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy. PMID:16113242

Properties of uvrE mutants of Escherichia coli K12. Part 1. Effects of uv irradiation on DNA metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vansluis, C.A.; Mattern, I.E.; Paterson, M.C.

1974-01-01

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet radiation. In an attempt to determine the underlying molecular basis for the UV sensitivity, a mutant and an isogenic wild type strain were compared with regard to several metabolic responses to 254 nm radiation. The introduction of single strand breaks into intracellular DNA after irradiation is normal; however, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain. These data suggest that the uvrEmore » gene product may be involved in a reaction between the incision and excision steps in the excision repair process. (Author) (GRA)« less

Expression and function of AtMBD4L, the single gene encoding the nuclear DNA glycosylase MBD4L in Arabidopsis.

PubMed

Nota, Florencia; Cambiagno, Damián A; Ribone, Pamela; Alvarez, María E

2015-06-01

DNA glycosylases recognize and excise damaged or incorrect bases from DNA initiating the base excision repair (BER) pathway. Methyl-binding domain protein 4 (MBD4) is a member of the HhH-GPD DNA glycosylase superfamily, which has been well studied in mammals but not in plants. Our knowledge on the plant enzyme is limited to the activity of the Arabidopsis recombinant protein MBD4L in vitro. To start evaluating MBD4L in its biological context, we here characterized the structure, expression and effects of its gene, AtMBD4L. Phylogenetic analysis indicated that AtMBD4L belongs to one of the seven families of HhH-GPD DNA glycosylase genes existing in plants, and is unique on its family. Two AtMBD4L transcripts coding for active enzymes were detected in leaves and flowers. Transgenic plants expressing the AtMBD4L:GUS gene confined GUS activity to perivascular leaf tissues (usually adjacent to hydathodes), flowers (anthers at particular stages of development), and the apex of immature siliques. MBD4L-GFP fusion proteins showed nuclear localization in planta. Interestingly, overexpression of the full length MBD4L, but not a truncated enzyme lacking the DNA glycosylase domain, induced the BER gene LIG1 and enhanced tolerance to oxidative stress. These results suggest that endogenous MBD4L acts on particular tissues, is capable of activating BER, and may contribute to repair DNA damage caused by oxidative stress. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Base Excision Repair of Tandem Modifications in a Methylated CpG Dinucleotide*

PubMed Central

Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S.; Beard, William A.; Wilson, Samuel H.

2014-01-01

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3′-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5′-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide. PMID:24695738

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea

PubMed Central

Jones, Daniel L.; Baxter, Bonnie K.

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a “first line of defense,” and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms. PMID:29033920

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

PubMed

Gupta, Rakesh Kumar; Bajpai, Deepti; Singh, Neeta

2015-01-01

Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. SiHa cells were cultured and treated with 10% Noni, 10 μg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea.

PubMed

Jones, Daniel L; Baxter, Bonnie K

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a "first line of defense," and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms.

Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells

PubMed Central

Weng, Mao-wen; Hu, Yu; Chen, Wei-sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C.; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A.; Tang, Moon-shong

2014-01-01

Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS. PMID:24939871

[DNA damage in human pleural mesothelial cells induced by exposure to carbon nanotubes].

PubMed

Ogasawara, Yuki; Umezu, Noriaki; Ishii, Kazuyuki

2012-01-01

Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

PubMed

Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

2017-06-19

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

PubMed Central

2017-01-01

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54(34), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER. PMID:28460163

A significant role of non-thermal equilibrated electrons in the formation of deleterious complex DNA damage.

PubMed

Kai, Takeshi; Yokoya, Akinari; Ukai, Masatoshi; Fujii, Kentaro; Toigawa, Tomohiro; Watanabe, Ritsuko

2018-01-24

Although most of the radiation damage to genomic DNA could be rendered harmless using repair enzymes in a living cell, a certain fraction of the damage is persistent resulting in serious genetic effects, such as mutation induction. In order to understand the mechanisms of the deleterious DNA damage formation in terms of its earliest physical stage at the radiation track end, dynamics of low energy electrons and their thermalization processes around DNA molecules were investigated using a dynamic Monte Carlo code. The primary incident (1 keV) electrons multiply collide within 1 nm (equivalent to three DNA-base-pairs, 3bp) and generate secondary electrons which show non-Gaussian and non-thermal equilibrium distributions within 300 fs. On the other hand, the secondary electrons are mainly distributed within approximately 10 nm from their parent cations although approximately 5% of the electrons are localized within 1 nm of the cations owing to the interaction of their Coulombic fields. The mean electron energy is 0.7 eV; however, more than 10% of the electrons fall into a much lower-energy region than 0.1 eV at 300 fs. These results indicate that pre-hydrated electrons are formed from the extremely decelerated electrons over a few nm from the cations. DNA damage sites comprising multiple nucleobase lesions or single strand breaks can therefore be formed by multiple collisions of these electrons within 3bp. This multiple damage site is hardly processed by base excision repair enzymes. However, pre-hydrated electrons can also be produced resulting in an additional base lesion (or a strand break) more than 3bp away from the multi-damage site. These damage sites may be finally converted into a double strand break (DSB) when base excision enzymes process the additional base lesions. This DSB includes another base lesion(s) at their termini, and may introduce miss-rejoining by DSB repair enzymes, and hence may result in biological effects such as mutation in surviving cells.

Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

PubMed Central

Goellner, Eva M.; Grimme, Bradford; Brown, Ashley R.; Lin, Ying-Chih; Wang, Xiao-Hong; Sugrue, Kelsey F.; Mitchell, Leah; Trivedi, Ram N.; Tang, Jiang-bo; Sobol, Robert W.

2011-01-01

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O6-methylguanine (O6-MeG) due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. Based on our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of NAD+, we have hypothesized that combined BER and NAD+ biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD+ biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD+ biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM. PMID:21406402

Communication: Site-selective bond excision of adenine upon electron transfer

NASA Astrophysics Data System (ADS)

Cunha, T.; Mendes, M.; Ferreira da Silva, F.; Eden, S.; García, G.; Limão-Vieira, P.

2018-01-01

This work demonstrates that selective excision of hydrogen atoms at a particular site of the DNA base adenine can be achieved in collisions with electronegative atoms by controlling the impact energy. The result is based on analysing the time-of-flight mass spectra yields of potassium collisions with a series of labeled adenine derivatives. The production of dehydrogenated parent anions is consistent with neutral H loss either from selective breaking of C-H or N-H bonds. These unprecedented results open up a new methodology in charge transfer collisions that can initiate selective reactivity as a key process in chemical reactions that are dominant in different areas of science and technology.

Base excision repair, the redox environment and therapeutic implications.

PubMed

Storr, S J; Woolston, C M; Martin, S G

2012-01-01

Control of redox homeostasis is crucial for a number of cellular processes with deregulation leading to a number of serious consequences including oxidative damage such induction of DNA base lesions. The DNA lesions caused by oxidative damage are principally repaired by the base excision repair (BER) pathway. Pharmacological inhibition of BER is becoming an increasingly active area of research with the emergence of PARP inhibitors in cancer therapy. The redox status of the cell is modulated by a number of systems, including a large number of anti-oxidant enzymes who function in the control of superoxide and hydrogen peroxide, and ultimately in the release of the damaging hydroxyl radical. Here we provide an overview of reactive oxygen species (ROS) production and its modulation by antioxidant enzymes. The review also discusses the effect of ROS on the BER pathway, particularly in relation to cancer. Finally, as the modulation of the redox environment is of interest in cancer therapy, with certain agents having the potential to reverse chemo- and radiotherapy resistance or treat therapy related toxicity, we discuss redox modulating agents currently under development.

The Fpg/Nei family of DNA glycosylases: substrates, structures, and search for damage.

PubMed

Prakash, Aishwarya; Doublié, Sylvie; Wallace, Susan S

2012-01-01

During the initial stages of the base excision DNA repair pathway, DNA glycosylases are responsible for locating and removing the majority of endogenous oxidative base lesions. The bifunctional formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of the Fpg/Nei family, one of the two families of glycosylases that recognize oxidized DNA bases, the other being the HhH/GPD (or Nth) superfamily. Structural and biochemical developments over the past decades have led to novel insights into the mechanism of damage recognition by the Fpg/Nei family of enzymes. Despite the overall structural similarity among members of this family, these enzymes exhibit distinct features that make them unique. This review summarizes the current structural knowledge of the Fpg/Nei family members, emphasizes their substrate specificities, and describes how these enzymes search for lesions. Copyright © 2012 Elsevier Inc. All rights reserved.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

USGS Publications Warehouse

Kienzler, Aude; Mahler, Barbara J.; Van Metre, Peter C.; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-01-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line.

PubMed

Kienzler, Aude; Mahler, Barbara J; Van Metre, Peter C; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-07-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermodynamic Signature of DNA Damage: Characterization of DNA with a 5-Hydroxy-2'-deoxycytidine•2'-Deoxyguanosine Base Pair

PubMed Central

Ganguly, Manjori; Szulik, Marta W.; Donahue, Patrick S.; Clancy, Kate; Stone, Michael P.; Gold, Barry

2012-01-01

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2'-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2'-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and NMR results presented herein, describe the effect of a 5-hydroxy-2'-deoxycytidine•2'-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2'-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that require the lesion to be extrahelical. PMID:22332945

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

PubMed Central

Sobol, Robert W.; Watson, David E.; Nakamura, Jun; Yakes, F. Michael; Hou, Esther; Horton, Julie K.; Ladapo, Joseph; Van Houten, Bennett; Swenberg, James A.; Tindall, Kenneth R.; Samson, Leona D.; Wilson, Samuel H.

2002-01-01

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N′-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure. PMID:11983862

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

PubMed

Morris, Lydia P; Conley, Andrew B; Degtyareva, Natalya; Jordan, I King; Doetsch, Paul W

2017-11-01

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

PubMed Central

Le May, Nicolas; Egly, Jean-Marc; Coin, Frédéric

2010-01-01

Nucleotide excision repair (NER) is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α) and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process. PMID:20725631

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

PubMed Central

Gonzalez, Kaisha; Faustoferri, Roberta C.; Quivey, Robert G.

2012-01-01

Summary The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defenses for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2’-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY, or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the G. mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared to the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism. PMID:22651851

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation,more » providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.« less

Newly identified CHO ERCC3/XPB mutations and phenotype characterization

PubMed Central

Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

2010-01-01

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

DOE PAGES

Cunniffe, Siobhan; O’Neill, Peter; Greenberg, Marc M.; ...

2014-04-01

A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repairmore » is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.« less

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins.

PubMed

Bowen, Nikki; Kolodner, Richard D

2017-04-04

Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using S. cerevisiae proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

PubMed

Cazzalini, Ornella; Scovassi, A Ivana; Savio, Monica; Stivala, Lucia A; Prosperi, Ennio

2010-01-01

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response. 2010 Elsevier B.V. All rights reserved.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases.

PubMed

Boiteux, Serge; Coste, Franck; Castaing, Bertrand

2017-06-01

Oxidatively damaged DNA results from the attack of sugar and base moieties by reactive oxygen species (ROS), which are formed as byproducts of normal cell metabolism and during exposure to endogenous or exogenous chemical or physical agents. Guanine, having the lowest redox potential, is the DNA base the most susceptible to oxidation, yielding products such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2-6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). In DNA, 8-oxoG was shown to be mutagenic yielding GC to TA transversions upon incorporation of dAMP opposite this lesion by replicative DNA polymerases. In prokaryotic and eukaryotic cells, 8-oxoG is primarily repaired by the base excision repair pathway (BER) initiated by a DNA N-glycosylase, Fpg and OGG1, respectively. In Escherichia coli, Fpg cooperates with MutY and MutT to prevent 8-oxoG-induced mutations, the "GO-repair system". In Saccharomyces cerevisiae, OGG1 cooperates with nucleotide excision repair (NER), mismatch repair (MMR), post-replication repair (PRR) and DNA polymerase η to prevent mutagenesis. Human and mouse cells mobilize all these pathways using OGG1, MUTYH (MutY-homolog also known as MYH), MTH1 (MutT-homolog also known as NUDT1), NER, MMR, NEILs and DNA polymerases η and λ, to prevent 8-oxoG-induced mutations. In fact, mice deficient in both OGG1 and MUTYH develop cancer in different organs at adult age, which points to the critical impact of 8-oxoG repair on genetic stability in mammals. In this review, we will focus on Fpg and OGG1 proteins, their biochemical and structural properties as well as their biological roles. Other DNA N-glycosylases able to release 8-oxoG from damaged DNA in various organisms will be discussed. Finally, we will report on the role of OGG1 in human disease and the possible use of 8-oxoG DNA N-glycosylases as therapeutic targets. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansson, J.; Keyse, S.M.; Lindahl, T.

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurementsmore » of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.« less

The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae

PubMed Central

Koch, Melissa R.; House, Nealia C. M.; Cosetta, Casey M.; Jong, Robyn M.; Salomon, Christelle G.; Joyce, Cailin E.; Philips, Elliot A.; Su, Xiaofeng A.; Freudenreich, Catherine H.

2018-01-01

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair. PMID:29305386

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Hui; Shi, Qiong; Song, Xiufang

2015-07-01

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observedmore » phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.« less

Structure–Function Studies of DNA Polymerase λ

PubMed Central

2015-01-01

DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ. PMID:24716527

Synthesis of Deoxyribonucleic Acid After Ultraviolet Irradiation of Sensitive and Resistant Haemophilus influenzae

PubMed Central

Modak, Sohan P.; Setlow, Jane K.

1969-01-01

Synthesis of deoxyribonucleic acid (DNA) has been measured as a function of ultraviolet (UV) radiation dose in wild-type and seven UV-sensitive strains of Haemophilus influenzae. At the UV doses used, all strains were able to resume DNA synthesis, even those which are unable to excise pyrimidine dimers from their DNA. These excisionless strains showed longer UV-induced delays in DNA synthesis than all but one of the other strains. The longest delay was shown by DB117, a strain which can excise dimers but which is recombination deficient and unable to rejoin X ray-induced single-strand breaks. All strains showed a progressive decrease in sensitivity as they approached the stationary phase. PMID:5305934

International congress on DNA damage and repair: Book of abstracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging

PubMed Central

Buechner, Claudia N.; Maiti, Atanu; Drohat, Alexander C.; Tessmer, Ingrid

2015-01-01

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG–DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases. PMID:25712093

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

PubMed Central

Younger, Ellen; Fernando, Booshini D; Khaleel, Thanafez; Stark, W Marshall; Smith, Margaret C M

2018-01-01

Abstract To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase. PMID:29228292

Molecular mechanisms of DNA repair inhibition by caffeine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selby, C.P.; Sancar, A.

1990-05-01

Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, includingmore » acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.« less

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

PubMed

Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

2017-10-13

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and specificity of FEN-1 from Methanopyrus kandleri

DOE PAGES

Shah, Santosh; Dunten, Pete; Stiteler, Amanda; ...

2014-11-18

DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Ultimately, we found both to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

PubMed

Lenz, Stefan A P; Wetmore, Stacey D

2016-02-09

Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

Ugene, a newly identified protein that is commonly over-expressed in cancer, and that binds uracil DNA-glycosylase

PubMed Central

Guo, Chunguang; Zhang, Xiaodong; Fink, Stephen P; Platzer, Petra; Wilson, Keith; Willson, James K. V.; Wang, Zhenghe; Markowitz, Sanford D

2008-01-01

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including, breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA-glycosylase 2 (UNG2), an important enzyme in the base excision repair pathway, was identified as a partner protein that binds to Ugene. Co-immunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH2-terminus. We suggest Ugene induction in cancer may contribute to the cancer phenotype by interacting with the base excision repair pathway. PMID:18676834

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

PubMed Central

Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

2015-01-01

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation

PubMed Central

Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N.; Chen, Lung-Chi; Tang, Moon-shong

2015-01-01

Second-hand smoke (SHS) is associated with 20–30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers. PMID:26431382

The λ Integrase Site-specific Recombination Pathway

PubMed Central

LANDY, ARTHUR

2017-01-01

The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in Mobile DNA II, there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics. PMID:26104711

RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, E.; Prakash, L.; Guzder, S.N.

1992-12-01

Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M[sub r] 95,356). Themore » RAD25 (SSL2)- and XPCX-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UN sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys[sup 392] residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD 25 (SSL2) ATPase/DNA helicase activity in viability. 40 refs., 3 figs., 1 tab.« less

Coupling between nucleotide excision repair and gene expression.

PubMed

Cambindo Botto, Adrián E; Muñoz, Juan C; Muñoz, Manuel J

2018-05-17

Gene expression and DNA repair are fundamental processes for life. During the last decade, accumulating experimental evidence point towards different modes of coupling between these processes. Here we discuss the molecular mechanisms by which RNAPII-dependent transcription affects repair by the Nucleotide Excision Repair system (NER) and how NER activity, through the generation of single stranded DNA intermediates and activation of the DNA damage response kinase ATR, drives gene expression in a genotoxic scenario. Since NER-dependent repair is compromised in Xeroderma Pigmentosum (XP) patients, and having in mind that these patients present a high degree of clinical heterogeneity, we speculate that some of the clinical features of XP patients can be explained by misregulation of gene expression.

A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

PubMed

Scarbrough, Peter M; Weber, Rachel Palmieri; Iversen, Edwin S; Brhane, Yonathan; Amos, Christopher I; Kraft, Peter; Hung, Rayjean J; Sellers, Thomas A; Witte, John S; Pharoah, Paul; Henderson, Brian E; Gruber, Stephen B; Hunter, David J; Garber, Judy E; Joshi, Amit D; McDonnell, Kevin; Easton, Doug F; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A; Schildkraut, Joellen M

2016-01-01

DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P < 5.09 × 10(-6)), MSH5 (P < 5.09 × 10(-6)), and BRCA2 (P = 5.70 × 10(-6)). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. ©2015 American Association for Cancer Research.

In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz

1997-08-01

Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs.

PubMed

Majumdar, Chandrima; Nuñez, Nicole N; Raetz, Alan G; Khuu, Cindy; David, Sheila S

2018-01-01

Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease. © 2018 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such thatmore » a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.« less

BRCA1 is Required for Post-replication Repair After UV-induced DNA Damage

PubMed Central

Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J.; Scully, Ralph; Feunteun, Jean; Livingston, David M.

2011-01-01

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent, but nucleotide excision repair- independent manner. More specifically, at UV- stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and post- replicative repair. These BRCA1 functions differ from those required for DSBR. PMID:21963239

Interactions of Human Nucleotide Excision Repair Protein XPA with DNA and RPA70 Delta c327: Chemical Shift Mapping and N-15 NMR Relaxation Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Daughdrill, Gary W.; De Lorimier, Robert

1999-12-28

Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence.

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2009-09-01

binding ETS domain) and five type II (without ETS domain). Fusion-positive type I– and type II–containing phages were amplified with T3 and T7 primers...will be performed to identify the authentic 3’ UTRs from the mRNA pool from CaP patient specimens. Using phage excision strategy, we will use to... phage DNA sequences plasmids (cDNA) clones were generated by using phage excision strategy. Figure 1. ERG splice variants in prostate cancer

Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi's anemia.

PubMed Central

Remsen, J F; Cerutti, P A

1976-01-01

The capacity of preparations of skin fibroblasts from normal individuals and patients with Fanconi's anemia to excise gamma-ray products of the 5,6-dihydroxydihydrothymine type from exogenous DNA was investigated. The excision capacity of whole-cell homogenates of fibroblasts from two of four patients with Fanconi's anemia was substantially below normal. This repair deficiency was further pronounced in nuclear preparations from cells of the same two patients. PMID:1065896

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

PubMed Central

Maltseva, Ekaterina A.

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA. PMID:29320546

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

PubMed

Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

EPA Science Inventory

The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

Lower nucleotide excision repair capacity in newborns compared to their mothers: a pilot study.

PubMed

Vande Loock, Kim; Decordier, Ilse; Plas, Gina; Ciardelli, Roberta; Haumont, Dominique; Kirsch-Volders, Micheline

2014-01-01

Recognition of the potential vulnerability of children and newborns and protection of their health is essential, especially regarding to genotoxic compounds. Benzo(a)pyrene B(a)P a commonly found carcinogen, and its metabolite BPDE, are known to cross the placenta. To investigate how well newborns are able to cope with BPDE-induced DNA damage, a recent developed nucleotide excision repair cell phenotype assay was applied in a pilot study of 25 newborn daughters and their mothers, using the Alkaline Comet Assay and taking demographic data into account. Newborns seemed to be less able to repair BPDE-induced DNA damage since lower repair capacity levels were calculated compared to their mothers although statistical significance was not reached. Assessment of repair capacity in combination with genotypes will provide important information to support preventive strategies in neonatal care and to define science based exposure limits for pregnant women and children. Copyright © 2013 Elsevier Inc. All rights reserved.

Xeroderma pigmentosum and other diseases of human premature aging and DNA repair: Molecules to patients

PubMed Central

Niedernhofer, Laura J.; Bohr, Vilhelm A.; Sander, Miriam; Kraemer, Kenneth H.

2012-01-01

A workshop1 to share, consider and discuss the latest developments in understanding xeroderma pigmentosum and other human diseases caused by defects in nucleotide excision repair (NER) of DNA damage was held on September 21–24, 2010 in Virginia. It was attended by approximately 100 researchers and clinicians, as well as several patients and representatives of patient support groups. This was the third in a series of workshops with similar design and goals: to emphasize discussion and interaction among participants as well as open exchange of information and ideas. The participation of patients, their parents and physicians was an important feature of this and the preceding two workshops. Topics discussed included the natural history and clinical features of the diseases, clinical and laboratory diagnosis of these rare diseases, therapeutic strategies, mouse models of neurodegeneration, molecular analysis of accelerated aging, impact of transcriptional defects and mitochondrial dysfunction on neurodegeneration, and biochemical insights into mechanisms of NER and base excision repair. PMID:21708183

Rational Inhibitors of DNA Base Excision Repair Enzymes: New Tools for Elucidating the Role of BER in Cancer Chemotherapy. Addendum

DTIC Science & Technology

2006-11-01

30. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) The role of electrophilic and base catalysis in the mechanism of Escherichia...based on a duplex previously used in rapid kinetic studies of base flipping by UDG (Figure 2) (5). The 2′ fluorinated deoxyuridine substrate analogue...Boca Raton, FL. 32. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) Role of electrophilic and general base catalysis in the

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kao, Shyan-Yuan, E-mail: shyan-yuan_kao@meei.harvard.edu

Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant.more » Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.« less

Potential of the homeopathic remedy, Arnica Montana 30C, to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes.

PubMed

Das, Sreemanti; Saha, Santu Kumar; De, Arnab; Das, Durba; Khuda-Bukhsh, Anisur Rahman

2012-03-01

To examine to what degree an ultra-highly diluted homeopathic remedy, Arnica Montana 30C (AM-30C), used in the treatment of shock and injury, can modulate the expression of nucleotide excision repair genes in Escherichia coli exposed to ultraviolet (UV) irradiation. E. coli were cultured to their log phase in a standard Luria-Bertani medium and then exposed to sublethal doses of UV irradiation at 25 and 50 J/m(2) for 22.5 and 45 s, respectively. The UV-exposed bacteria were then supplemented with either AM-30C (drug) or placebo (P-30C). The drug-treated and placebo-treated bacteria were subjected to assay for DNA damage and oxidative stress 90 min after UV exposure. Several protocols like comet assay, gel electrophoresis for DNA ladder and intracellular reactive oxygen species (ROS) generation, and biomarker measurement like superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were conducted. The mRNA expressions of the excision repair genes like ultraviolet repair uvrA, B and C genes (or also known as excision repair genes) were estimated by reverse transcription-polymerase chain reaction method. The UV-exposed bacteria showed DNA damage and oxidative stress, as revealed by an increase in ROS generation, and a decrease in SOD, CAT and GSH activities. As compared to placebo, the AM-30C-treated bacteria showed less DNA damage and oxidative stress as manifested by a decrease in ROS generation, and an increase in SOD, CAT and GSH activities. AM-30C also up-regulated the expression of repair genes as compared to the control. AM-30C helped repair the DNA damage through up-regulation of repair genes and also ameliorated the oxidative stress through the reduction of ROS generation and suitable modulation of anti-oxidative stress enzymes.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

PubMed

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer’s disease

PubMed Central

Gredilla, Ricardo; Weissman, Lior; Yang, Jenq-Lin; Bohr, Vilhelm A.; Stevnsner, Tinna

2010-01-01

Brain aging is associated with synaptic decline and cognitive impairment. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). In mitochondria, base excision repair (BER) is the main DNA repair pathway for base modifications such as deamination and oxidation, and constitutes an important mechanism to avoid accumulation of mtDNA mutations. Synaptic function is highly dependent on mitochondria, and in the current study we have investigated BER in synaptosomes of mouse brain during normal aging and in an AD model. Synaptosomes are isolated synapses in membranous structures produced by subcellular fractionation of brain tissue. They include the whole presynaptic terminal as well as portions of the postsynaptic terminal. Synaptosomes contain the molecular machinery necessary for uptake, storage, and release of neurotransmitters, including synaptic vesicles and mitochondria. BER activities were measured in synaptosomal fractions from young and old mice and from pre-symptomatic and symptomatic AD mice harboring mutated APP, Tau and PS1 (3xTgAD). During normal aging, a reduction in the BER capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of pre-symptomatic and symptomatic AD mice. Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed. PMID:20708822

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase*

PubMed Central

Xia, Bo; Liu, Yinling; Li, Wei; Brice, Allyn R.; Dominy, Brian N.; Cao, Weiguo

2014-01-01

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily. PMID:24838246

A DNA 3′-phosphatase functions in active DNA demethylation in Arabidopsis

PubMed Central

Martínez-Macías, María Isabel; Qian, Weiqiang; Miki, Daisuke; Pontes, Olga; Liu, Yunhua; Tang, Kai; Liu, Renyi; Morales-Ruiz, Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

2012-01-01

SUMMARY DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway. PMID:22325353

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

PubMed Central

Sentürker, S; Auffret van der Kemp, P; You, H J; Doetsch, P W; Dizdaroglu, M; Boiteux, S

1998-01-01

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells. PMID:9826748

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

PubMed Central

Bogani, Federica; Boehmer, Paul E.

2008-01-01

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5′-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3′ side of abasic sites and 5′-dRP residues that remain after cleavage by 5′-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency. PMID:18695225

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

Regulation and function of DNA methylation in plants and animals

PubMed Central

He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

2011-01-01

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

PubMed Central

Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; SWE-BRCA; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gómez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collée, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; HEBON; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th.; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Investigators, kConFab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

2014-01-01

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10−3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10−3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. PMID:24698998

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli.

PubMed

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-09-23

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48% of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA(-) strain were G:C --> T:A transversions, occurring within the sequence which in recA(+) strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C --> A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

PubMed Central

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-01-01

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA- strain were G:C → T:A transversions, occurring within the sequence which in recA+ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations. PMID:19834545

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

PubMed

Bj Rås, Karine Ø; Sousa, Mirta M L; Sharma, Animesh; Fonseca, Davi M; S Gaard, Caroline K; Bj Rås, Magnar; Otterlei, Marit

2017-08-21

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of Immunoglobulin Class-Switch Recombination: Choreography of Noncoding Transcription, Targeted DNA Deamination, and Long-Range DNA Repair

PubMed Central

Matthews, Allysia J.; Zheng, Simin; DiMenna, Lauren J.; Chaudhuri, Jayanta

2014-01-01

Upon encountering antigens, mature IgM-positive B lymphocytes undergo class-switch recombination (CSR) wherein exons encoding the default Cμ constant coding gene segment of the immunoglobulin (Ig) heavy-chain (Igh) locus are excised and replaced with a new constant gene segment (referred to as “Ch genes”, e.g., Cγ, Cε, or Cα). The B cell thereby changes from expressing IgM to one producing IgG, IgE, or IgA, with each antibody isotype having a different effector function during an immune reaction. CSR is a DNA deletional-recombination reaction that proceeds through the generation of DNA double-strand breaks (DSBs) in repetitive switch (S) sequences preceding each Ch gene and is completed by end-joining between donor Sμ and acceptor S regions. CSR is a multistep reaction requiring transcription through S regions, the DNA cytidine deaminase AID, and the participation of several general DNA repair pathways including base excision repair, mismatch repair, and classical nonhomologous end-joining. In this review, we discuss our current understanding of how transcription through S regions generates substrates for AID-mediated deamination and how AID participates not only in the initiation of CSR but also in the conversion of deaminated residues into DSBs. Additionally, we review the multiple processes that regulate AID expression and facilitate its recruitment specifically to the Ig loci, and how deregulation of AID specificity leads to oncogenic translocations. Finally, we summarize recent data on the potential role of AID in the maintenance of the pluripotent stem cell state during epigenetic reprogramming. PMID:24507154

Epigenetic role for the conserved Fe-S cluster biogenesis protein AtDRE2 in Arabidopsis thaliana.

PubMed

Buzas, Diana Mihaela; Nakamura, Miyuki; Kinoshita, Tetsu

2014-09-16

On fertilization in Arabidopsis thaliana, one maternal gamete, the central cell, forms a placenta-like tissue, the endosperm. The DNA glycosylase DEMETER (DME) excises 5-methylcytosine via the base excision repair pathway in the central cell before fertilization, creating patterns of asymmetric DNA methylation and maternal gene expression across DNA replications in the endosperm lineage (EDL). Active DNA demethylation in the central cell is essential for transcriptional activity in the EDL of a set of genes, including FLOWERING WAGENINGEN (FWA). A DME-binding motif for iron-sulfur (Fe-S) cluster cofactors is indispensable for its catalytic activity. We used an FWA-GFP reporter to find mutants defective in maternal activation of FWA-GFP in the EDL, and isolated an allele of the yeast Dre2/human antiapoptotic factor CIAPIN1 homolog, encoding an enzyme previously implicated in the cytosolic Fe-S biogenesis pathway (CIA), which we named atdre2-2. We found that AtDRE2 acts in the central cell to regulate genes maternally activated in the EDL by DME. Furthermore, the FWA-GFP expression defect in atdre2-2 was partially suppressed genetically by a mutation in the maintenance DNA methyltransferase MET1; the DNA methylation levels at four DME targets increased in atdre2-2 seeds relative to WT. Although atdre2-2 shares zygotic seed defects with CIA mutants, it also uniquely manifests dme phenotypic hallmarks. These results demonstrate a previously unidentified epigenetic function of AtDRE2 that may be separate from the CIA pathway.

Entrapment and Structure of an Extrahelical Guanine Attempting to Enter the Active Site of a Bacterial DNA Glycosylase, MutM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qi, Yan; Spong, Marie C.; Nam, Kwangho

2010-09-21

MutM, a bacterial DNA glycosylase, protects genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions, thereby initiating base excision DNA repair. The process of searching for and locating oxoG lesions is especially challenging, because of the close structural resemblance of oxoG to its million-fold more abundant progenitor, G. Extrusion of the target nucleobase from the DNA double helix to an extrahelical position is an essential step in lesion recognition and catalysis by MutM. Although the interactions between the extruded oxoG and the active site of MutM have been well characterized, little is known in structural detail regarding themore » interrogation of extruded normal DNA bases by MutM. Here we report the capture and structural elucidation of a complex in which MutM is attempting to present an undamaged G to its active site. The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general.« less

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

PubMed Central

Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D.; Malinsky, Sophie; Bétermier, Mireille

2011-01-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics. PMID:21533177

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

PubMed

Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

2011-04-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

Rational Inhibitors of DNA Base Excision Repair Enzymes: New Tools for Elucidating the Role of BER in Cancer Chemotherapy

DTIC Science & Technology

2006-05-01

2005, 7, 493-500. (30) Nilsen, H.; Rosewell , I.; Robins, P.; Skjelbred, C. F.; Andersen, S.; Slupphaug, G.; Daly, G.; Krokan, H. E.; Lindahl, T.; Barnes...Cell Biol. 2005, 7, 493–500. 31. Nilsen, H.; Rosewell , I.; Robins, P.; Skjelbred, C. F.; Andersen, S.; Slupphaug, G.; Daly, G.; Krokan, H. E

The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

PubMed Central

Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing

2016-01-01

Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142

Heat shock protein 70 enhanced deoxyribonucleic acid base excision repair in human leukemic cells after ionizing radiation

PubMed Central

Bases, Robert

2006-01-01

Base excision repair (BER) of DNA damage in irradiated THP1 human leukemic cells was stimulated by pretreating the cells with exogenous recombinant Hsp70. The treatment of THP1 cells with recombinant Hsp70 in cell culture promoted repair by reducing the frequency of apurinic, apyrimidinic (AP) sites in DNA before and after 1.3 Gy of radiation. However, by 30 minutes after 2.6 Gy, accelerated repair of abasic sites supervened, which may contribute to the loss of the very-low-dose cell hypersensitivity seen in clonogenic studies of other laboratories. After irradiation with 2.6 Gy, the crucial initial glycosylase step was markedly incomplete when cells had been transfected 24 hours before with a small interfering RNA (siRNA) designed to inhibit synthesis of Hsp70. In confirmation, lysates from irradiated siRNA-treated cells after 2.6 Gy were deficient in uracil glycosylase activity (UDG). Transfection with a scrambled RNA of the same size did not interfere with the glycosylase step, ie, the prompt conversion of damaged pyrimidine sites to abasic sites as well as the subsequent repair of those sites. BER measured by reduction of DNA AP sites before and after low-dose radiation was also deficient in THP1 cells that had been transfected with the siRNA designed to inhibit synthesis of Hsp70. These results implicate BER and the participation of Hsp70 in the repair of DNA in human leukemic cells with the doses of ionizing radiation used in clinical regimens. PMID:17009597

Balancing repair and tolerance of DNA damage caused by alkylating agents.

PubMed

Fu, Dragony; Calvo, Jennifer A; Samson, Leona D

2012-01-12

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.

SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

PubMed Central

Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

2013-01-01

Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity. PMID:22237395

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

PubMed

Holton, Nathaniel W; Andrews, Joel F; Gassman, Natalie R

2017-09-05

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

PubMed Central

Keith, Brian J.; Jozwiakowski, Stanislaw K.; Connolly, Bernard A.

2013-01-01

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated–naphthoylated DEAE–cellulose, resulting in a low background mutation frequency (∼1 × 10−4). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system. PMID:23098700

AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

PubMed

Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

2006-05-01

The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability.

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair.

PubMed

Sterpone, Silvia; Cozzi, Renata

2010-07-25

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2003-05-01

Alkylating minor groove DNA binder adozelesin is capable of inhibiting DNA replication in treated cells through a trans-acting mechanism. The trans... replication in vitro. Using purified proteins in DNA replication initiation assays, we found that RPA purified from cells treated with adozelesin in not...adozelesin has the same single-stranded DNA binding activity and support nucleotide excision repair as normal RPA, but is not able to support SV40 DNA

Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

PubMed Central

Catania, Francesco; McGrath, Casey L.; Doak, Thomas G.; Lynch, Michael

2013-01-01

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation. PMID:23737328

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA

USDA-ARS?s Scientific Manuscript database

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise ...

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

PubMed

Sun, Cheng; Wyngaard, Grace; Walton, D Brian; Wichman, Holly A; Mueller, Rachel Lockridge

2014-03-11

Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

PubMed Central

2014-01-01

Background Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Results Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Conclusions Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms. PMID:24618421

Genetic polymorphisms in 19q13.3 genes associated with alteration of repair capacity to BPDE-DNA adducts in primary cultured lymphocytes.

PubMed

Xiao, Mingyang; Xiao, Sha; Straaten, Tahar van der; Xue, Ping; Zhang, Guopei; Zheng, Xiao; Zhang, Qianye; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Zhu, Guolian; Lu, Xiaobo

2016-12-01

Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage in cells caused by BPDE is normally repaired by Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Genetic variations in NER and BER can change individual DNA repair capacity to DNA damage induced by BPDE. In the present study we determined the number of in vitro induced BPDE-DNA adducts in lymphocytes, to reflect individual susceptibility to Polycyclic aromatic hydrocarbons (PAHs)-induced carcinogenesis. The BPDE-DNA adduct level in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 281 randomly selected participants. We genotyped for 9 single nucleotide polymorphisms (SNPs) in genes involved in NER (XPB rs4150441, XPC rs2228001, rs2279017 and XPF rs4781560), BER (XRCC1 rs25487, rs25489 and rs1799782) and genes located on chromosome 19q13.2-3 (PPP1R13L rs1005165 and CAST rs967591). We found that 3 polymorphisms in chromosome 19q13.2-3 were associated with lower levels of BPDE-DNA adducts (MinorT allele in XRCC1 rs1799782, minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571). In addition, a modified comet assay was performed to further confirm the above conclusions. We found both minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571 were associated with the lower levels of BPDE-adducts. Our data suggested that the variant genotypes of genes in chromosome 19q13.2-3 are associated with the alteration of repair efficiency to DNA damage caused by Benzo[a]pyrene, and may contribute to enhance predictive value for individual's DNA repair capacity in response to environmental carcinogens. Copyright © 2016 Elsevier B.V. All rights reserved.

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

PubMed

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cleaver, J.E.

1989-01-01

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were notmore » significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.« less

Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

PubMed

Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

2012-05-25

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

A novel function of adenomatous polyposis coli (APC) in regulating DNA repair

PubMed Central

Jaiswal, Aruna S.; Narayan, Satya

2008-01-01

Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase β (Pol-β) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis and chemotherapy by determining whether cells with DNA damage survive or undergo apoptosis. In this review, we summarize the evidence supporting this novel concept and suggest that these results will have implications for the development of more effective strategies for chemoprevention, prognosis, and chemotherapy of certain types of tumors. PMID:18662849

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors*

PubMed Central

Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H.; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

2015-01-01

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position −3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position −6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position −19 to +8. XPG and XPF/ERCC1 endo-nucleases then cut the damaged DNA at the limit of the opened structure that was previously “labeled” by the positioning of XPC/HR23B and TFIIH. PMID:14981083

Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA

PubMed Central

Luch, Andreas; Glas, Andreas; Carell, Thomas; Naegeli, Hanspeter

2011-01-01

How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin. PMID:22039351

NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi

2015-06-05

Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated inmore » the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.« less

Sequential cloning of chromosomes

DOEpatents

Lacks, Sanford A.

1995-07-18

A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism's chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

PubMed Central

2014-01-01

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis.

PubMed

Morita, Masashi; Stamp, Gordon; Robins, Peter; Dulic, Anna; Rosewell, Ian; Hrivnak, Geza; Daly, Graham; Lindahl, Tomas; Barnes, Deborah E

2004-08-01

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.

Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity.

PubMed

Lee, Chang Yeol; Park, Ki Soo; Park, Hyun Gyu

2017-12-15

We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

PubMed Central

Dubrana, K; Le Mouël, A; Amar, L

1997-01-01

Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae.

PubMed

Morey, Natalie J; Doetsch, Paul W; Jinks-Robertson, Sue

2003-06-01

Cellular metabolic processes constantly generate reactive species that damage DNA. To counteract this relentless assault, cells have developed multiple pathways to resist damage. The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal. Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways. In this study, we examine the consequences of simultaneously compromising three or more of these pathways. Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal. In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth. In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

Base excision repair imbalance in colorectal cancer has prognostic value and modulates response to chemotherapy

PubMed Central

Leguisamo, Natalia M.; Gloria, Helena C.; Kalil, Antonio N.; Martins, Talita V.; Azambuja, Daniel B.

2017-01-01

Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC. PMID:28903334

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

PubMed Central

Guffanti, Federica; Fratelli, Maddalena; Ganzinelli, Monica; Bolis, Marco; Ricci, Francesca; Bizzaro, Francesca; Chilà, Rosaria; Sina, Federica Paola; Fruscio, Robert; Lupia, Michela; Cavallaro, Ugo; Cappelletti, Maria Rosa; Generali, Daniele; Giavazzi, Raffaella; Damia, Giovanna

2018-01-01

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients. PMID:29872499

Nucleotide excision repair is a potential therapeutic target in multiple myeloma

PubMed Central

Szalat, R; Samur, M K; Fulciniti, M; Lopez, M; Nanjappa, P; Cleynen, A; Wen, K; Kumar, S; Perini, T; Calkins, A S; Reznichenko, E; Chauhan, D; Tai, Y-T; Shammas, M A; Anderson, K C; Fermand, J-P; Arnulf, B; Avet-Loiseau, H; Lazaro, J-B; Munshi, N C

2018-01-01

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM. PMID:28588253

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

PubMed

Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

2015-10-01

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

PubMed Central

Fonseca, A.S.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.

2015-01-01

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337

Functional characterization of polymorphisms in DNA repair genes using cytogenetic challenge assays.

PubMed

Au, William W; Salama, Salama A; Sierra-Torres, Carlos H

2003-11-01

A major barrier to understanding the role of polymorphic DNA repair genes for environmental cancer is that the functions of variant genotypes are largely unknown. Using our cytogenetic challenge assays, we conducted an investigation to address the deficiency. Using X-rays or ultraviolet (UV) light, we irradiated blood lymphocytes from 80 nonsmoking donors to challenge the cells to repair the induced DNA damage, and we analyzed expression of chromosome aberrations (CA) specific to the inducing agents. We have genotyped polymorphic DNA repair genes preferentially involved with base excision repair (BER) and nucleotide excision repair (NER) activities (XRCC1, XRCC3, APE1, XPD) corresponding to the repair of X-ray- and UV light-induced DNA damage, respectively. We expected that defects in specific DNA repair pathways due to polymorphisms would cause corresponding increases of specific CA. From our data, XRCC1 399Gln and XRCC3 241Met were associated with significant increases in chromosome deletions compared with the corresponding homozygous wild types (18.27 1.1 vs 14.79 1.2 and 18.22 0.99 vs 14.20 1.39, respectively); XPD 312Asn and XPD 751Gln were associated with significant increases in chromatid breaks compared with wild types (16.09 1.36 vs 11.41 0.98 and 16.87 1.27 vs 10.54 0.87, respectively), p < 0.05. The data indicate that XRCC1 399Gln and XRCC3 241Met are significantly defective in BER, and the XPD 312Asn and XPD 751Gln are significantly defective in NER. In addition, the variant genotypes interact significantly, with limited overlap of the two different repair pathways.

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

PubMed Central

Georg, Jens; Schomacher, Lars; Chong, James P. J.; Majerník, Alan I.; Raabe, Monika; Urlaub, Henning; Müller, Sabine; Ciirdaeva, Elena; Kramer, Wilfried; Fritz, Hans-Joachim

2006-01-01

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity. PMID:17012282

Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

PubMed

Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

2006-01-01

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents

PubMed Central

Negishi, Tomoe

2013-01-01

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O 6-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the Fʹprolac from strain CC102 (FʹCC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O 6-alkylguanine. The results showed the repair of O 6-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O 6-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells. PMID:23446177

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

PubMed Central

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

DNA repair in Chromobacterium violaceum.

PubMed

Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

2004-03-31

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Sequential cloning of chromosomes

DOEpatents

Lacks, S.A.

1995-07-18

A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

PubMed

Gavande, Navnath S; VanderVere-Carozza, Pamela; Mishra, Akaash K; Vernon, Tyler L; Pawelczak, Katherine S; Turchi, John J

2017-10-12

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC 50 of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.

Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartman, P.S.; Hevelone, J.; Dwarakanath, V.

1989-06-01

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). Thismore » correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.« less

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

Insight in the multilevel regulation of NER

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dijk, Madelon; Typas, Dimitris; Mullenders, Leon, E-mail: l.mullenders@lumc.nl

2014-11-15

Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of themore » core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.« less

Developing an in silico model of the modulation of base excision repair using methoxyamine for more targeted cancer therapeutics.

PubMed

Gurkan-Cavusoglu, Evren; Avadhani, Sriya; Liu, Lili; Kinsella, Timothy J; Loparo, Kenneth A

2013-04-01

Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair.

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

PubMed Central

Lu, Duo; Silhan, Jan; MacDonald, James T.; Carpenter, Elisabeth P.; Jensen, Kirsten; Tang, Christoph M.; Baldwin, Geoff S.; Freemont, Paul S.

2012-01-01

Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures. PMID:23035246

Oxidized C5-methyl cytosine bases in DNA: 5-Hydroxymethylcytosine; 5-formylcytosine; and 5-carboxycytosine.

PubMed

Klungland, Arne; Robertson, Adam B

2017-06-01

Recent reports suggest that the Tet enzyme family catalytically oxidize 5-methylcytosine in mammalian cells. The oxidation of 5-methylcytosine can result in three chemically distinct species - 5-hydroxymethylcytsine, 5-formylcytosine, and 5-carboxycytosine. While the base excision repair machinery processes 5-formylcytosine and 5-carboxycytosine rapidly, 5-hydroxymethylcytosine is stable under physiological conditions. As a stable modification 5-hydroxymethylcytosine has a broad range of functions, from stem cell pluriopotency to tumorigenesis. The subsequent oxidation products, 5-formylcytosine and 5-carboxycytosine, are suggested to be involved in an active DNA demethylation pathway. This review provides an overview of the biochemistry and biology of 5-methylcytosine oxidation products. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

PubMed

Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

2018-01-30

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

PubMed

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA.

PubMed

Gantchev, Tsvetan G; Hunting, Darel J

2010-12-28

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.

Endonuclease G promotes mitochondrial genome cleavage and replication

PubMed Central

Wiehe, Rahel Stefanie; Gole, Boris; Chatre, Laurent; Walther, Paul; Calzia, Enrico; Ricchetti, Miria; Wiesmüller, Lisa

2018-01-01

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis. PMID:29719607

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

PubMed Central

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5′ incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix–hairpin–helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded–double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop (‘bubble DNA’). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine–valine–glycine residues followed by lysine–arginine–arginine, a positively charged surface patch and the second hairpin region consisting of glycine–isoleucine–serine. A model for the protein– DNA complex is proposed that accounts for this specificity. PMID:12426397

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

PubMed

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-09-20

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

DNA Damage Related Crosstalk Between the Nucleus and Mitochondria

PubMed Central

Saki, Mohammad; Prakash, Aishwarya

2017-01-01

The electron transport chain is the primary pathway by which a cell generates energy in the form of ATP. Byproducts of this process produce reactive oxygen species that can cause damage to mitochondrial DNA. If not properly repaired, the accumulation of DNA damage can lead to mitochondrial dysfunction linked to several human disorders including neurodegenerative diseases and cancer. Mitochondria are able to combat oxidative DNA damage via repair mechanisms that are analogous to those found in the nucleus. Of the repair pathways currently reported in the mitochondria, the base excision repair pathway is the most comprehensively described. Proteins that are involved with the maintenance of mtDNA are encoded by nuclear genes and translocate to the mitochondria making signaling between the nucleus and mitochondria imperative. In this review, we discuss the current understanding of mitochondrial DNA repair mechanisms and also highlight the sensors and signaling pathways that mediate crosstalk between the nucleus and mitochondria in the event of mitochondrial stress. PMID:27915046

XPD polymorphisms: effects on DNA repair proficiency.

PubMed

Lunn, R M; Helzlsouer, K J; Parshad, R; Umbach, D M; Harris, E L; Sanford, K K; Bell, D A

2000-04-01

XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Animal Studies in the Mode of Action of Agents, That Are Antitransformers in Cell Cultures.

DTIC Science & Technology

1987-10-28

The oel- let was hydrolysed at 90 C in 6% PCA for 30 min. The DNA content (ootical density at 260 nm and 290 nm) and the radioactivitv ( liquid ...required: DNA damage, excision of the damage and DNA-strand polimerization and ligation. The misrepair or incomplete repair of DNA damage may be an ini...with non ionic deter- gents in the ?resence of high salt concentration. The secondary and tertiary structure (supercoils) of DNA remains intact under

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

PubMed Central

Zucca, Elisa; Bertoletti, Federica; Wimmer, Ursula; Ferrari, Elena; Mazzini, Giuliano; Khoronenkova, Svetlana; Grosse, Nicole; van Loon, Barbara; Dianov, Grigory; Hübscher, Ulrich; Maga, Giovanni

2013-01-01

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells. PMID:23118481

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms.

PubMed

Kladova, Olga A; Krasnoperov, Lev N; Kuznetsov, Nikita A; Fedorova, Olga S

2018-03-30

Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tC O ), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.

Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

PubMed

Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

2014-10-10

Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

The Chemical and Biological Effects of cis-Dichlorodiammineplatinum (II), an Antitumor Agent, on DNA

PubMed Central

Munchausen, Linda L.

1974-01-01

cis-Dichlorodiammineplatinum (II) binds irreversibly to the bases in DNA; the amount of platinum complex bound can be determined from changes in the ultraviolet absorption spectrum. As the ratio of platinum to phosphate is increased, an increasing inactivation of bacterial transforming DNA is observed. At a ratio that corresponds to spectrometric saturation, transforming activity is inactivated >105-fold. The trans isomer of the platinum complex, which is not effective against tumors, induces a similar inactivation of transforming DNA but with half the efficiency, indicating a different mode of binding. The sensitivity to inactivation by cis isomer varies slightly with the genetic marker assayed but is not dependent on the excision repair system. Uptake of DNA by competent cells is unaffected by bound platinum complex; however, integration of platinum-bound transforming DNA into the host genome decreases as the mole fraction of platinum increases. This loss of integration parallels the decreased transforming activity of the DNA. Although the drug induces interstrand crosslinks in DNA in vitro, these crosslinks are relatively rare events and cannot account for the observed inactivation. PMID:4548188

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

8-Oxoguanine DNA glycosylase1-driven DNA repair-A paradoxical role in lung aging.

PubMed

German, Peter; Saenz, David; Szaniszlo, Peter; Aguilera-Aguirre, Leopoldo; Pan, Lang; Hegde, Muralidhar L; Bacsi, Attila; Hajas, Gyorgy; Radak, Zsolt; Ba, Xueqing; Mitra, Sankar; Papaconstantinou, John; Boldogh, Istvan

2017-01-01

Age-associated changes in lung structure and function are some of the most important predictors of overall health, cognitive activities and longevity. Common to all aging cells is an increase in oxidatively modified DNA bases, primarily 8-oxo-7,8-dihydroguanine (8-oxoG). It is repaired via DNA base excision repair pathway driven by 8-oxoguanine DNA glycosylase-1 (OGG1-BER), whose role in aging has been the focus of many studies. This study hypothesizes that signaling and consequent gene expression during cellular response to OGG1-BER "wires" senescence/aging processes. To test OGG1-BER was mimicked by repeatedly exposing diploid lung fibroblasts cells and airways of mice to 8-oxoG base. Results showed that repeated exposures led to G1 cell cycle arrest and pre-matured senescence of cultured cells in which over 1000 genes were differentially expressed -86% of them been identical to those in naturally senesced cells. Gene ontology analysis of gene expression displayed biological processes driven by small GTPases, phosphoinositide 3-kinase and mitogen activated kinase cascades both in cultured cells and lungs. These results together, points to a new paradigm about the role of DNA damage and repair by OGG1 in aging and age-associated disease processes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Purification, crystallization and preliminary X-ray analysis of uracil-DNA glycosylase from Sulfolobus tokodaii strain 7

PubMed Central

Kawai, Akito; Higuchi, Shigesada; Tsunoda, Masaru; Nakamura, Kazuo T.; Miyamoto, Shuichi

2012-01-01

Uracil-DNA glycosylase (UDG) specifically removes uracil from DNA by catalyzing hydrolysis of the N-glycosidic bond, thereby initiating the base-excision repair pathway. Although a number of UDG structures have been determined, the structure of archaeal UDG remains unknown. In this study, a deletion mutant of UDG isolated from Sulfolobus tokodaii strain 7 (stoUDGΔ) and stoUDGΔ complexed with uracil were crystallized and analyzed by X-ray crystallography. The crystals were found to belong to the orthorhombic space group P212121, with unit-cell parameters a = 52.2, b = 52.3, c = 74.7 Å and a = 52.1, b = 52.2, c = 74.1 Å for apo stoUDGΔ and stoUDGΔ complexed with uracil, respectively. PMID:22949205

Population-specific variation in haplotype composition and heterozygosity at the POLB locus.

PubMed

Yamtich, Jennifer; Speed, William C; Straka, Eva; Kidd, Judith R; Sweasy, Joann B; Kidd, Kenneth K

2009-05-01

DNA polymerase beta plays a central role in base excision repair (BER), which removes large numbers of endogenous DNA lesions from each cell on a daily basis. Little is currently known about germline polymorphisms within the POLB locus, making it difficult to study the association of variants at this locus with human diseases such as cancer. Yet, approximately thirty percent of human tumor types show variants of DNA polymerase beta. We have assessed the global frequency distributions of coding and common non-coding SNPs in and flanking the POLB gene for a total of 14 sites typed in approximately 2400 individuals from anthropologically defined human populations worldwide. We have found a marked difference between haplotype frequencies in African populations and in non-African populations.

Formation and processing of DNA damage substrates for the hNEIL enzymes.

PubMed

Fleming, Aaron M; Burrows, Cynthia J

2017-06-01

Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

PubMed

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Systemically administered gp100 encoding DNA vaccine for melanoma using water-in-oil-in-water multiple emulsion delivery systems.

PubMed

Kalariya, Mayurkumar; Amiji, Mansoor M

2013-09-10

The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity. Copyright © 2013 Elsevier B.V. All rights reserved.

Mapping Ribonucleotides Incorporated into DNA by Hydrolytic End-Sequencing.

PubMed

Orebaugh, Clinton D; Lujan, Scott A; Burkholder, Adam B; Clausen, Anders R; Kunkel, Thomas A

2018-01-01

Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.

Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum | Center for Cancer Research

Cancer.gov

Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with

Genetic variation in the base excision repair pathway and bladder cancer risk.

PubMed

Figueroa, Jonine D; Malats, Núria; Real, Francisco X; Silverman, Debra; Kogevinas, Manolis; Chanock, Stephen; Welch, Robert; Dosemeci, Mustafa; Tardón, Adonina; Serra, Consol; Carrato, Alfredo; García-Closas, Reina; Castaño-Vinyals, Gemma; Rothman, Nathaniel; García-Closas, Montserrat

2007-04-01

Genetic polymorphisms in DNA repair genes may impact individual variation in DNA repair capacity and alter cancer risk. In order to examine the association of common genetic variation in the base-excision repair (BER) pathway with bladder cancer risk, we analyzed 43 single nucleotide polymorphisms (SNPs) in 12 BER genes (OGG1, MUTYH, APEX1, PARP1, PARP3, PARP4, XRCC1, POLB, POLD1, PCNA, LIG1, and LIG3). Using genotype data from 1,150 cases of urinary bladder transitional cell carcinomas and 1,149 controls from the Spanish Bladder Cancer Study we estimated odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, region and smoking status. SNPs in three genes showed significant associations with bladder cancer risk: the 8-oxoG DNA glycosylase gene (OGG1), the Poly (ADP-ribose) polymerase family member 1 (PARP1) and the major gap filling polymerase-beta (POLB). Subjects who were heterozygous or homozygous variant for an OGG1 SNP in the promoter region (rs125701) had significantly decreased bladder cancer risk compared to common homozygous: OR (95%CI) 0.78 (0.63-0.96). Heterozygous or homozygous individuals for the functional SNP PARP1 rs1136410 (V762A) or for the intronic SNP POLB rs3136717 were at increased risk compared to those homozygous for the common alleles: 1.24 (1.02-1.51) and 1.30 (1.04-1.62), respectively. In summary, data from this large case-control study suggested bladder cancer risk associations with selected BER SNPs, which need to be confirmed in other study populations.

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

PubMed

Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2012-11-01

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER. Copyright © 2012 Elsevier B.V. All rights reserved.

The interplay of mutations and electronic properties in disease-related genes

NASA Astrophysics Data System (ADS)

Shih, Chi-Tin; Wells, Stephen A.; Hsu, Ching-Ling; Cheng, Yun-Yin; Römer, Rudolf A.

2012-02-01

Electronic properties of DNA are believed to play a crucial role in many phenomena in living organisms, for example the location of DNA lesions by base excision repair (BER) glycosylases and the regulation of tumor-suppressor genes such as p53 by detection of oxidative damage. However, the reproducible measurement and modelling of charge migration through DNA molecules at the nanometer scale remains a challenging and controversial subject even after more than a decade of intense efforts. Here we show, by analysing 162 disease-related genes from a variety of medical databases with a total of almost 20,000 observed pathogenic mutations, a significant difference in the electronic properties of the population of observed mutations compared to the set of all possible mutations. Our results have implications for the role of the electronic properties of DNA in cellular processes, and hint at the possibility of prediction, early diagnosis and detection of mutation hotspots.

Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

PubMed

Caldecott, K W

2001-05-01

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

Universal Readers Based on Hydrogen Bonding or π-π Stacking for Identification of DNA Nucleotides in Electron Tunnel Junctions.

PubMed

Biswas, Sovan; Sen, Suman; Im, JongOne; Biswas, Sudipta; Krstic, Predrag; Ashcroft, Brian; Borges, Chad; Zhao, Yanan; Lindsay, Stuart; Zhang, Peiming

2016-12-27

A reader molecule, which recognizes all the naturally occurring nucleobases in an electron tunnel junction, is required for sequencing DNA by a recognition tunneling (RT) technique, referred to as a universal reader. In the present study, we have designed a series of heterocyclic carboxamides based on hydrogen bonding and a large-sized pyrene ring based on a π-π stacking interaction as universal reader candidates. Each of these compounds was synthesized to bear a thiolated linker for attachment to metal electrodes and examined for their interactions with naturally occurring DNA nucleosides and nucleotides by 1 H NMR, ESI-MS, computational calculations, and surface plasmon resonance. RT measurements were carried out in a scanning tunnel microscope. All of these molecules generated electrical signals with DNA nucleotides in tunneling junctions under physiological conditions (phosphate buffered aqueous solution, pH 7.4). Using a support vector machine as a tool for data analysis, we found that these candidates distinguished among naturally occurring DNA nucleotides with the accuracy of pyrene (by π-π stacking interactions) > azole carboxamides (by hydrogen-bonding interactions). In addition, the pyrene reader operated efficiently in a larger tunnel junction. However, the azole carboxamide could read abasic (AP) monophosphate, a product from spontaneous base hydrolysis or an intermediate of base excision repair. Thus, we envision that sequencing DNA using both π-π stacking and hydrogen-bonding-based universal readers in parallel should generate more comprehensive genome sequences than sequencing based on either reader molecule alone.

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

Oxidized Guanine Base Lesions Function in 8-Oxoguanine DNA Glycosylase-1-mediated Epigenetic Regulation of Nuclear Factor κB-driven Gene Expression*

PubMed Central

Pan, Lang; Hao, Wenjing; Ba, Xueqing

2016-01-01

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression. PMID:27756845

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

PubMed Central

Ormeño, Fernando; Barrientos, Camila; Ramirez, Santiago; Ponce, Iván; Valenzuela, Lucía; Sepúlveda, Sofía; Bitar, Mainá; Kemmerling, Ulrike; Machado, Carlos Renato; Cabrera, Gonzalo; Galanti, Norbel

2016-01-01

Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing. PMID:27284968

Evidence for conformational capture mechanism for damage recognition by NER protein XPC/Rad4.

NASA Astrophysics Data System (ADS)

Chakraborty, Sagnik; Steinbach, Peter J.; Paul, Debamita; Min, Jung-Hyun; Ansari, Anjum

Altered flexibility of damaged DNA sites is considered to play an important role in damage recognition by DNA repair proteins. Characterizing lesion-induced DNA dynamics has remained a challenge. We have combined ps-resolved fluorescence lifetime measurements with cytosine analog FRET pair uniquely sensitive to local unwinding/twisting to analyze DNA conformational distributions. This innovative approach maps out with unprecedented sensitivity the alternative conformations accessible to a series of DNA constructs containing 3-base-pair mismatch, suitable model lesions for the DNA repair protein xeroderma pigmentosum C (XPC) complex. XPC initiates eukaryotic nucleotide excision repair by recognizing various DNA lesions primarily through DNA deformability. Structural studies show that Rad4 (yeast ortholog of XPC) unwinds DNA at the lesion site and flips out two nucleotide pairs. Our results elucidate a broad range of conformations accessible to mismatched DNA even in the absence of the protein. Notably, the most severely distorted conformations share remarkable resemblance to the deformed conformation seen in the crystal structure of the Rad4-bound ``recognition'' complex supporting for the first time a possible ``conformational capture'' mechanism for damage recognition by XPC/Rad4. NSF Univ of Illinois-Chicago.

An AP Endonuclease Functions in Active DNA Demethylation and Gene Imprinting in Arabidopsis

PubMed Central

Li, Yan; Córdoba-Cañero, Dolores; Qian, Weiqiang; Zhu, Xiaohong; Tang, Kai; Zhang, Huiming; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

2015-01-01

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/−zdp−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis. PMID:25569774

Human OGG1 activity in nucleosomes is facilitated by transient unwrapping of DNA and is influenced by the local histone environment.

PubMed

Bilotti, Katharina; Kennedy, Erin E; Li, Chuxuan; Delaney, Sarah

2017-11-01

If unrepaired, damage to genomic DNA can cause mutations and/or be cytotoxic. Single base lesions are repaired via the base excision repair (BER) pathway. The first step in BER is the recognition and removal of the nucleobase lesion by a glycosylase enzyme. For example, human oxoguanine glycosylase 1 (hOGG1) is responsible for removal of the prototypic oxidatively damaged nucleobase, 8-oxo-7,8-dihydroguanine (8-oxoG). To date, most studies of glycosylases have used free duplex DNA substrates. However, cellular DNA is packaged as repeating nucleosome units, with 145 base pair segments of DNA wrapped around histone protein octamers. Previous studies revealed inhibition of hOGG1 at the nucleosome dyad axis and in the absence of chromatin remodelers. In this study, we reveal that even in the absence of chromatin remodelers or external cofactors, hOGG1 can initiate BER at positions off the dyad axis and that this activity is facilitated by spontaneous and transient unwrapping of DNA from the histones. Additionally, we find that solution accessibility as determined by hydroxyl radical footprinting is not fully predictive of glycosylase activity and that histone tails can suppress hOGG1 activity. We therefore suggest that local nuances in the nucleosome environment and histone-DNA interactions can impact glycosylase activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

PubMed

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria

PubMed Central

Hilton, Jason A.; Meeks, John C.; Zehr, Jonathan P.

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria. PMID:27206019

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

PubMed

Hilton, Jason A; Meeks, John C; Zehr, Jonathan P

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria.

Ancestor of land plants acquired the DNA-3-methyladenine glycosylase (MAG) gene from bacteria through horizontal gene transfer.

PubMed

Fang, Huimin; Huangfu, Liexiang; Chen, Rujia; Li, Pengcheng; Xu, Shuhui; Zhang, Enying; Cao, Wei; Liu, Li; Yao, Youli; Liang, Guohua; Xu, Chenwu; Zhou, Yong; Yang, Zefeng

2017-08-24

The origin and evolution of land plants was an important event in the history of life and initiated the establishment of modern terrestrial ecosystems. From water to terrestrial environments, plants needed to overcome the enhanced ultraviolet (UV) radiation and many other DNA-damaging agents. Evolving new genes with the function of DNA repair is critical for the origin and radiation of land plants. In bacteria, the DNA-3-methyladenine glycosylase (MAG) recognizes of a variety of base lesions and initiates the process of the base excision repair for damaged DNA. The homologs of MAG gene are present in all major lineages of streptophytes, and both the phylogenic and sequence similarity analyses revealed that green plant MAG gene originated through an ancient horizontal gene transfer (HGT) event from bacteria. Experimental evidence demonstrated that the expression of the maize ZmMAG gene was induced by UV and zeocin, both of which are known as DNA-damaging agents. Further investigation revealed that Streptophyta MAG genes had undergone positive selection during the initial evolutionary period in the ancestor of land plants. Our findings demonstrated that the ancient HGT of MAG to the ancestor of land plants probably played an important role in preadaptation to DNA-damaging agents in terrestrial environments.

Xeroderma pigmentosum complementation group E and UV-damaged DNA-binding protein

PubMed Central

Tang, Jean; Chu, Gilbert

2010-01-01

UV-damaged DNA-binding protein (UV-DDB) is composed of two subunits, DDB1 (p127) and DDB2 (p48). Mutations in the DDB2 gene inactivate UV-DDB in individuals from complementation group E of xeroderma pigmentosum (XP-E), an autosomal recessive disease characterized by sun sensitivity, severe risk for skin cancer and defective nucleotide excision repair. UV-DDB is also deficient in many rodent tissues, exposing a shortcoming in rodent models for cancer. In vitro, UV-DDB binds to cyclobutane pyrimidine dimers (CPDs), 6–4 photoproducts and other DNA lesions, stimulating the excision of CPDs, and to a lesser extent, of 6–4 photoproducts. In vivo, UV-DDB plays an important role in the p53-dependent response of mammalian cells to DNA damage. When cells are exposed to UV, the resulting accumulation of p53 activates DDB2 transcription, which leads to increased levels of UV-DDB. Binding of UV-DDB to CPDs targets these lesions for global genomic repair, suppressing mutations without affecting UV survival. Apparently, cells are able to survive with unrepaired CPDs because of the activity of bypass DNA polymerases. Finally, there is evidence that UV-DDB may have roles in the cell that are distinct from DNA repair. PMID:12509284

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

PubMed Central

Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

2014-01-01

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

Growth and Synthesis of Nucleic Acid and Protein by Excised Radish Cotyledons 1

PubMed Central

Nieman, R. H.; Poulsen, L. L.

1967-01-01

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO3. The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division. PMID:16656601

APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast.

PubMed

Saini, Natalie; Roberts, Steven A; Sterling, Joan F; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

2017-05-01

Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis. Published by Elsevier B.V.

Nucleotide Excision Repair Proteins Rapidly Accumulate but Fail to Persist in Human XP-E (DDB2 Mutant) Cells

PubMed Central

Oh, Kyu-Seon; Imoto, Kyoko; Emmert, Steffen; Tamura, Deborah; DiGiovanna, John J.; Kraemer, Kenneth. H.

2011-01-01

The XP-E DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from 4 newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA, and XPF) co-localized to CPD and 6-4PP positive regions immediately (< 0.1h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal. PMID:21388382

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

PubMed Central

Copertino, D W; Christopher, D A; Hallick, R B

1991-01-01

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts. Images PMID:1721702

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

PubMed

Grasso, Francesca; Ruggieri, Vitalba; De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-04-10

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

Effect of mitomycin C on keloid fibroblasts: an in vitro study.

PubMed

Simman, Richard; Alani, Hashim; Williams, Frances

2003-01-01

Keloids are the result of aberrant wound healing of human skin after dermal injury. Therapeutic options include excision followed by radiation therapy, steroid injection, and compression with silicone sheets among others. Local invasion and recurrence after excision has provoked interest in treating keloids as neoplasms. The purpose of this study was to determine the effect of mitomycin C (MMC) on keloid fibroblasts. Keloid fibroblasts obtained from five different patients were exposed to MMC. A control group of normal and keloid cells was treated with phosphate buffered saline only. Contrast microscopy showed a decrease of fibroblast density during the 3 weeks after exposure for normal and keloid fibroblasts relative to untreated fibroblasts. This was confirmed by total cell counts ( = 0.1) and measurement of DNA synthesis. By the third week, there was a recovery in DNA synthesis and increased cell count for some of the treated fibroblasts. We concluded that at an appropriate concentration, MMC shows proliferation of keloid fibroblasts in vitro for a period of 3 weeks. This agent may be considered in clinical trials after surgical excision of keloids.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zahn, Karl E.; Averill, April; Wallace, Susan S.

5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C {yields} T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, amore » high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC {approx}5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.« less

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA.

PubMed

Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A

2006-07-13

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

PubMed Central

Kothandapani, Anbarasi; Sawant, Akshada; Dangeti, Venkata Srinivas Mohan Nimai; Sobol, Robert W.; Patrick, Steve M.

2013-01-01

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin. PMID:23761438

Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae▿ ‡

PubMed Central

Czurda, Stefan; Jechlinger, Wolfgang; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

2010-01-01

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery. PMID:20562305

PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome

PubMed Central

2010-01-01

Background The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe. Results In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing. Conclusion The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants. PMID:20178628

The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

PubMed

Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

2016-05-19

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

PubMed Central

Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

2016-01-01

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

DTIC Science & Technology

2014-03-01

in BRCA1, which is required for homologous recombination, or in any of the Fanconi anaemia complementation group ( FANC ) genes , which excise DNA...The Brca1 gene is required for DNA repair by homologous recombination and normal embryonic development. The protein 53BP1 promotes ligation and...in the right panel. Key Research Accomplishments • Deletion of the DNA damage response gene RIF1 mimics 53BP1 deficiency with respect to

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

PubMed Central

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-01-01

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites is differentially Governed by Replication

PubMed Central

Shen, Xi; Do, Huong; Li, Yongjian; Chung, Woo-Hyun; Tomasz, Maria; de Winter, Johan P.; Xia, Bing; Elledge, Stephen J.; Wang, Weidong; Li, Lei

2009-01-01

Summary Fanconi anemia (FA) is characterized by cellular hypersensivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remains unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Inter-dependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. PMID:19748364

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA*

PubMed Central

Rashid, Ishtiaque; Tomkinson, Alan E.; Pederson, David S.

2017-01-01

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin. PMID:28184006

In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

PubMed Central

Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

2012-01-01

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.

PubMed

Lindsey-Boltz, Laura A; Reardon, Joyce T; Wold, Marc S; Sancar, Aziz

2012-10-19

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2

PubMed Central

Malfatti, Matilde Clarissa; Balachander, Sathya; Antoniali, Giulia; Koh, Kyung Duk; Saint-Pierre, Christine; Gasparutto, Didier; Chon, Hyongi; Crouch, Robert J.

2017-01-01

Abstract Ribonucleoside 5′-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3′-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA. PMID:28977421

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

EMBRYONIC DEVELOPMENT AND A QUANTITATIVE MODEL OF PROGRAMMED DNA ELIMINATION IN MESOCYCLOPS EDAX (S. A. FORBES, 1891) (COPEPODA: CYCLOPOIDA)

PubMed Central

Clower, Michelle K.; Holub, Ashton S.; Smith, Rebecca T.; Wyngaard, Grace A.

2016-01-01

The highly programmed fragmentation of chromosomes and elimination of large amounts of nuclear DNA from the presomatic cell lineages (i.e., chromatin diminution), occurs in the embryos of the freshwater zooplankton Mesocyclops edax (S. A. Forbes, 1891) (Crustacea: Copepoda). The somatic genome is reorganized and reduced to a size five times smaller even though the germline genome remains intact. We present the first comprehensive, quantitative model of DNA content throughout embryogenesis in a copepod that possesses embryonic DNA elimination. We used densitometric image analysis to measure the DNA content of polar bodies, germline and somatic nuclei, and excised DNA “droplets.” We report: 1) variable DNA contents of polar bodies, some of which do not contain the amount corresponding to the haploid germline genome size; 2) presence of pronuclei in newly laid embryo sacs; 3) gonomeric chromosomes in the second to fourth cleavage divisions and in the primordial germ cell and primordial endoderm cell during the fifth cleavage division; 4) timing of early embryonic cell stages, elimination of DNA, and divisions of the primordial germ cell and primordial endoderm cell at 22°C; and 5) persistence of a portion of the excised DNA “droplets” throughout embryogenesis. DNA elimination is a trait that spans multiple embryonic stages and a knowledge of the timing and variability of the associated cytological events with DNA elimination will promote the study of the molecular mechanisms involved in this trait. We propose the “genome yolk hypothesis” as a functional explanation for the persistence of the eliminated DNA that might serve as a resource during postdiminution cleavage divisions. PMID:27857452

Application of the microfluidic-assisted replication track analysis to measure DNA repair in human and mouse cells.

PubMed

Welcsh, Piri; Kehrli, Keffy; Lazarchuk, Pavlo; Ladiges, Warren; Sidorova, Julia

2016-10-01

Functional studies of the roles that DNA helicases play in human cells have benefited immensely from DNA fiber (or single molecule) technologies, which enable us to discern minute differences in behaviors of individual replication forks in genomic DNA in vivo. DNA fiber technologies are a group of methods that use different approaches to unravel and stretch genomic DNA to its contour length, and display it on a glass surface in order to immuno-stain nucleoside analog incorporation into DNA to reveal tracks (or tracts) of replication. We have previously adopted a microfluidic approach to DNA stretching and used it to analyze DNA replication. This method was introduced under the moniker maRTA or microfluidic-assisted Replication Track Analysis, and we have since used it to analyze roles of the RECQ helicases WRN and BLM, and other proteins in normal and perturbed replication. Here we describe a novel application of maRTA to detect and measure repair of DNA damage produced by three different agents relevant to etiology or therapy of cancer: methyl-methanesulfonate, UV irradiation, and mitomycin C. Moreover, we demonstrate the utility of this method by analyzing DNA repair in cells with reduced levels of WRN or of the base excision repair protein XRCC1. Copyright © 2016 Elsevier Inc. All rights reserved.

Evidence for a Role of FEN1 in Maintaining Mitochondrial DNA Integrity

PubMed Central

Kalifa, Lidza; Beutner, Gisela; Phadnis, Naina; Sheu, Shey-Shing; Sia, Elaine A.

2009-01-01

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle’s high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. PMID:19699691

A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion.

PubMed

Hook, Ch D; Samsonov, V V; Ublinskaya, A A; Kuvaeva, T M; Andreeva, E V; Gorbacheva, L Yu; Stoynova, N V

2016-11-01

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable Cm R marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the Cm R -marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Mechanism of Adenomatous Polyposis Coli (APC)-mediated Blockage of Longpatch Base Excision Repair†

PubMed Central

Jaiswal, Aruna S.; Balusu, Ramesh; Armas, Melissa L.; Kundu, Chanakya N.; Narayan, Satya

2008-01-01

Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase β (pol-β) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC−/− cells than in APC+/+ cells. In addition to pol-β, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-β and Fen-1, we performed a domain-mapping of APC and found that both pol-β and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-β-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Our further studies demonstrated that APC blocks 5′-flap endonuclease as well as 5′-3′ exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results we concluded that APC can have three different effects in the LP-BER pathway. First, APC can block pol-β-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking “Hit and Run” synthesis. These studies will have important implications of APC in DNA damage-induced carcinogenesis and chemoprevention. PMID:17176113

Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines.

PubMed

Karmakar, P; Balajee, A S; Natarajan, A T

2001-05-01

Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase delta and epsilon, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.

Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

PubMed Central

Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

1998-01-01

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions. PMID:9560228

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair.

PubMed

Sutherland, Betsy M; Hacham, Haim; Bennett, Paula; Sutherland, John C; Moran, Michael; Gange, R W

2002-06-01

Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stark, M.; Naiman, T.; Canaani, D.

1989-08-15

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

Nucleotide excision repair by dual incisions in plants.

PubMed

Canturk, Fazile; Karaman, Muhammet; Selby, Christopher P; Kemp, Michael G; Kulaksiz-Erkmen, Gulnihal; Hu, Jinchuan; Li, Wentao; Lindsey-Boltz, Laura A; Sancar, Aziz

2016-04-26

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.

COMPREHENSIVE ANALYSES OF DNA REPAIR PATHWAYS, SMOKING, AND BLADDER CANCER RISK IN LOS ANGELES AND SHANGHAI

PubMed Central

Corral, Roman; Lewinger, Juan Pablo; Berg, David Van Den; Joshi, Amit D.; Yuan, Jian-Min; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Pike, Malcolm C.; Conti, David V.; Thomas, Duncan C.; Edlund, Christopher K.; Gao, Yu-Tang; Xiang, Yong-Bing; Zhang, Wei; Su, Yu-Chen; Stern, Mariana C.

2014-01-01

Tobacco smoking is a bladder cancer risk factor and a source of carcinogens that induce DNA damage to urothelial cells. Using data and samples from 988 cases and 1,004 controls enrolled in the Los Angeles County Bladder Cancer Study and the Shanghai Bladder Cancer Study we investigated associations between bladder cancer risk and 632 tagSNPs that comprehensively capture genetic variation in 28 DNA repair genes from four DNA repair pathways: base excision repai, nucleotide excision repair (NER), non-homologous end-joining (NHEJ), and homologous recombination repair (HHR). Odds ratios (ORs) and 95% confidence intervals (CIs) for each tagSNP were corrected for multiple testing for all SNPs within each gene using pACT, and for genes within each pathway and across pathways with Bonferroni. Gene and pathway summary estimates were obtained using ARTP. We observed an association between bladder cancer and POLB rs7832529 (BER) (pACT = 0.003; ppathway = 0.021) among all, and SNPs in XPC (NER) and OGG1 (BER) among Chinese men and women, respectively. The NER pathway showed an overall association with risk among Chinese males (ARTP NER p = 0.034). The XRCC6 SNP rs2284082 (NHEJ), also in LD with SREBF2, showed an interaction with smoking (Smoking status interaction pgene = 0.001, ppathway = 0.008, poverall = 0.034). Our findings support a role in bladder carcinogenesis for regions that map close to or within BER (POLB, OGG1) and NER genes (XPC). A SNP that tags both the XRCC6 and SREBF2 genes strongly modifies the association between bladder cancer risk and smoking. PMID:24382701

Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vermeulen, W.; Kleijer, W.J.; Bootsma, D.

1994-02-01

The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with the remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in themore » vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here the authors report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of >40 years. Analysis of the frequency of hprt[sup [minus

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

PubMed Central

Kadyk, L. C.; Hartwell, L. H.

1993-01-01

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. PMID:8454200

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Ultraviolet-Induced Decrease in Integration of Haemophilus influenzae Transforming Deoxyribonucleic Acid in Sensitive and Resistant Cells

PubMed Central

Muhammed, Amir; Setlow, Jane K.

1970-01-01

The decrease in integration of transforming deoxyribonucleic acid (DNA) caused by ultraviolet irradiation of the DNA was found to be independent of the presence or absence of excision repair in the recipient cell. Much of the ultraviolet-induced inhibition of integration resulted from the presence in the transforming DNA of pyrimidine dimers, as judged by the photoreactivability of the inhibition with yeast photoreactivating enzyme. The inhibition of integration made only a small contribution to the inactivation of transforming ability of the DNA by ultraviolet radiation. PMID:5308769

The Balancing Act of Ribonucleotides in DNA

PubMed Central

Cerritelli, Susana M.; Crouch, Robert J.

2016-01-01

The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the Ribonucleotides Excision Repair pathway, a process similar to Okazaki fragment processing after incision by RNase H2. All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA and review the data that indicate that removal of ribonucleotides plays an important role in maintaining genome stability. PMID:26996833

Molecular Cloning and 3D Structure Modeling of APEX1, DNA Base Excision Repair Enzyme from the Camel, Camelus dromedarius

PubMed Central

Ataya, Farid Shokry; Fouad, Dalia; Malik, Ajamaluddin; Saeed, Hesham Mahmoud

2012-01-01

The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, producing the highly mutagenic apurinic/apyrimidinicsites (AP sites). Therefore, recognition of AP sites is fundamental to cell/organism survival. In the present work, the full coding sequence of a putative cAPEX1 gene was amplified for the first time from C. dromedarius by RT-PCR and cloned (NCBI accession number are HM209828 and ADJ96599 for nucleotides and amino acids, respectively). cDNA sequencing was deduced to be 1041 nucleotides, of which 954 nucleotides encode a protein of 318 amino acids, similar to the coding region of the APEX1 gene and the protein from many other species. The calculated molecular weight and isoelectric point of cAPEX1 using Bioinformatics tools was 35.5 kDa and 8.11, respectively. The relative expressions of cAPEX1 in camel kidney, spleen, lung and testis were examined using qPCR and compared with that of the liver using a 18S ribosomal subunit as endogenous control. The highest level of cAPEX1 transcript was found in the testis; 325% higher than the liver, followed by spleen (87%), kidney (20%) and lung (5%), respectively. The cAPEX1 is 94%–97% similar to their mammalian counterparts. Phylogenetic analysis revealed that cAPEX1 is grouped together with that of S. scrofa. The predicted 3D structure of cAPEX1 has similar folds and topology with the human (hAPEX1). The root-mean-square deviation (rmsd) between cAPEX1 and hAPEX1 was 0.582 and the Q-score was 0.939. PMID:22942721

DNA-PKcs deficiency leads to persistence of oxidatively-induced clustered DNA lesions in human tumor cells

PubMed Central

Peddi, Prakash; Loftin, Charles W.; Dickey, Jennifer S.; Hair, Jessica M.; Burns, Kara J.; Aziz, Khaled; Francisco, Dave C.; Panayiotidis, Mihalis I.; Sedelnikova, Olga A.; Bonner, William M.; Winters, Thomas A.; Georgakilas, Alexandros G.

2010-01-01

DNA-dependent protein kinase (DNA-PK) is a key non-homologous end joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. In order to examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three different models of DNA-PK deficiency i.e. chemical inactivation of its kinase activity by novel inhibitors IC86621 and NU7026, knock-down and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). Compromised DNA-PK repair pathway has lead to accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively-induced non-DSB clustered DNA lesions measured using a modified version of pulsed field gel electrophoresis or single cell gel electrophoresis (Comet assay). In all cases, DNA-PK inactivation lead to a higher level of lesion persistence even after 24–72 hrs of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters by first compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions due to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important not only for understating cancer etiology in the presence of a NHEJ deficiency but also lead to a better understanding of cancer treatments based on the induction of oxidative stress and inhibition of cluster repair. PMID:20193758

Mutations in Cockayne Syndrome-Associated Genes (Csa and Csb) Predispose to Cisplatin-Induced Hearing Loss in Mice

PubMed Central

Rainey, Robert N.; Ng, Sum-yan; Llamas, Juan; van der Horst, Gijsbertus T. J.

2016-01-01

Cisplatin is a common and effective chemotherapeutic agent, yet it often causes permanent hearing loss as a result of sensory hair cell death. The causes of sensitivity to DNA-damaging agents in nondividing cell populations, such as cochlear hair and supporting cells, are poorly understood, as are the specific DNA repair pathways that protect these cells. Nucleotide excision repair (NER) is a conserved and versatile DNA repair pathway for many DNA-distorting lesions, including cisplatin-DNA adducts. Progressive sensorineural hearing loss is observed in a subset of NER-associated DNA repair disorders including Cockayne syndrome and some forms of xeroderma pigmentosum. We investigated whether either of the two overlapping branches that encompass NER, transcription-coupled repair or global genome repair, which are implicated in Cockayne syndrome and xeroderma pigmentosum group C, respectively, modulates cisplatin-induced hearing loss and cell death in the organ of Corti, the auditory sensory epithelium of mammals. We report that cochlear hair cells and supporting cells in transcription-coupled repair-deficient Cockayne syndrome group A (Csa−/−) and group B (Csb−/−) mice are hypersensitive to cisplatin, in contrast to global genome repair-deficient Xpc−/− mice, both in vitro and in vivo. We show that sensory hair cells in Csa−/− and Csb−/− mice fail to remove cisplatin-DNA adducts efficiently in vitro; and unlike Xpc−/− mice, Csa−/− and Csb−/− mice lose hearing and manifest outer hair cell degeneration after systemic cisplatin treatment. Our results demonstrate that Csa and Csb deficiencies predispose to cisplatin-induced hearing loss and hair/supporting cell damage in the mammalian organ of Corti, and emphasize the importance of transcription-coupled DNA repair in the protection against cisplatin ototoxicity. SIGNIFICANCE STATEMENT The utility of cisplatin in chemotherapy remains limited due to serious side effects, including sensorineural hearing loss. We show that mouse models of Cockayne syndrome, a progeroid disorder resulting from a defect in the transcription-coupled DNA repair (TCR) branch of nucleotide excision repair, are hypersensitive to cisplatin-induced hearing loss and sensory hair cell death in the organ of Corti, the mammalian auditory sensory epithelium. Our work indicates that Csa and Csb, two genes involved in TCR, are preferentially required to protect against cisplatin ototoxicity, relative to global genome repair-specific elements of nucleotide excision repair, and suggests that TCR is a major force maintaining DNA integrity in the cochlea. The Cockayne syndrome mice thus represent a model for testing the contribution of DNA repair mechanisms to cisplatin ototoxicity. PMID:27122034

DNA repair pathways and mitochondrial DNA mutations in gastrointestinal carcinogenesis.

PubMed

Basso, Daniela; Navaglia, Filippo; Fogar, Paola; Zambon, Carlo-Federico; Greco, Eliana; Schiavon, Stefania; Fasolo, Michela; Stranges, Alessia; Falda, Alessandra; Padoan, Andrea; Fadi, Elisa; Pedrazzoli, Sergio; Plebani, Mario

2007-05-01

This work focuses on the main DNA repair pathways, highlighting their role in gastrointestinal carcinogenesis and the role of mitochondrial DNA (mtDNA), mutations being described in several tumor types, including those of the gastrointestinal tract. The mismatch repair (MMR) system is inherently altered in patients with hereditary non-polyposis colorectal cancer, and plays a role in carcinogenesis in a subset of sporadic colorectal, gastric and esophageal cancers. Alterations in homologous recombination (HR) and non-homologous end-joining (NHEJ) also contribute to the development of pancreatic cancer. Gene polymorphisms of some X-ray cross-complementing (XRCCs), cofactor proteins involved in the base excision repair pathway, have been investigated in relation to gastric, colorectal and pancreatic cancer. Yet only one polymorphism, XRCC1 Arg194Trp, appears to be involved in smoking-related cancers and in early onset pancreatic cancer. Although evidence in the literature indicates that mtDNA somatic mutations play a role in gastric and colorectal carcinogenesis, no sound conclusions have yet been drawn regarding this issue in pancreatic cancer, although an mtDNA variant at 16519 is believed to worsen the outcome of pancreatic cancer patients, possibly because it is involved in altering cellular metabolism.

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation

PubMed Central

De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-01-01

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG). Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh−/−mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh−/− mice whereas similarly treated wild-type animals remained tumor-free. PMID:25638157

Canonical DNA Repair Pathways Influence R-Loop-Driven Genome Instability.

PubMed

Stirling, Peter C; Hieter, Philip

2017-10-27

DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation of DNA single-strand displacement by compromised nucleotide excision repair

PubMed Central

Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

2012-01-01

Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

PubMed Central

Peacock, Matthew; Brem, Reto; Macpherson, Peter; Karran, Peter

2014-01-01

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage. PMID:25414333

Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

PubMed

Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

2015-11-01

Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk.

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

PubMed Central

Qian, Liangyue; Yuan, Fenghua; Rodriguez-Tello, Paola; Padgaonkar, Suyog; Zhang, Yanbin

2013-01-01

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability. PMID:24349332

Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells1

PubMed Central

Jaiswal, Aruna S; Panda, Harekrushna; Pampo, Christine A; Siemann, Dietmar W; Gairola, C Gary; Hromas, Robert; Narayan, Satya

2013-01-01

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model. PMID:23555190

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

PubMed Central

Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

2004-01-01

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2.

PubMed

Ali, Seikh Imtiaz; Shin, Jae-Sun; Bae, Sung-Hun; Kim, Byoungkook; Choi, Byong-Seok

2010-07-01

The 32kDa subunit of replication protein A (RPA32) is involved in various DNA repair systems such as nucleotide excision repair, base excision repair, and homologous recombination. In these processes, RPA32 interacts with different binding partners via its C-terminal domain (RPA32C; residues 172-270). It has been reported recently that RPA32C also interacts with TIPIN during the intra-S checkpoint. To determine the significance of the interaction of RPA32C with TIPIN, we have examined the interaction mode using NMR spectroscopy and an in silico modeling approach. Here, we show that TIPIN(185-218), which shares high sequence similarity with XPA(10-43) and UNG2(56-89), is less ordered in the free state and then forms a longer alpha-helix upon binding to RPA32C. The binding interface between TIPIN(185-218) and RPA32C is similar to those of XPA and UNG2, but its mode of interaction is different. The results suggest that RPA32 is an exchange point for multiple proteins involved in DNA repair, homologous recombination, and checkpoint processes and that it binds to different partners with comparable binding affinity using a single site. Copyright 2010 Elsevier Ltd. All rights reserved.

An Aromatic Sensor with Aversion to Damaged Strands Confers Versatility to DNA Repair

PubMed Central

Maillard, Olivier; Solyom, Szilvia; Naegeli, Hanspeter

2007-01-01

It was not known how xeroderma pigmentosum group C (XPC) protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix. PMID:17355181

Antimutagenic, antigenotoxic and antioxidant activities of Acacia salicina extracts (ASE) and modulation of cell gene expression by H2O2 and ASE treatment.

PubMed

Bouhlel, Ines; Valenti, Kita; Kilani, Soumaya; Skandrani, Ines; Ben Sghaier, Mohamed; Mariotte, Anne-Marie; Dijoux-Franca, Marie-Genevieve; Ghedira, Kamel; Hininger-Favier, Isabelle; Laporte, François; Chekir-Ghedira, Leila

2008-08-01

The total oligomers flavonoids (TOF), chloroform, petroleum ether and aqueous extracts from Acacia salicina, were investigated for the antioxidative, cytotoxic, antimutagenic and antigenotoxic activities. The viability of K562 cells were affected by all extracts after 48 h exposure. Our results showed that A. salicina extracts have antigenotoxic and/or antimutagenic activities. TOF and chloroform extracts exhibit antioxidant properties, expressed by the capacity of these extracts to inhibit xanthine oxidase activity. To further explore the mechanism of action of A. salicina extracts, we characterized expression profiles of genes involved in antioxidant protection and DNA repair in the human lymphoblastic cell line K562 exposed to H2O2. Transcription of several genes related to the thioredoxin antioxidant system and to the DNA base-excision repair pathway was up-regulated after incubation with chloroform, TOF and petroleum ether extracts. Moreover genes involved in the nucleotide-excision repair pathway and genes coding for catalase and Mn-superoxide-dismutase, two important antioxidant enzymes, were induced after incubation with the chloroform extract. Taken together, these observations provide evidence that the chloroform and TOF extracts of A. salicina leaves contain bioactive compounds that are able to protect cells against the consequences of an oxidative stress.

The versatile DNA nucleotide excision repair (NER) and its medical significance.

PubMed

Falik-Zaccai, Tzipora C; Keren, Zohar; Slor, Hanoch

2009-12-01

Two of DNA's worst enemies, ultraviolet light and chemical carcinogens, can cause damage to the molecule by mutating individual nucleotides or changing its physical structure. In most cases, genomic integrity is restored by specialized suites of proteins dedicated to repairing specific types of injuries. One restoration mechanism, called nucleotide excision repair (NER), recruits and coordinates the services of 20-30 proteins to recognize and remove structure-impairing lesions, including those induced by ultraviolet (UV) light. Mutations in a gene that encodes a protein from the NER machinery might cause a wide variety of rare inherited human disorders. Sun sensitivity, cancer, developmental retardation, neurodegeneration and premature aging characterize these syndromes. Identification of the causative genes and proteins in affected families in Israel allowed us to establish accurate molecular diagnosis of couples at risk, and provide them with better genetic counseling.

Excision of thymine dimers in vitro by extracts of bacteriophage-infected Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedberg, E.C.; Minton, K.; Pawl, G.

1974-05-01

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from uv- irradiated DNA previously nicked with T4 uv endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 uv-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. Amore » similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7. (auth)« less

Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea.

PubMed Central

Margison, G P; Kleihues, P

1975-01-01

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds. PMID:1200992

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

PubMed

Perucca, Paola; Mocchi, Roberto; Guardamagna, Isabella; Bassi, Elisabetta; Sommatis, Sabrina; Nardo, Tiziana; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

2018-06-01

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2 Wt and DDB2 PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2 PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity. Copyright © 2018 Elsevier B.V. All rights reserved.

Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

PubMed Central

Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

2015-01-01

DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

PubMed

You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

2014-03-01

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Distant neighbor base sequence context effects in human nucleotide excision repair of a benzo[a]pyrene-derived DNA lesion

PubMed Central

Cai, Yuqin; Kropachev, Konstantin; Xu, Rong; Tang, Yijin; Kolbanovskii, Marina; Kolbanovskii, Alexander; Amin, Shantu; Patel, Dinshaw J.; Broyde, Suse; Geacintov, Nicholas E.

2010-01-01

Summary The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N2-dG (G*) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G*-C-T-A-C-C-3′ (CG*C-I), and 5′-C-A-C3-A4-C5-G*-C-A-C-A-C-3′ (CG*C-II). We utilized gel electrophoresis to compare the extent of DNA bending, and molecular dynamics (MD) simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6 ± 0.2 times greater in the case of the CG*C-II than the CG*C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG*C-II duplex is more bent than the CG*C-I duplex, while MD simulations showed that the unique -C3-A4-C5- segment in the CG*C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, namely, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG*C-II than in CG*C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG*C-II sequence. These structural properties fit well with our prior findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N2-dG lesions, flexible bends and enlarged minor groove widths (Cai et al. (2009) J. Mol. Biol., 385: 30–44) constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion. PMID:20399214

Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H‐NS of Escherichia coli

PubMed Central

Hong, Seok Hoon; Wang, Xiaoxue; Wood, Thomas K.

2010-01-01

Summary The global regulator H‐NS of Escherichia coli controls genes related to stress response, biofilm formation and virulence by recognizing curved DNA and by silencing acquired genes. Here, we rewired H‐NS to control biofilm formation using protein engineering; H‐NS variant K57N was obtained that reduces biofilm formation 10‐fold compared with wild‐type H‐NS (wild‐type H‐NS increases biofilm formation whereas H‐NS K57N reduces it). Whole‐transcriptome analysis revealed that H‐NS K57N represses biofilm formation through its interaction with the nucleoid‐associated proteins Cnu and StpA and in the absence of these proteins, H‐NS K57N was unable to reduce biofilm formation. Significantly, H‐NS K57N enhanced the excision of defective prophage Rac while wild‐type H‐NS represses excision, and H‐NS controlled only Rac excision among the nine resident E. coli K‐12 prophages. Rac prophage excision not only led to the change in biofilm formation but also resulted in cell lysis through the expression of toxin HokD. Hence, the H‐NS regulatory system may be evolved through a single‐amino‐acid change in its N‐terminal oligomerization domain to control biofilm formation, prophage excision and apoptosis. PMID:21255333

Alar base reduction: the boomerang-shaped excision.

PubMed

Foda, Hossam M T

2011-04-01

A boomerang-shaped alar base excision is described to narrow the nasal base and correct the excessive alar flare. The boomerang excision combined the external alar wedge resection with an internal vestibular floor excision. The internal excision was inclined 30 to 45 degrees laterally to form the inner limb of the boomerang. The study included 46 patients presenting with wide nasal base and excessive alar flaring. All cases were followed for a mean period of 18 months (range, 8 to 36 months). The laterally oriented vestibular floor excision allowed for maximum preservation of the natural curvature of the alar rim where it meets the nostril floor and upon its closure resulted in a considerable medialization of alar lobule, which significantly reduced the amount of alar flare and the amount of external alar excision needed. This external alar excision measured, on average, 3.8 mm (range, 2 to 8 mm), which is significantly less than that needed when a standard vertical internal excision was used ( P < 0.0001). Such conservative external excisions eliminated the risk of obliterating the natural alar-facial crease, which did not occur in any of our cases. No cases of postoperative bleeding, infection, or vestibular stenosis were encountered. Keloid or hypertrophic scar formation was not encountered; however, dermabrasion of the scars was needed in three (6.5%) cases to eliminate apparent suture track marks. The boomerang alar base excision proved to be a safe and effective technique for narrowing the nasal base and elimination of the excessive flaring and resulted in a natural, well-proportioned nasal base with no obvious scarring. © Thieme Medical Publishers.

Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions

PubMed Central

Tomanicek, Stephen J.; Hughes, Ronny C.; Ng, Joseph D.; Coates, Leighton

2010-01-01

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-­hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-­ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily. PMID:20823514

Delayed repair of radiation induced clustered DNA damage: Friend or foe?

PubMed Central

Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

2011-01-01

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts

PubMed Central

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-01-01

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

PubMed

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-11-24

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.

Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

PubMed

Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

2008-12-01

Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

PubMed

Malfatti, Matilde Clarissa; Balachander, Sathya; Antoniali, Giulia; Koh, Kyung Duk; Saint-Pierre, Christine; Gasparutto, Didier; Chon, Hyongi; Crouch, Robert J; Storici, Francesca; Tell, Gianluca

2017-11-02

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The role of RNase H2 in processing ribonucleotides incorporated during DNA replication.

PubMed

Williams, Jessica S; Gehle, Daniel B; Kunkel, Thomas A

2017-05-01

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 (rnh201-RED; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε (pol2-M644G) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 (rnh201Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2-5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication. Published by Elsevier B.V.

Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

PubMed Central

Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

2010-01-01

The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

PubMed

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

PubMed Central

Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff S.; Tang, Christoph M.

2013-01-01

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. PMID:22296581

Contribution of indirect effects to clustered damage in DNA irradiated with protons.

PubMed

Pachnerová Brabcová, K; Štěpán, V; Karamitros, M; Karabín, M; Dostálek, P; Incerti, S; Davídková, M; Sihver, L

2015-09-01

Protons are the dominant particles both in galactic cosmic rays and in solar particle events and, furthermore, proton irradiation becomes increasingly used in tumour treatment. It is believed that complex DNA damage is the determining factor for the consequent cellular response to radiation. DNA plasmid pBR322 was irradiated at U120-M cyclotron with 30 MeV protons and treated with two Escherichia coli base excision repair enzymes. The yields of SSBs and DSBs were analysed using agarose gel electrophoresis. DNA has been irradiated in the presence of hydroxyl radical scavenger (coumarin-3-carboxylic acid) in order to distinguish between direct and indirect damage of the biological target. Pure scavenger solution was used as a probe for measurement of induced OH· radical yields. Experimental OH· radical yield kinetics was compared with predictions computed by two theoretical models-RADAMOL and Geant4-DNA. Both approaches use Geant4-DNA for description of physical stages of radiation action, and then each of them applies a distinct model for description of the pre-chemical and chemical stage. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

PubMed

Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

2005-04-15

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

The Influence of Local DNA Sequence and DNA Repair Background on the Mutational Specificity of 1-Nitroso-8-Nitropyrene in Escherichia Coli: Inferences for Mutagenic Mechanisms

PubMed Central

Lambert, I. B.; Gordon, AJE.; Glickman, B. W.; McCalla, D. R.

1992-01-01

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/-ΔuvrB) and MucA/B-mediated error-prone translesion synthesis (+/-pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (>92%) which occurred at G·C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G·C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G·C -> T·A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G·C -> T·A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G·C -> T·A transversions by a factor of 30-60. In contrast, the -(G·C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G·C -> T·A transversions and -(G·C) frameshifts. Deletion mutations were induced in the Δuvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G·C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell. PMID:1459443

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

PubMed Central

Haley, Joshua A.; Stark, W. Marshall

2016-01-01

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo. The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research. PMID:28003200

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system

PubMed Central

Verhoeven, Esther E. A.; van Kesteren, Marian; Turner, John J.; van der Marel, Gijs A.; van Boom, Jacques H.; Moolenaar, Geri F.; Goosen, Nora

2002-01-01

Nucleotide excision repair in Escherichia coli involves formation of the UvrB–DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB–DNA complex. Both stable and unstable UvrB–DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB–DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix–hairpin–helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)2 domain. This (HhH)2 domain was previously shown to be important for the 5′ incision on a substrate containing a (cis-Pt)·GG adduct, but not for 3′ incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)2 domain can be important for 3′ and/or 5′ incision. We propose that the (HhH)2 domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system. PMID:12034838

Arsenic Exposure Is Associated with Decreased DNA Repair in Vitro and in Individuals Exposed to Drinking Water Arsenic

PubMed Central

Andrew, Angeline S.; Burgess, Jefferey L.; Meza, Maria M.; Demidenko, Eugene; Waugh, Mary G.; Hamilton, Joshua W.; Karagas, Margaret R.

2006-01-01

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action. PMID:16882524

Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

PubMed

Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

2016-08-16

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

A single-layer platform for Boolean logic and arithmetic through DNA excision in mammalian cells

PubMed Central

Weinberg, Benjamin H.; Hang Pham, N. T.; Caraballo, Leidy D.; Lozanoski, Thomas; Engel, Adrien; Bhatia, Swapnil; Wong, Wilson W.

2017-01-01

Genetic circuits engineered for mammalian cells often require extensive fine-tuning to perform their intended functions. To overcome this problem, we present a generalizable biocomputing platform that can engineer genetic circuits which function in human cells with minimal optimization. We used our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform to build more than 100 multi-input-multi-output circuits. We devised a quantitative metric to evaluate the performance of the circuits in human embryonic kidney and Jurkat T cells. Of 113 circuits analysed, 109 functioned (96.5%) with the correct specified behavior without any optimization. We used our platform to build a three-input, two-output Full Adder and six-input, one-output Boolean Logic Look Up Table. We also used BLADE to design circuits with temporal small molecule-mediated inducible control and circuits that incorporate CRISPR/Cas9 to regulate endogenous mammalian genes. PMID:28346402

Polymorphisms in RAI and in genes of nucleotide and base excision repair are not associated with risk of testicular cancer.

PubMed

Laska, Magdalena J; Nexø, Bjørn A; Vistisen, Kirsten; Poulsen, Henrik Enghusen; Loft, Steffen; Vogel, Ulla

2005-07-28

Testicular cancer has been suggested to be primed in utero and there is familiar occurrence, particularly brothers and sons of men with testicular cancer have increased risk. Although no specific causative genotoxic agents have been identified, variations in DNA repair capacity could be associated with the risk of testicular cancer. A case-control study of 184 testicular cancer cases and 194 population-based controls living in the Copenhagen Greater Area in Denmark was performed. We found that neither polymorphisms in several DNA repair genes nor alleles of several polymorphisms in the chromosomal of region 19q13.2-3, encompassing the genes ASE, ERCC1, RAI and XPD, were associated with risk of testicular cancer in Danish patients. This is in contrast to other cancers, where we reported strong associations between polymorphisms in ERCC1, ASE and RAI and occurrence of basal cell carcinoma, breast cancer and lung. To our knowledge this is the first study of DNA repair gene polymorphisms and risk of testicular cancer.

Repair of naturally occurring mismatches can induce mutations in flanking DNA

PubMed Central

Chen, Jia; Miller, Brendan F; Furano, Anthony V

2014-01-01

‘Normal’ genomic DNA contains hundreds of mismatches that are generated daily by the spontaneous deamination of C (U/G) and methyl-C (T/G). Thus, a mutagenic effect of their repair could constitute a serious genetic burden. We show here that while mismatches introduced into human cells on an SV40-based episome were invariably repaired, this process induced mutations in flanking DNA at a significantly higher rate than no mismatch controls. Most mutations involved the C of TpC, the substrate of some single strand-specific APOBEC cytidine deaminases, similar to the mutations that can typify the ‘mutator phenotype’ of numerous tumors. siRNA knockdowns and chromatin immunoprecipitation showed that TpC preferring APOBECs mediate the mutagenesis, and siRNA knockdowns showed that both the base excision and mismatch repair pathways are involved. That naturally occurring mispairs can be converted to mutators, represents an heretofore unsuspected source of genetic changes that could underlie disease, aging, and evolutionary change. DOI: http://dx.doi.org/10.7554/eLife.02001.001 PMID:24843013

Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

PubMed

Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

2016-04-06

Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A

PubMed Central

Rapić Otrin, Vesna; Kuraoka, Isao; Nardo, Tiziana; McLenigan, Mary; Eker, A. P. M.; Stefanini, Miria; Levine, Arthur S.; Wood, Richard D.

1998-01-01

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB− XP-E cell extracts, but microinjection of the protein into DDB− XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin. PMID:9584159

Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells.

PubMed

Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

2012-09-15

Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

Synthesis and structure of duplex DNA containing the genotoxic nucleobase lesion N7-methylguanine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, S.; Bowman, B.R.; Ueno, Y.

2008-11-03

The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2{prime}-deoxyguanosine (m{sup 7}dG). M{sup 7}dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans. Structural studies of m{sup 7}dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile m{sup 7}dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2{prime}-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in themore » DNA deprotection reaction. Duplex DNA containing 2{prime}-fluoro-m{sup 7}dG (Fm{sup 7}dG) cocrystallized with AlkA as a host-guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of Fm{sup 7}dG:C is nearly identical to that of G:C, and Fm{sup 7}dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m{sup 7}dG lesions.« less

The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation.

PubMed

Guo, Yan; Cai, Qiuyin; Samuels, David C; Ye, Fei; Long, Jirong; Li, Chung-I; Winther, Jeanette F; Tawn, E Janet; Stovall, Marilyn; Lähteenmäki, Päivi; Malila, Nea; Levy, Shawn; Shaffer, Christian; Shyr, Yu; Shu, Xiao-Ou; Boice, John D

2012-05-15

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children. Copyright © 2012 Elsevier B.V. All rights reserved.

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

PubMed

Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

2016-04-01

Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.

PubMed

Musharova, Olga; Klimuk, Evgeny; Datsenko, Kirill A; Metlitskaya, Anastasia; Logacheva, Maria; Semenova, Ekaterina; Severinov, Konstantin; Savitskaya, Ekaterina

2017-04-07

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

PubMed

Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

2014-09-01

A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

PubMed Central

Isogawa, Asako; Fujii, Shingo

2017-01-01

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication. PMID:28686598

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease.

PubMed

O'Brien, Travis J; Jiang, Guohui; Chun, Gina; Mandel, H George; Westphal, Craig S; Kahen, Kaveh; Montaser, Akbar; States, J Christopher; Patierno, Steven R

2006-11-07

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.

Adenine specific DNA chemical sequencing reaction.

PubMed Central

Iverson, B L; Dervan, P B

1987-01-01

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Images PMID:3671067

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera).

PubMed

Park, Bokri; Kim, Yonggyun

2011-06-01

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.

Cockayne syndrome: Clinical features, model systems and pathways

PubMed Central

Karikkineth, Ajoy C.; Scheibye-Knudsen, Morten; Fivenson, Elayne; Croteau, Deborah L.; Bohr, Vilhelm A.

2016-01-01

Cockayne syndrome (CS) is a disorder characterized by a variety of clinical features including cachectic dwarfism, severe neurological manifestations including microcephaly and cognitive deficits, pigmentary retinopathy, cataracts, sensorineural deafness, and ambulatory and feeding difficulties, leading to death by 12 years of age on average. It is an autosomal recessive disorder, with a prevalence of approximately 2.5 per million. There are several phenotypes (1, 2 and 3) and complementation groups (CSA and CSB), and overlaps with xeroderma pigmentosum (XP). It has been considered a progeria, and many of the clinical features resemble accelerated aging. As such, the study of CS affords an opportunity to better understand the underlying mechanisms of aging. The molecular basis of CS has traditionally been considered to be due to defects in transcription and transcription-coupled nucleotide excision repair (TC-NER). However, recent work suggests that defects in base excision DNA repair and mitochondrial functions may also play key roles. This opens up the possibility of molecular interventions in CS, and by extrapolation, possibly in aging. PMID:27507608

Glycosylases utilize ``stop and go'' motion to locate DNA damage

NASA Astrophysics Data System (ADS)

Nelson, Shane

2015-03-01

Oxidative damage to DNA results in alterations that are mutagenic or even cytotoxic. Base excision repair is a mechanism that functions to identify and correct these lesions, and is present in organisms ranging from bacteria to humans. DNA glycosylases are the first enzymes in this pathway and function to locate and remove oxidatively damaged bases, and do so utilizing only thermal energy. However, the question remains of how these enzymes locate and recognize a damaged base among millions of undamaged bases. Utilizing fluorescence video microscopy with high spatial and temporal resolution, we have observed a number of different fluorescently labeled glycosylases (including bacterial FPG, NEI, and NTH as well as mammalian MutyH and OGG). These enzymes diffuse along DNA tightropes at approximately 0.01 +/- 0.005 μm2/s with binding lifetimes ranging from one second to several minutes. Chemically induced damage to the DNA substrate causes a ~ 50% reduction in diffusion coefficients and a ~ 400% increase in binding lifetimes, while mutation of the key ``wedge residue'' - which has been shown to be responsible for damage detection - results in a 200% increase in the diffusion coefficient. Utilizing a sliding window approach to measure diffusion coefficients within individual trajectories, we observe that distributions of diffusion coefficients are bimodal, consistent with periods of diffusive motion interspersed with immobile periods. Utilizing a unique chemo-mechanical simulation approach, we demonstrate that the motion of these glycosylases can be explained as free diffusion along the helical pitch of the DNA, punctuated with two different types of pauses: 1) rapid, short-lived pauses as the enzyme rapidly probes DNA bases to interrogate for damage and, 2) less frequent, longer lived pauses that reflect the enzyme bound to and catalytically removing a damaged base. These simulations also indicate that the wedge residue is critical for interrogation and recognition of damage, and thus enzymes missing this residue diffuse faster. Similarly, chemically induced damage increases the frequency with which the enzymes encounter damaged bases, resulting in slower diffusion.

Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

PubMed Central

Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

2014-01-01

Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

Base excision repair: a critical player in many games.

PubMed

Wallace, Susan S

2014-07-01

This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person's view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field. Copyright © 2014 Elsevier B.V. All rights reserved.

Association of Leukotrichia in Vitiligo and Asp148Glu Polymorphism of Apurinic/Apyrimidinic Endonuclease 1.

PubMed

Aydin, A Fatih; Aydıngöz, İkbal Esen; Doğru-Abbasoğlu, Semra; Vural, Pervin; Uysal, Müjdat

2017-01-01

Oxidative stress and increased DNA damage have been implicated in the etiopathogenesis of vitiligo. Oxidative DNA damage is mainly repaired by the base excision repair (BER) pathway. We sought to determine whether polymorphisms in DNA repair genes may have a role in the pathogenesis of vitiligo. We conducted a study including 100 patients with vitiligo and age- and sex-matched 193 control subjects to examine the role of single-nucleotide polymorphisms of BER genes, human 8-oxoG DNA N-glycosylase 1 (codon 326), apurinic/apyrimidinic endonuclease 1 (APE1) (codon 148), and X-ray repair cross-complementing group 1 (codon 399) as risk factors for vitiligo. These polymorphisms were determined by quantitative real-time polymerase chain reaction and melting curve analysis. No significant association was observed between the variant alleles of studied genes and vitiligo. However, we showed that the presence of APE1 148Glu variant allele is associated with leukotrichia. This preliminary study suggests that APE1 (codon 148) polymorphism may play a role in vitiligo pathogenesis.

Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs.

PubMed

Tessitore, Marion Vaglio; Sottini, Alessandra; Roccaro, Aldo M; Ghidini, Claudia; Bernardi, Simona; Martellosio, Giovanni; Serana, Federico; Imberti, Luisa

2017-04-05

A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. DNA was prepared from blood of 203 healthy adults (range: 18-91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.

Requirement of the Saccharomyces cerevisiae APN1 Gene for the Repair of Mitochondrial DNA Alkylation Damage

PubMed Central

Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.

2010-01-01

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988

Age and exposure to arsenic alter base excision repair transcript levels in mice.

PubMed

Osmond, Megan J; Kunz, Bernard A; Snow, Elizabeth T

2010-09-01

Arsenic (As) induces DNA-damaging reactive oxygen species. Most oxidative DNA damage is countered by base excision repair (BER), the capacity for which may be reduced in older animals. We examined whether age and consumption of As in lactational milk or drinking water influences BER gene transcript levels in mice. Lactating mothers and 24-week-old mice were exposed (24 h or 2 weeks) to As (2 or 50 p.p.m.) in drinking water. Lung tissue was harvested from adults, neonates (initially 1 week old) feeding from lactating mothers and untreated animals 1-26 weeks old. Transcripts encoding BER proteins were quantified. BER transcript levels decreased precipitously with age in untreated mice but increased in neonates whose mothers were exposed to 50 p.p.m. As for 24 h or 2 weeks. Treatment of 24-week-old mice with 2 or 50 p.p.m. As for 2 weeks decreased all transcript levels measured. Exposure to As attenuated the age-related transcript level decline for only one BER gene. We conclude that aging is associated with a rapid reduction of BER transcript levels in mice, which may contribute to decreased BER activity in older animals. Levels of As that can alter gene expression are transmitted to neonatal mice in lactational milk produced by mothers drinking water containing As, raising concerns about breastfeeding in countries having As-contaminated groundwater. Reduction of BER transcript levels in 24-week-old mice exposed to As for 2 weeks suggests As may potentiate sensitivity to itself in older animals.

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ.

PubMed

Klvaňa, Martin; Bren, Urban; Florián, Jan

2016-12-29

Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3'-OH group of the primer DNA strand, and the subsequent formation and cleavage of P-O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol -1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P-O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pK a of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

PubMed Central

2016-01-01

Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3′-OH group of the primer DNA strand, and the subsequent formation and cleavage of P–O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol–1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P–O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3′-OH group as a predictor of the catalytic rate of X-family DNA polymerases. PMID:27992186

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Jung-Hoon; Qiu Junzhuan; Cai Sheng

2006-05-01

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding proteinmore » that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.« less

The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

PubMed Central

Gros, Laurent; Ishchenko, Alexander A.; Ide, Hiroshi; Elder, Rhoderick H.; Saparbaev, Murat K.

2004-01-01

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5′-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2′-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2′-deoxyuridine, alpha-2′-deoxyadenosine and alpha-thymidine adducts, generating 3′-hydroxyl and 5′-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed. PMID:14704345

Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

NASA Astrophysics Data System (ADS)

Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

2014-11-01

Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

PubMed Central

Newman, Matthew; Murray-Rust, Judith; Lally, John; Rudolf, Jana; Fadden, Andrew; Knowles, Philip P; White, Malcolm F; McDonald, Neil Q

2005-01-01

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3′ flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes. PMID:15719018

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

PubMed

Tsuda, Masataka; Cho, Kosai; Ooka, Masato; Shimizu, Naoto; Watanabe, Reiko; Yasui, Akira; Nakazawa, Yuka; Ogi, Tomoo; Harada, Hiroshi; Agama, Keli; Nakamura, Jun; Asada, Ryuta; Fujiike, Haruna; Sakuma, Tetsushi; Yamamoto, Takashi; Murai, Junko; Hiraoka, Masahiro; Koike, Kaoru; Pommier, Yves; Takeda, Shunichi; Hirota, Kouji

2017-01-01

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

Whole-genome landscape of pancreatic neuroendocrine tumours.

PubMed

Scarpa, Aldo; Chang, David K; Nones, Katia; Corbo, Vincenzo; Patch, Ann-Marie; Bailey, Peter; Lawlor, Rita T; Johns, Amber L; Miller, David K; Mafficini, Andrea; Rusev, Borislav; Scardoni, Maria; Antonello, Davide; Barbi, Stefano; Sikora, Katarzyna O; Cingarlini, Sara; Vicentini, Caterina; McKay, Skye; Quinn, Michael C J; Bruxner, Timothy J C; Christ, Angelika N; Harliwong, Ivon; Idrisoglu, Senel; McLean, Suzanne; Nourse, Craig; Nourbakhsh, Ehsan; Wilson, Peter J; Anderson, Matthew J; Fink, J Lynn; Newell, Felicity; Waddell, Nick; Holmes, Oliver; Kazakoff, Stephen H; Leonard, Conrad; Wood, Scott; Xu, Qinying; Nagaraj, Shivashankar Hiriyur; Amato, Eliana; Dalai, Irene; Bersani, Samantha; Cataldo, Ivana; Dei Tos, Angelo P; Capelli, Paola; Davì, Maria Vittoria; Landoni, Luca; Malpaga, Anna; Miotto, Marco; Whitehall, Vicki L J; Leggett, Barbara A; Harris, Janelle L; Harris, Jonathan; Jones, Marc D; Humphris, Jeremy; Chantrill, Lorraine A; Chin, Venessa; Nagrial, Adnan M; Pajic, Marina; Scarlett, Christopher J; Pinho, Andreia; Rooman, Ilse; Toon, Christopher; Wu, Jianmin; Pinese, Mark; Cowley, Mark; Barbour, Andrew; Mawson, Amanda; Humphrey, Emily S; Colvin, Emily K; Chou, Angela; Lovell, Jessica A; Jamieson, Nigel B; Duthie, Fraser; Gingras, Marie-Claude; Fisher, William E; Dagg, Rebecca A; Lau, Loretta M S; Lee, Michael; Pickett, Hilda A; Reddel, Roger R; Samra, Jaswinder S; Kench, James G; Merrett, Neil D; Epari, Krishna; Nguyen, Nam Q; Zeps, Nikolajs; Falconi, Massimo; Simbolo, Michele; Butturini, Giovanni; Van Buren, George; Partelli, Stefano; Fassan, Matteo; Khanna, Kum Kum; Gill, Anthony J; Wheeler, David A; Gibbs, Richard A; Musgrove, Elizabeth A; Bassi, Claudio; Tortora, Giampaolo; Pederzoli, Paolo; Pearson, John V; Waddell, Nicola; Biankin, Andrew V; Grimmond, Sean M

2017-03-02

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

ATM regulates 3-Methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents

PubMed Central

Agnihotri, Sameer; Burrell, Kelly; Buczkowicz, Pawel; Remke, Marc; Golbourn, Brian; Chornenkyy, Yevgen; Gajadhar, Aaron; Fernandez, Nestor A.; Clarke, Ian D.; Barszczyk, Mark S.; Pajovic, Sanja; Ternamian, Christian; Head, Renee; Sabha, Nesrin; Sobol, Robert W.; Taylor, Michael D; Rutka, James T.; Jones, Chris; Dirks, Peter B.; Zadeh, Gelareh; Hawkins, Cynthia

2014-01-01

Alkylating agents are a frontline therapy for the treatment of several aggressive cancers including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed; increasing therapeutic response while minimizing toxicity. Using a siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM) were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. PMID:25100205

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair*

PubMed Central

Bélanger, François; Angers, Jean-Philippe; Fortier, Émile; Hammond-Martel, Ian; Costantino, Santiago; Drobetsky, Elliot; Wurtele, Hugo

2016-01-01

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response. PMID:26578521

Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes

PubMed Central

Fujita, Masashi; Watanabe, Shun; Yoshizawa, Mariko; Yamamoto, Junpei; Iwai, Shigenori

2015-01-01

Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4) photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4) photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA. PMID:25679955

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

PubMed

Miropolskaya, Nataliya; Petushkov, Ivan; Kulbachinskiy, Andrey; Makarova, Alena V

2017-08-31

Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ι, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ι, with possible involvement of residues from the finger domain in the dRP group cleavage.

THE ROLE OF THE RETINOBLASTOMA/E2F1 TUMOR SUPPRESSOR PATHWAY IN THE LESION RECOGNITION STEP OF NUCLEOTIDE EXCISION REPAIR

PubMed Central

Lin, Patrick S.; McPherson, Lisa A.; Chen, Aubrey Y.; Sage, Julien; Ford, James M.

2009-01-01

The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb−/−;p107−/−;p130−/− MEFs repaired both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1−/− MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1−/− cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair. PMID:19376752

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

PubMed Central

Robu, Mihaela; Shah, Rashmi G.; Purohit, Nupur K.; Zhou, Pengbo; Naegeli, Hanspeter

2017-01-01

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV–DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1–XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV–DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins. PMID:28760956

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

PubMed

Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair

PubMed Central

Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

2015-01-01

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

Differences in DNA-damage in non-smoking men and women exposed to environmental tobacco smoke (ETS).

PubMed

Collier, Abby C; Dandge, Sachin D; Woodrow, James E; Pritsos, Chris A

2005-07-28

There is much data implicating environmental tobacco smoke (ETS) in the development and progression of disease, notably cancer, yet the mechanisms for this remain unclear. As ETS is both a pro-oxidant stressor and carcinogen, we investigated the relationship of ETS exposure to intracellular and serum levels of DNA-damage, both oxidative 8-hydroxy-2-deoxyguanosine (8OHdG) and general, in non-smokers from non-smoking households, occupationally exposed to ETS. General DNA-damage consisting of single and double strand breaks, alkali-labile sites and incomplete base-excision repair, increased significantly in a dose-dependent manner with ETS exposure in men (P=0.015, n=32, Pearson) but not women (P=0.736, n=17). Intracellular 8OHdG-DNA-damage and general DNA-damage were both greater in men than women (P=0.0005 and 0.016, respectively) but 8OHdG serum levels did not differ between the genders. Neither 8OHdG-DNA-damage nor serum levels correlated with increasing ETS exposure. This is the first study to demonstrate dose-dependent increases in DNA-damage from workplace ETS exposure. Perhaps most interesting was that despite equivalent ETS exposure, significantly greater DNA-damage occurred in men than women. These data may begin to provide a mechanistic rationale for the generally higher incidence of some diseases in males due to tobacco smoke and/or other genotoxic stressors.

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

PubMed Central

Santos, M. E.; Drake, J. W.

1994-01-01

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

N-methylpurine DNA glycosylase and DNA polymerase β modulate BER inhibitor potentiation of glioma cells to temozolomide

PubMed Central

Tang, Jiang-bo; Svilar, David; Trivedi, Ram N.; Wang, Xiao-hong; Goellner, Eva M.; Moore, Briana; Hamilton, Ronald L.; Banze, Lauren A.; Brown, Ashley R.; Sobol, Robert W.

2011-01-01

Temozolomide (TMZ) is the preferred chemotherapeutic agent in the treatment of glioma following surgical resection and/or radiation. Resistance to TMZ is attributed to efficient repair and/or tolerance of TMZ-induced DNA lesions. The majority of the TMZ-induced DNA base adducts are repaired by the base excision repair (BER) pathway and therefore modulation of this pathway can enhance drug sensitivity. N-methylpurine DNA glycosylase (MPG) initiates BER by removing TMZ-induced N3-methyladenine and N7-methylguanine base lesions, leaving abasic sites (AP sites) in DNA for further processing by BER. Using the human glioma cell lines LN428 and T98G, we report here that potentiation of TMZ via BER inhibition [methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG] is greatly enhanced by over-expression of the BER initiating enzyme MPG. We also show that methoxyamine-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme DNA polymerase β (Polβ), suggesting that cells proficient for BER readily repair AP sites in the presence of MX. Further, depletion of Polβ increases PARP inhibitor-induced potentiation in the MPG over-expressing glioma cells, suggesting that expression of Polβ modulates the cytotoxic effect of combining increased repair initiation and BER inhibition. This study demonstrates that MPG overexpression, together with inhibition of BER, sensitizes glioma cells to the alkylating agent TMZ in a Polβ-dependent manner, suggesting that the expression level of both MPG and Polβ might be used to predict the effectiveness of MX and PARP-mediated potentiation of TMZ in cancer treatment. PMID:21377995

Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia

PubMed Central

Lepère, Gersende; Bétermier, Mireille; Meyer, Eric; Duharcourt, Sandra

2008-01-01

The germline genome of ciliates is extensively rearranged during the development of a new somatic macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal macronucleus, regulates rearrangements of the zygotic genome. PMID:18519642

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

PubMed

Haskett, Timothy L; Terpolilli, Jason J; Ramachandran, Vinoy K; Verdonk, Callum J; Poole, Phillip S; O'Hara, Graham W; Ramsay, Joshua P

2018-03-01

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

PubMed

Bausinger, Julia; Schütz, Petra; Piberger, Ann Liza; Speit, Günter

2016-03-01

The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1−/− mice

PubMed Central

Prasher, Joanna M; Lalai, Astrid S; Heijmans-Antonissen, Claudia; Ploemacher, Robert E; Hoeijmakers, Jan H J; Touw, Ivo P; Niedernhofer, Laura J

2005-01-01

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1−/− mice was compared to that in young and old wild-type mice. Ercc1−/− mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1−/− mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa−/− mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging. PMID:15692571

Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

PubMed

Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

2012-09-01

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

POLD1: central mediator of DNA replication and repair, and implication in cancer and other pathologies

PubMed Central

Nicolas, Emmanuelle; Golemis, Erica A.; Arora, Sanjeevani

2016-01-01

The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5’–3’ DNA polymerase and 3’–5’ exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions. PMID:27320729

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

PubMed Central

Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

2006-01-01

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan.

PubMed

Schroeder, Elizabeth A; Shadel, Gerald S

2014-01-01

Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A method for detecting genetic toxicity using the RNA synthesis response to DNA damage.

PubMed

Morita, Yoko; Iwai, Shigenori; Kuraoka, Isao

2011-10-01

To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

PubMed

Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

2017-06-01

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis. Copyright © 2017 American Society for Microbiology.

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex*

PubMed Central

Das, Devashish; Faridounnia, Maryam; Kovacic, Lidija; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

2017-01-01

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble. PMID:28028171

[Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification].

PubMed

Dovgerd, A P; Zharkov, D O

2014-01-01

PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples.

DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair.

PubMed

Bajinskis, Ainars; Natarajan, Adayapalam T; Erixon, Klaus; Harms-Ringdahl, Mats

2013-08-30

The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by (137)Cs γ-rays or radon progeny α-particles. Irradiation was also performed in the presence of 2M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with γ-rays or α-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

Proximity to AGCT sequences dictates MMR-independent versus MMR-dependent mechanisms for AID-induced mutation via UNG2

PubMed Central

Thientosapol, Eddy Sanchai; Sharbeen, George; Lau, K.K. Edwin; Bosnjak, Daniel; Durack, Timothy; Stevanovski, Igor; Weninger, Wolfgang

2017-01-01

Abstract AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BER-resistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCT-distal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites. PMID:28039326

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

PubMed

Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

2016-01-08

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

PubMed Central

Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

2016-01-01

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

Pdsg1 and Pdsg2, Novel Proteins Involved in Developmental Genome Remodelling in Paramecium

PubMed Central

Hoehener, Cristina; Singh, Aditi; Swart, Estienne C.; Nowacki, Mariusz

2014-01-01

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization. PMID:25397898

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

PubMed

Arambasic, Miroslav; Sandoval, Pamela Y; Hoehener, Cristina; Singh, Aditi; Swart, Estienne C; Nowacki, Mariusz

2014-01-01

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

PubMed

Arczewska, Katarzyna D; Tomazella, Gisele G; Lindvall, Jessica M; Kassahun, Henok; Maglioni, Silvia; Torgovnick, Alessandro; Henriksson, Johan; Matilainen, Olli; Marquis, Bryce J; Nelson, Bryant C; Jaruga, Pawel; Babaie, Eshrat; Holmberg, Carina I; Bürglin, Thomas R; Ventura, Natascia; Thiede, Bernd; Nilsen, Hilde

2013-05-01

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

PubMed Central

Kozmin, Stanislav G.; Sedletska, Yuliya; Reynaud-Angelin, Anne; Gasparutto, Didier; Sage, Evelyne

2009-01-01

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells. PMID:19174565

A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene.

PubMed

Hill, J; McGraw, P; Tzagoloff, A

1985-03-25

The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.

Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity

PubMed Central

Coin, Frédéric; Auriol, Jérome; Tapias, Angel; Clivio, Pascale; Vermeulen, Wim; Egly, Jean-Marc

2004-01-01

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. The xeroderma pigmentosum group B (XPB) helicase subunit of TFIIH functions in NER and transcription. The serine 751 (S751) residue of XPB was found to be phosphorylated in vivo. This phosphorylation inhibits NER and the microinjection of a phosphomimicking XPB-S751E mutant is unable to correct the NER defect of XP-B cells. Conversely, XPB-S751 dephosphorylation or its substitution with alanine (S751A) restores NER both in vivo and in vitro. Surprisingly, phospho/dephosphorylation of S751 spares TFIIH-dependent transcription. Finally, the phosphorylation of XPB-S751 does not impair the TFIIH unwinding of the DNA around the lesion, but rather prevents the 5′ incision triggered by the ERCC1-XPF endonuclease. These data support an additional role for XPB in promoting the incision of the damaged fragment and reveal a point of NER regulation on TFIIH without interference in its transcription activity. PMID:15549133

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flejter, W.L.; McDaniel, L.D.; Johns, D.

1992-01-01

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2)more » gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.« less

Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha

Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposedmore » to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in nucleotide excision repair after AFB{sub 1} exposure.« less

Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

PubMed

Fowler, Anna-Kate; Hewetson, Aveline; Agrawal, Rajiv G; Dagda, Marisela; Dagda, Raul; Moaddel, Ruin; Balbo, Silvia; Sanghvi, Mitesh; Chen, Yukun; Hogue, Ryan J; Bergeson, Susan E; Henderson, George I; Kruman, Inna I

2012-12-21

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Interplay of DNA repair with transcription: from structures to mechanisms.

PubMed

Deaconescu, Alexandra M; Artsimovitch, Irina; Grigorieff, Nikolaus

2012-12-01

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum | Center for Cancer Research

Cancer.gov

Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with XP. As more abnormalities form in DNA, cells malfunction and eventually become cancerous or die. XP patients have more than a 10,000-fold increased risk of developing skin cancer. Kenneth Kraemer, M.D., in CCR’s Dermatology Branch, has been studying XP patients at the Clinical Center for more than 40 years.

Predictive role of repair enzymes in the efficacy of Cisplatin combinations in pancreatic and lung cancer.

PubMed

Peters, Godefridus J; Avan, Abolfazl; Ruiz, Marielle Gallegos; Orsini, Vanessa; Avan, Amir; Giovannetti, Elisa; Smit, Egbert F

2014-01-01

Platinum combinations are the mainstay of treatment for non-small cell lung cancer (NSCLC), while for pancreatic cancer platinum combinations are being given to good-performance status patients. These platinum combinations consist of cis- or carboplatin with gemcitabine, while, for non-squamous NSCLC and mesothelioma, of pemetrexed. The combination of gemcitabine and cisplatin is based on gemcitabine-induced increased formation and retention of DNA-platinum adducts, which can be explained by a decrease of excision repair cross-complementing group-1 (ERCC1)-mediated DNA repair. In these patients, survival and response is prolonged when ERCC1 has a low protein or mRNA expression. A low expression of ribonucleotide reductase (RR) is related to a better treatment outcome after both gemcitabine and gemcitabine-platinum combinations. For pemetrexed combinations, ERCC1 expression was not related to survival. For both NSCLC and pancreatic cancer, polymorphisms in ERCC1 (C118T) and Xeroderma pigmentosum group D (XPD) (A751C) were related to survival. In currently ongoing and future prospective studies, patients should be selected based on their DNA repair status, but it still has to be determined whether this should be by immunohistochemistry, mRNA expression, or a polymorphism.

Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes.

PubMed

Leontovich, Alexey A; Intine, Robert V; Sarras, Michael P

2016-01-01

Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6-13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

PubMed

Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

2008-12-15

Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines that have been investigated in the past 50 years. Our goal is to emphasize the importance of these neglected lesions in many biological and disease processes.

NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

PubMed Central

Sarker, Altaf H.; Chatterjee, Arpita; Williams, Monique; Lin, Sabrina; Havel, Christopher; Jacob III, Peyton; Boldogh, Istvan; Hazra, Tapas K.; Talbot, Prudence; Hang, Bo

2014-01-01

Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon–quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer. PMID:24595271

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.

PubMed

Fogg, Paul C M; Haley, Joshua A; Stark, W Marshall; Smith, Margaret C M

2017-03-01

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces , most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor , by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae , an emerging model bacterium in Streptomyces research. Copyright © 2017 Fogg et al.

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

PubMed

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory proteins guiding distinct BER sub-pathways.

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

PubMed

Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

2012-06-01

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

DTIC Science & Technology

1988-02-02

the bands excised, and the DNA extracted with phenol for cloning in M13 . 6 Nuclotida sequence analysis. The two fragments were each cloned into phages ...DNA; and strain JM103 (29) was used to propagate M13 ph&ge derivatives. -1 Subcloning and detection of PA-producing rsccmbinants. The isolation of...method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 19. Lauben, J. 0., and J. 2. K. Nielsen. 1982. Penicillinase and

Loss of Nucleotide Excision Repair as a Source of Genomic Instability in Breast Cancer

DTIC Science & Technology

2005-06-01

prone syndrome xeroderma pigmentosum (XP), and reminiscent of p48 deficiency in XP-E cells, and we have in result in the developmental and...photoproduct; UV-DDB, UV-damaged DNA binding factor; wt, wild-type; well as damaged DNA itself, thus suggesting that it may act as XP, xeroderma pigmentosum . a...the potential for mutagenesis. If UV-DDB is a chromatin the classification of five cell strains of xeroderma pigmentosum group E remodeling factor

Genetic polymorphisms in 85 DNA repair genes and bladder cancer risk.

PubMed

Michiels, Stefan; Laplanche, Agnès; Boulet, Thomas; Dessen, Philippe; Guillonneau, Bertrand; Méjean, Arnaud; Desgrandchamps, François; Lathrop, Mark; Sarasin, Alain; Benhamou, Simone

2009-05-01

Several defense mechanisms have been developed and maintained during the evolution to protect human cells against damage produced from exogenous or endogenous sources. We examined the associations between bladder cancer and a panel of 652 polymorphisms from 85 genes involved in maintenance of genetic stability [base excision repair, nucleotide excision repair, double-strand break repair (DSBR) and mismatch repair, as well as DNA synthesis and cell cycle regulation pathways] in 201 incident bladder cancer cases and 326 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value the expected proportion of false discoveries (q-value). Haplotype analysis revealed significant associations (P < 0.01) between bladder cancer and two genes (POLB and FANCA) with an associated q-value of 24%. A permutation test was also used to determine whether, in each pathway analyzed, there are more variants whose allelic frequencies are different between cases and controls as compared with what would be expected by chance. Differences were found for cell cycle regulation (P = 0.02) and to a lesser extent for DSBR (P = 0.05) pathways. These results hint to a few potential candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations. It is anticipated that genome-wide association studies will open new perspectives for interpretation of the results of extensive candidate gene studies such as ours.