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Sample records for dsb repair capacity

  1. The impact of heterochromatin on DSB repair.

    PubMed

    Goodarzi, Aaron A; Noon, Angela T; Jeggo, Penny A

    2009-06-01

    DNA NHEJ (non-homologous end-joining) is the major DNA DSB (double-strand break) repair pathway in mammalian cells. Although NHEJ-defective cell lines show marked DSB-repair defects, cells defective in ATM (ataxia telangiectasia mutated) repair most DSBs normally. Thus NHEJ functions independently of ATM signalling. However, approximately 15% of radiation-induced DSBs are repaired with slow kinetics and require ATM and the nuclease Artemis. DSBs persisting in the presence of an ATM inhibitor, ATMi, localize to heterochromatin, suggesting that ATM is required for repairing DSBs arising within or close to heterochromatin. Consistent with this, we show that siRNA (small interfering RNA) of key heterochromatic proteins, including KAP-1 [KRAB (Krüppel-associated box) domain-associated protein 1], HP1 (heterochromatin protein 1) and HDAC (histone deacetylase) 1/2, relieves the requirement for ATM for DSB repair. Furthermore, ATMi addition to cell lines with genetic alterations that have an impact on heterochromatin, including Suv39H1/2 (suppressor of variegation 3-9 homologue 1/2)-knockout, ICFa (immunodeficiency, centromeric region instability, facial anomalies syndrome type a) and Hutchinson-Guilford progeria cell lines, fails to have an impact on DSB repair. KAP-1 is a highly dose-dependent, transient and ATM-specific substrate, and mutation of the ATM phosphorylation site on KAP-1 influences DSB repair. Collectively, the findings show that ATM functions to overcome the barrier to DSB repair posed by heterochromatin. However, even in the presence of ATM, gamma-H2AX (phosphorylated histone H2AX) foci form on the periphery rather than within heterochromatic centres. Finally, we show that KAP-1's association with heterochromatin is diminished as cells progress through mitosis. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications to promote DSB repair and mitotic progression in a manner that allows localized and transient

  2. Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair.

    PubMed

    Lemaître, Charlène; Soutoglou, Evi

    2014-07-01

    Chromosomal translocations are a hallmark of cancer cells and they represent a major cause of tumorigenesis. To avoid chromosomal translocations, faithful repair of DNA double strand breaks (DSBs) has to be ensured in the context of high ordered chromatin structure. However, chromatin compaction is proposed to represent a barrier for DSB repair. Here we review the different mechanisms cells use to alleviate the heterochromatic barrier for DNA repair. At the same time, we discuss the activating role of heterochromatin-associated proteins in this process, therefore proposing that chromatin structure, more than being a simple barrier, is a key modulator of DNA repair.

  3. An approach to estimate radioadaptation from DSB repair efficiency.

    PubMed

    Yatagai, Fumio; Sugasawa, Kaoru; Enomoto, Shuichi; Honma, Masamitsu

    2009-09-01

    In this review, we would like to introduce a unique approach for the estimation of radioadaptation. Recently, we proposed a new methodology for evaluating the repair efficiency of DNA double-strand breaks (DSB) using a model system. The model system can trace the fate of a single DSB, which is introduced within intron 4 of the TK gene on chromosome 17 in human lymphoblastoid TK6 cells by the expression of restriction enzyme I-SceI. This methodology was first applied to examine whether repair of the DSB (at the I-SceI site) can be influenced by low-dose, low-dose rate gamma-ray irradiation. We found that such low-dose IR exposure could enhance the activity of DSB repair through homologous recombination (HR). HR activity was also enhanced due to the pre-IR irradiation under the established conditions for radioadaptation (50 mGy X-ray-6 h-I-SceI treatment). Therefore, radioadaptation might account for the reduced frequency of homozygous loss of heterozygosity (LOH) events observed in our previous experiment (50 mGy X-ray-6 h-2 Gy X-ray). We suggest that the present evaluation of DSB repair using this I-SceI system, may contribute to our overall understanding of radioadaptation.

  4. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

    PubMed

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  5. Cancer TARGETases: DSB repair as a pharmacological target.

    PubMed

    Samadder, Pounami; Aithal, Rakesh; Belan, Ondrej; Krejci, Lumir

    2016-05-01

    Cancer is a disease attributed to the accumulation of DNA damages due to incapacitation of DNA repair pathways resulting in genomic instability and a mutator phenotype. Among the DNA lesions, double stranded breaks (DSBs) are the most toxic forms of DNA damage which may arise as a result of extrinsic DNA damaging agents or intrinsic replication stress in fast proliferating cancer cells. Accurate repair of DSBs is therefore paramount to the cell survival, and several classes of proteins such as kinases, nucleases, helicases or core recombinational proteins have pre-defined jobs in precise execution of DSB repair pathways. On one hand, the proper functioning of these proteins ensures maintenance of genomic stability in normal cells, and on the other hand results in resistance to various drugs employed in cancer therapy and therefore presents a suitable opportunity for therapeutic targeting. Higher relapse and resistance in cancer patients due to non-specific, cytotoxic therapies is an alarming situation and it is becoming more evident to employ personalized treatment based on the genetic landscape of the cancer cells. For the success of personalized treatment, it is of immense importance to identify more suitable targetable proteins in DSB repair pathways and also to explore new synthetic lethal interactions with these pathways. Here we review the various alternative approaches to target the various protein classes termed as cancer TARGETases in DSB repair pathway to obtain more beneficial and selective therapy.

  6. DNA DSB repair pathway choice: an orchestrated handover mechanism.

    PubMed

    Kakarougkas, A; Jeggo, P A

    2014-03-01

    DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.

  7. DSB (Im)mobility and DNA repair compartmentalization in mammalian cells.

    PubMed

    Lemaître, Charlène; Soutoglou, Evi

    2015-02-13

    Chromosomal translocations are considered as causal in approximately 20% of cancers. Therefore, understanding their mechanisms of formation is crucial in the prevention of carcinogenesis. The first step of translocation formation is the concomitant occurrence of double-strand DNA breaks (DSBs) in two different chromosomes. DSBs can be repaired by different repair mechanisms, including error-free homologous recombination (HR), potentially error-prone non-homologous end joining (NHEJ) and the highly mutagenic alternative end joining (alt-EJ) pathways. Regulation of DNA repair pathway choice is crucial to avoid genomic instability. In yeast, DSBs are mobile and can scan the entire nucleus to be repaired in specialized DNA repair centers or if they are persistent, in order to associate with the nuclear pores or the nuclear envelope where they can be repaired by specialized repair pathways. DSB mobility is limited in mammals; therefore, raising the question of whether the position at which a DSB occurs influences its repair. Here, we review the recent literature addressing this question. We first present the reports describing the extent of DSB mobility in mammalian cells. In a second part, we discuss the consequences of non-random gene positioning on chromosomal translocations formation. In the third part, we discuss the mobility of heterochromatic DSBs in light of our recent data on DSB repair at the nuclear lamina, and finally, we show that DSB repair compartmentalization at the nuclear periphery is conserved from yeast to mammals, further pointing to a role for gene positioning in the outcome of DSB repair. When regarded as a whole, the different studies reviewed here demonstrate the importance of nuclear architecture on DSB repair and reveal gene positioning as an important parameter in the study of tumorigenesis.

  8. Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

    PubMed

    Zhang, Ye; Rohde, Larry H; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish K; Jeevarajan, Antony S; Pierson, Duane L; Wu, Honglu

    2008-11-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that

  9. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    PubMed

    Lloyd, Andrew H; Wang, Dong; Timmis, Jeremy N

    2012-01-01

    DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  10. Stochastic properties of radiation-induced DSB: DSB distributions in large scale chromatin loops, the HPRT gene and within the visible volumes of DNA repair foci.

    PubMed

    Ponomarev, Artem L; Costes, Sylvain V; Cucinotta, Francis A

    2008-11-01

    We computed probabilities to have multiple double-strand breaks (DSB), which are produced in DNA on a regional scale, and not in close vicinity, in volumes matching the size of DNA damage foci, of a large chromatin loop, and in the physical volume of DNA containing the HPRT (human hypoxanthine phosphoribosyltransferase) locus. The model is based on a Monte Carlo description of DSB formation by heavy ions in the spatial context of the entire human genome contained within the cell nucleus, as well as at the gene sequence level. We showed that a finite physical volume corresponding to a visible DNA repair focus, believed to be associated with one DSB, can contain multiple DSB due to heavy ion track structure and the DNA supercoiled topography. A corrective distribution was introduced, which was a conditional probability to have excess DSB in a focus volume, given that there was already one present. The corrective distribution was calculated for 19.5 MeV/amu N ions, 3.77 MeV/amu alpha-particles, 1000 MeV/amu Fe ions, and X-rays. The corrected initial DSB yield from the experimental data on DNA repair foci was calculated. The DSB yield based on the corrective function converts the focus yield into the DSB yield, which is comparable with the DSB yield based on the earlier PFGE experiments. The distribution of DSB within the physical limits of the HPRT gene was analyzed by a similar method as well. This corrective procedure shows the applicability of the model and empowers the researcher with a tool to better analyze focus statistics. The model enables researchers to analyze the DSB yield based on focus statistics in real experimental situations that lack one-to-one focus-to-DSB correspondance.

  11. 53BP1 regulates DSB repair using Rif1 to control 5' end resection.

    PubMed

    Zimmermann, Michal; Lottersberger, Francisca; Buonomo, Sara B; Sfeir, Agnel; de Lange, Titia

    2013-02-08

    The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

  12. Higher-order chromatin structure in DSB induction, repair and misrepair.

    PubMed

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    2010-01-01

    Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.

  13. Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle.

    PubMed

    Taleei, Reza; Nikjoo, Hooshang

    2013-08-30

    The paper presents a model of double strand breaks (DSB) repair in G1 and early S phases of the cell cycle. The model is based on a plethora of published information on biochemical modification of DSB induced by ionizing radiation. So far, three main DSB repair pathways have been identified, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). During G1 and early S phases of the cell cycle, NHEJ and MMEJ repair pathways are activated dependent on the type of double strand breaks. Simple DSB are a substrate for NHEJ, while complex DSB and DSB in heterochromatin require further end processing. Repair of all DSB start with NHEJ presynaptic processes, and depending on the type of DSB pursue simple ligation, further end processing prior to ligation, or resection. Using law of mass action the model is translated into a mathematical formalism. The solution of the formalism provides the step by step and overall repair kinetics. The overall repair kinetics are compared with the published experimental measurements. Our calculations are in agreement with the experimental results and show that the complex types of DSBs are repaired with slow repair kinetics. The G1 and early S phase model could be employed to predict the kinetics of DSB repair for damage induced by high LET radiation.

  14. The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility.

    PubMed

    Keimling, Marlen; Deniz, Miriam; Varga, Dominic; Stahl, Andreea; Schrezenmeier, Hubert; Kreienberg, Rolf; Hoffmann, Isabell; König, Jochem; Wiesmüller, Lisa

    2012-05-01

    Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case-control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP-based test system using 3 pathway-specific substrates. We found increases of microhomology-mediated nonhomologous end joining (mmNHEJ) and nonconservative single-strand annealing (SSA) in women with familial risk vs. controls (P=0.0001-0.0022) and patients with breast cancer vs. controls (P=0.0004-0.0042). Young age (<50) at initial diagnosis of breast cancer, which could be indicative of genetic predisposition, was associated with elevated SSA using two different substrates, amounting to similar odds ratios (ORs=2.54-4.46, P=0.0059-0.0095) as for familial risk (ORs=2.61-4.05, P=0.0007-0.0045). These findings and supporting validation data underscore the great potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.

  15. UbcH7 regulates 53BP1 stability and DSB repair.

    PubMed

    Han, Xiangzi; Zhang, Lei; Chung, Jinsil; Mayca Pozo, Franklin; Tran, Amanda; Seachrist, Darcie D; Jacobberger, James W; Keri, Ruth A; Gilmore, Hannah; Zhang, Youwei

    2014-12-09

    DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.

  16. Artemis is required to improve the accuracy of repair of double-strand breaks with 5'-blocked termini generated from non-DSB-clustered lesions.

    PubMed

    Malyarchuk, Svitlana; Castore, Reneau; Shi, Runhua; Harrison, Lynn

    2013-05-01

    Clustered DNA lesions are defined as ≥2 damage events within 20 bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART(-/-)) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5' termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART(-/-) cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5'-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.

  17. Artemis is required to improve the accuracy of repair of double-strand breaks with 5′-blocked termini generated from non-DSB-clustered lesions

    PubMed Central

    Harrison, Lynn

    2013-01-01

    Clustered DNA lesions are defined as ≥2 damage events within 20bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART−/−) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5′ termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART−/− cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5′-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence. PMID:23448902

  18. Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

    PubMed

    Batta, Kiran; Yokokawa, Masatoshi; Takeyasu, Kunio; Kundu, Tapas K

    2009-01-23

    Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

  19. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    PubMed Central

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  20. ATM prevents DSB formation by coordinating SSB repair and cell cycle progression.

    PubMed

    Khoronenkova, Svetlana V; Dianov, Grigory L

    2015-03-31

    DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.

  1. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase

    PubMed Central

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R.; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J.; Chen, David J.

    2016-01-01

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells. PMID:26712563

  2. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

    PubMed

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J; Chen, David J

    2016-02-29

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

  3. An RNA polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair.

    PubMed

    Jha, Deepak Kumar; Strahl, Brian D

    2014-06-09

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered chromatin architecture and inappropriate resection during G1 near break sites. Surprisingly, Set2 and RNA polymerase II are programmed for destruction after DSBs in a temporal manner--resulting in H3K36me3 to H3K36me2 transition that may be linked to DSB repair. Finally, we show a requirement of Set2 in DSB repair in transcription units--thus underscoring the importance of transcription-dependent H3K36me in DSB repair.

  4. DSB repair model for mammalian cells in early S and G1 phases of the cell cycle: application to damage induced by ionizing radiation of different quality.

    PubMed

    Taleei, Reza; Girard, Peter M; Nikjoo, Hooshang

    2015-02-01

    The purpose of this work is to test the hypothesis that kinetics of double strand breaks (DSB) repair is governed by complexity of DSB. To test the hypothesis we used our recent published mechanistic mathematical model of DSB repair for DSB induced by selected protons, deuterons, and helium ions of different energies representing radiations of different qualities. In light of recent advances in experimental and computational techniques, the most appropriate method to study cellular responses in radiation therapy, and exposures to low doses of ionizing radiations is using mechanistic approaches. To this end, we proposed a 'bottom-up' approach to study cellular response that starts with the DNA damage. Monte Carlo track structure method was employed to simulate initial damage induced in the genomic DNA by direct and indirect effects. Among the different types of DNA damage, DSB are known to be induced in simple and complex forms. The DSB repair model in G1 and early S phases of the cell cycle was employed to calculate the repair kinetics. The model considers the repair of simple and complex DSB, and the DSB produced in the heterochromatin. The inverse sampling method was used to calculate the repair kinetics for each individual DSB. The overall repair kinetics for 500 DSB induced by single tracks of the radiation under test were compared with experimental results. The results show that the model is capable of predicting the repair kinetics for the DSB induced by radiations of different qualities within an accepted range of uncertainty.

  5. 53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair.

    PubMed

    Lottersberger, Francisca; Karssemeijer, Roos Anna; Dimitrova, Nadya; de Lange, Titia

    2015-11-05

    Increased mobility of chromatin surrounding double-strand breaks (DSBs) has been noted in yeast and mammalian cells but the underlying mechanism and its contribution to DSB repair remain unclear. Here, we use a telomere-based system to track DNA damage foci with high resolution in living cells. We find that the greater mobility of damaged chromatin requires 53BP1, SUN1/2 in the linker of the nucleoskeleton, and cytoskeleton (LINC) complex and dynamic microtubules. The data further demonstrate that the excursions promote non-homologous end joining of dysfunctional telomeres and implicated Nesprin-4 and kinesins in telomere fusion. 53BP1/LINC/microtubule-dependent mobility is also evident at irradiation-induced DSBs and contributes to the mis-rejoining of drug-induced DSBs in BRCA1-deficient cells showing that DSB mobility can be detrimental in cells with numerous DSBs. In contrast, under physiological conditions where cells have only one or a few lesions, DSB mobility is proposed to prevent errors in DNA repair.

  6. MutS homologue hMSH5: recombinational DSB repair and non-synonymous polymorphic variants.

    PubMed

    Wu, Xiling; Xu, Yang; Feng, Katey; Tompkins, Joshua D; Her, Chengtao

    2013-01-01

    Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.

  7. Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

    NASA Technical Reports Server (NTRS)

    O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

    2007-01-01

    When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy

  8. A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens.

    PubMed

    Keimling, Marlen; Kaur, Jatinder; Bagadi, Sarangadhara Appala Raju; Kreienberg, Rolf; Wiesmüller, Lisa; Ralhan, Ranju

    2008-08-01

    Increasing evidence indicates that breast cancer pathogenesis is linked with DNA double-strand break (DSB) repair dysfunction. This conclusion is based on advances in the study of functions of breast cancer susceptibility genes such as BRCA1 and BRCA2, on the identification of breast cancer-associated changes regarding the genetics, expression, and localization of multiple DSB repair factors, and on observations indicating enhanced radiation-induced chromosomal damage in cells from predisposed individuals and sporadic breast cancer patients. In this pilot study, we describe a sensitive method for the analysis of DSB repair functions in mammary carcinomas. Using this method we firstly document alterations in pathway-specific DSB repair activities in primary cells originating from familial as well as sporadic breast cancer. In particular, we identified increases in the mutagenic nonhomologous end joining and single-strand annealing mechanisms in sporadic breast cancers with wild-type BRCA1 and BRCA2, and, thus, similar phenotypes to tumors with mutant alleles of BRCA1 and BRCA2. This suggests that detection of error-prone DSB repair activities may be useful to extend the limits of genotypic characterization of high-risk susceptibility genes. This method may, therefore, serve as a marker for breast cancer risk assessment and, even more importantly, for the prediction of responsiveness to targeted therapies such as to inhibitors of poly(ADP-ribose)polymerase (PARP1).

  9. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  10. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  11. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

    PubMed Central

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-01-01

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse. PMID:28290521

  12. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells.

    PubMed

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-03-14

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3(+/-) germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

  13. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  14. Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

    PubMed

    Goglia, Alexander G; Delsite, Robert; Luz, Antonio N; Shahbazian, David; Salem, Ahmed F; Sundaram, Ranjini K; Chiaravalli, Jeanne; Hendrikx, Petrus J; Wilshire, Jennifer A; Jasin, Maria; Kluger, Harriet M; Glickman, J Fraser; Powell, Simon N; Bindra, Ranjit S

    2015-02-01

    Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules that selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits that potently inhibit DSB repair, and we have validated their functional activity in a comprehensive panel of orthogonal secondary assays. A selection of these inhibitors was found to radiosensitize cancer cell lines in vitro, which suggests that they may be useful as novel chemo- and radio sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker mibefradil dihydrochloride, that demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a phase I clinical trial testing mibefradil as a glioma radiosensitizer.

  15. Identification of Novel Radiosensitizers in a High-Throughput, Cell-Based Screen for DSB Repair Inhibitors

    PubMed Central

    Goglia, Alexander G.; Delsite, Robert; Luz, Antonio N.; Shahbazian, David; Salem, Ahmed F.; Sundaram, Ranjini K.; Chiaravalli, Jeanne; Hendrikx, Petrus J.; Wilshire, Jennifer A.; Jasin, Maria; Kluger, Harriet; Glickman, J. Fraser; Powell, Simon N.; Bindra, Ranjit S.

    2014-01-01

    Most cancer therapies involve a component of treatment which inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radio-sensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules which selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits which potently inhibit DSB repair, and we have validated their functional activity in comprehensive panel of orthogonal secondary assays. A selection of these inhibitors were found to radiosensitize cancer cell lines in vitro, which suggests they may be useful as novel chemo- and radio-sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker, mibefradil dihydrochloride, which demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a Phase I clinical trial testing mibefradil as glioma radiosensitizer. PMID:25512618

  16. Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage - when is a DSB not a DSB?

    PubMed

    Reynolds, Pamela; Botchway, Stanley W; Parker, Anthony W; O'Neill, Peter

    2013-08-30

    The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair.

  17. Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

    PubMed

    Gouge, Jérôme; Rosario, Sandrine; Romain, Félix; Poitevin, Frédéric; Béguin, Pierre; Delarue, Marc

    2015-04-15

    Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

  18. Participation of DNA-PKcs in DSB repair after exposure to high- and low-LET radiation.

    PubMed

    Anderson, Jennifer A; Harper, Jane V; Cucinotta, Francis A; O'Neill, Peter

    2010-08-01

    Cellular lesions (e.g. DSBs) are induced into DNA upon exposure to radiation, with DSB complexity increasing with radiation ionization density. Using M059K and M059J human glioblastoma cells (proficient and deficient in DNA-PKcs activity, respectively), we investigated the repair of DNA damage, including DSBs, induced by high- and low-LET radiation [gamma rays, alpha particles and high-charge and energy (HZE) ions]. In the absence of DNA-PKcs activity, less DSB repair and increased recruitment of RAD51 was seen at 24 h. After exposure to (56)Fe heavy ions, the number of cells with RAD51 tracks was less than the number of cells with gamma-H2AX at 24 h with both cell lines. Using alpha particles, comparable numbers of cells with visible gamma-H2AX and RAD51 were seen at 24 h in both cell lines. M059J cells irradiated with alpha particles accumulated in S phase, with a greater number of cyclin A and RAD51 co-stained cells seen at 24 h compared with M059K cells, where an S-phase block is absent. It is proposed that DNA-PKcs plays a role in the repair of some frank DSBs, which are longer-lived in NHEJ-deficient cells, and some non-DSB clustered damage sites that are converted into DSBs at replication as the cell cycles through to S phase.

  19. Topoisomerase degradation, DSB repair, p53 and IAPs in cancer cell resistance to camptothecin-like topoisomerase I inhibitors.

    PubMed

    Tomicic, Maja T; Kaina, Bernd

    2013-01-01

    Topoisomerase I (TOP1) inhibitors applied in cancer therapy such as topotecan and irinotecan are derivatives of the natural alkaloid camptothecin (CPT). The mechanism of CPT poisoning of TOP1 rests on inhibition of the re-ligation function of the enzyme resulting in the stabilization of the TOP1-cleavable complex. In the presence of CPTs this enzyme-DNA complex impairs transcription and DNA replication, resulting in fork stalling and the formation of DNA double-strand breaks (DSB) in proliferating cells. As with most chemotherapeutics, intrinsic and acquired drug resistance represents a hurdle that limits the success of CPT therapy. Preclinical data indicate that resistance to CPT-based drugs might be caused by factors such as (a) poor drug accumulation in the tumor, (b) high rate of drug efflux, (c) mutations in TOP1 leading to failure in CPT docking, or (d) altered signaling triggered by the drug-TOP1-DNA complex, (e) expression of DNA repair proteins, and (f) failure to activate cell death pathways. This review will focus on the issues (d-f). We discuss degradation of TOP1 as part of the repair pathway in the processing of TOP1 associated DNA damage, give a summary of proteins involved in repair of CPT-induced replication mediated DSB, and highlight the role of p53 and inhibitors of apoptosis proteins (IAPs), particularly XIAP and survivin, in cancer cell resistance to CPT-like chemotherapeutics.

  20. Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts.

    PubMed

    Constantinescu, Dan; Csoka, Antonei B; Navara, Christopher S; Schatten, Gerald P

    2010-10-15

    Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

  1. Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

    SciTech Connect

    Constantinescu, Dan; Csoka, Antonei B.; Navara, Christopher S.; Schatten, Gerald P.

    2010-10-15

    Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

  2. High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

    PubMed Central

    Sharma, Preety M.; Ponnaiya, Brian; Taveras, Maria; Shuryak, Igor; Turner, Helen; Brenner, David J.

    2015-01-01

    The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay. PMID:25794041

  3. High throughput measurement of γH2AX DSB repair kinetics in a healthy human population.

    PubMed

    Sharma, Preety M; Ponnaiya, Brian; Taveras, Maria; Shuryak, Igor; Turner, Helen; Brenner, David J

    2015-01-01

    The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

  4. RADIATION SENSITIVITY & PROCESSING OF DNA DAMAGE FOLLOWING LOW DOSES OF GAMMA-RAY ALPHA PARTICLES & HZE IRRADIATION OF NORMAL DSB REPAIR DEFICIENT CELLS

    SciTech Connect

    O'Neil, Peter

    2009-05-15

    Non-homologous end joining (NHEJ) predominates in the repair of DNA double strand breaks (DSB) over homologous recombination (HR). NHEJ occurs throughout the cell cycle whereas HR occurs in late S/G2 due to the requirement of a sister chromatid (Rothkamm et al, Mol Cell Biol 23 5706-15 [2003]). To date evidence obtained with DSB repair deficient cells using pulsed-field gel electrophoresis has revealed the major pathway throughout all phases of the cell cycle for processing high dose induced DSBs is NHEJ (Wang et al, Oncogene 20 2212-24 (2001); Pluth et al, Cancer Res. 61 2649-55 [2001]). These findings however were obtained at high doses when on average >> 20-30 DSBs are formed per cell. The contribution of the repair pathways (NHEJ and HR) induced in response to DNA damage during the various phases of the cell cycle may depend upon the dose (the level of initial DSBs) especially since low levels of DSBs are induced at low dose. To date, low dose studies using NHEJ and HR deficient mutants have not been carried out to address this important question with radiations of different quality. The work presented here leads us to suggest that HR plays a relatively minor role in the repair of radiation-induced prompt DSBs. SSBs lead to the induction of DSBs which are associated specifically with S-phase cells consistent with the idea that they are formed at stalled replication forks in which HR plays a major role in repair. That DNA-PKcs is in some way involved in the repair of the precursors to replication-induced DSB remains an open question. Persistent non-DSB oxidative damage also leads to an increase in RAD51 positive DSBs. Both simple and complex non-DSB DNA damage may therefore contribute to indirect DSBs induced by ionising radiation at replication forks.

  5. Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model.

    PubMed

    Delacôte, Fabien; Lopez, Bernard S

    2008-01-01

    A DNA double-strand break (DSB) is a highly harmful lesion that can lead to genome rearrangements. Two main pathways compete for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). Depending on the cell cycle phase, the choice of one DSB repair pathway over the other will secure genome stability maintenance or in contrast will increase the risk of genetic instability. HR with the sister chromatid is an efficient way to maintain genome stability, for damage occurring at a post-replication stage. However, in G(1) checkpoint-defective cells, DSBs produced in the G(1) phase and not repaired by NHEJ, can progress through S phase and be processed by HR in late S/G(2) phase. We propose the "trans-S DSB repair" model to account for these data. In this situation HR cannot use the sister chromatid (which is also broken at the same locus) and is thus forced to use ectopic homologous sequences dispersed through the genome, increasing the risk of genetic instability. This shows that the two DSB repair pathways can compete through the cell cycle and underlines the importance of the association between the cell cycle checkpoint and the appropriate DNA repair pathway for genome stability maintenance.

  6. Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

    PubMed

    Smith, Johanna A; Wang, Feng-Xiang; Zhang, Hui; Wu, Kou-Juey; Williams, Kevin Jon; Daniel, René

    2008-01-22

    Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

  7. Repair of I-SceI induced DSB at a specific site of chromosome in human cells: influence of low-dose, low-dose-rate gamma-rays.

    PubMed

    Yatagai, Fumio; Suzuki, Masao; Ishioka, Noriaki; Ohmori, Hitoshi; Honma, Masamitsu

    2008-11-01

    We investigated the influence of low-dose, low-dose-rate gamma-ray irradiation on DNA double strand break (DSB) repair in human lymphoblastoid TK6 cells. A single DSB was introduced at intron 4 of the TK+ allele (chromosome 17) by transfection with the I-SceI expression vector pCBASce. We assessed for DSB repair due to non-homologous end-joining (NHEJ) by determining the generation of TK-deficient mutants in the TK6 derivative TSCE5 (TK +/-) carrying an I-SceI recognition site. We similarly estimated DSB repair via homologous recombination (HR) at the same site in the derived compound heterozygote (TK-/-) cell line TSCER2 that carries an additional point mutation in exon 5. The NHEJ repair of DSB was barely influenced by pre-irradiation of the cells with 30 mGy gamma-rays at 1.2 mGy h(-1). DSB repair by HR, in contrast, was enhanced by approximately 50% after pre-irradiation of the cells under these conditions. Furthermore, when I-SceI digestion was followed by irradiation at a dose of 8.5 mGy, delivered at a dose rate of only 0.125 mGy h(-1), HR repair efficiency was enhanced by approximately 80%. This experimental approach can be applied to characterize DSB repair in the low-dose region of ionizing radiation.

  8. Genistein sensitizes sarcoma cells in vitro and in vivo by enhancing apoptosis and by inhibiting DSB repair pathways.

    PubMed

    Liu, X X; Sun, C; Jin, X D; Li, P; Zheng, X G; Zhao, T; Li, Q

    2016-06-01

    The aim of this work was to investigate the radiosensitization effects of genistein on mice sarcoma cells and the corresponding biological mechanisms in vitro and in vivo Using the non-toxic dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at 50% cell survival (IC50) was 1.45 for S180 cells. For mice cotreated with genistein and X-rays, the excised tumor tissues had reduced blood vessels and decreased size and volume compared with the control and irradiation-only groups. Moreover, a significant increase in apoptosis was accompanied by upregulation of Bax and downregulation of Bcl-2 in the mitochondria, and lots of cytochrome c being transferred to the cytoplasm. Furthermore, X-rays combined with genistein inhibited the activity of DNA-PKcs, so DNA-injured sites were dominated by Ku70/80, leading to incompleteness of homologous recombination (HR) and non-homologous end-joining (NHEJ) repairs and the eventual occurrence of cell apoptosis. Our study, for the first time, demonstrated that genistein sensitized sarcoma cells to X-rays and that this radiosensitizing effect depended on induction of the mitochondrial apoptosis pathway and inhibition of the double-strand break (DSB) repair pathways.

  9. Reduced DNA double-strand break repair capacity and risk of squamous cell carcinoma of the head and neck--A case-control study.

    PubMed

    Liu, Zhensheng; Liu, Hongliang; Gao, Fengqin; Dahlstrom, Kristina R; Sturgis, Erich M; Wei, Qingyi

    2016-04-01

    Tobacco smoke and alcohol use play important roles in the etiology of squamous cell carcinoma of the head and neck (SCCHN). Smoking causes DNA damage, including double-strand DNA breaks (DSBs), that leads to carcinogenesis. To test the hypothesis that suboptimal DSB repair capacity is associated with risk of SCCHN, we applied a flow cytometry-based method to detect the DSB repair phenotype first in four EBV-immortalized human lymphoblastoid cell lines and then in human peripheral blood T-lymphocytes (PBTLs). With this blood-based laboratory assay, we conducted a pilot case-control study of 100 patients with newly diagnosed, previously untreated SCCHN and 124 cancer-free controls of non-Hispanic whites. We found that the mean DSB repair capacity level was significantly lower in cases (42.1%) than that in controls (54.4%) (P<0.001). When we used the median DSB repair capacity level in the controls as the cutoff value for calculating the odds ratios (ORs) with adjustment for age, sex, smoking and drinking status, the cases were more likely than the controls to have a reduced DSB repair capacity (adjusted OR=1.93; 95% confidence interval, CI=1.04-3.56, P=0.037), especially for those subjects who were ever drinkers (adjusted OR=2.73; 95% CI=1.17-6.35, P=0.020) and had oropharyngeal tumors (adjusted OR=2.17; 95% CI=1.06-4.45, P=0.035). In conclusion, these findings suggest that individuals with a reduced DSB repair capacity may be at an increased risk of developing SCCHN. Larger studies are warranted to confirm these preliminary findings.

  10. Transcriptional elongation factor ENL phosphorylated by ATM recruits polycomb and switches off transcription for DSB repair.

    PubMed

    Ui, Ayako; Nagaura, Yuko; Yasui, Akira

    2015-05-07

    Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. DNA repair capacity of zebrafish.

    PubMed

    Sussman, Raquel

    2007-08-14

    Damage to the genome is unavoidable in living creatures, because of sunlight exposure as well as environmental chemicals present in food and drinking water. There is a need to monitor and purify the drinking water; therefore, several methods of detection have been developed. A very promising model system for this purpose is the zebrafish (Danio rerio), which is endowed with special qualities for detecting external as well as internal abnormalities. Grossman and Wei's assay [Grossman L, Wei Q (1995) Clin Chem 12:1854-1863], which measures the expression level of a nonreplicating recombinant plasmid DNA containing a UV-damaged luciferase reporter gene, shows that zebrafish can repair chromosomal lesions to a much greater extent than the human population. This vertebrate model is still very promising after possible down-regulation of the DNA repair enzymes.

  12. The RABiT: High Throughput Technology for Assessing Global DSB Repair

    PubMed Central

    Turner, H.C.; Sharma, P.; Perrier, J.R.; Bertucci, A.; Smilenov, L.; Johnson, Gary; Taveras, M.; Brenner, D.J.; Garty, G.

    2014-01-01

    At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry we have developed a Rapid Automated Biodosimetry Tool (RABiT); this is a completely automated, ultra-high throughput robotically-based biodosimetry workstation designed for use following a large scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7, 24 hours) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker. PMID:24477408

  13. Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage – When is a DSB not a DSB?☆

    PubMed Central

    Reynolds, Pamela; Botchway, Stanley W.; Parker, Anthony W.; O’Neill, Peter

    2013-01-01

    The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair. PMID:23688615

  14. Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity.

    PubMed

    Abramenkovs, Andris; Stenerlöw, Bo

    2017-09-27

    Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or γ-H2AX) or flow cytometric analysis of γ-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, γ-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than γ-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of γ-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of γ-H2AX.

  15. Variation in Base Excision Repair Capacity

    PubMed Central

    Wilson, David M.; Kim, Daemyung; Berquist, Brian R.; Sigurdson, Alice J.

    2010-01-01

    The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays. PMID:21167187

  16. KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-01

    Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear {gamma}-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.

  17. KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair.

    PubMed

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-01

    Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Identification and Analysis of MS5(d): A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

    PubMed

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2016-01-01

    Here, we report the identification of the Brassica-specific gene MS5(d), which is responsible for male sterility in Brassica napus. The MS5(d) gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5(d) gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5(d), encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5(d) likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

  19. Identification and Analysis of MS5d: A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes

    PubMed Central

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2017-01-01

    Here, we report the identification of the Brassica-specific gene MS5d, which is responsible for male sterility in Brassica napus. The MS5d gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5d gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5d, encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5d likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus. PMID:28101089

  20. Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.

    PubMed

    Darroudi, Firouz; Wiegant, Wouter; Meijers, Matty; Friedl, Anna A; van der Burg, Mirjam; Fomina, Janna; van Dongen, Jacques J M; van Gent, Dik C; Zdzienicka, Małgorzata Z

    2007-02-03

    We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of

  1. Plant γH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors.

    PubMed

    Lang, Julien; Smetana, Ondrej; Sanchez-Calderon, Lenin; Lincker, Frédéric; Genestier, Julie; Schmit, Anne-Catherine; Houlné, Guy; Chabouté, Marie-Edith

    2012-04-01

    Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (γH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of γH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses. We further establish the existence, restrictive to the G1/S transition, of specific DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

  2. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex.

    PubMed

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

  3. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response. PMID:26683461

  4. Base excision repair capacity in informing healthspan

    PubMed Central

    Brenerman, Boris M.; Illuzzi, Jennifer L.; Wilson, David M.

    2014-01-01

    Base excision repair (BER) is a frontline defense mechanism for dealing with many common forms of endogenous DNA damage, several of which can drive mutagenic or cell death outcomes. The pathway engages proteins such as glycosylases, abasic endonucleases, polymerases and ligases to remove substrate modifications from DNA and restore the genome back to its original state. Inherited mutations in genes related to BER can give rise to disorders involving cancer, immunodeficiency and neurodegeneration. Studies employing genetically defined heterozygous (haploinsufficient) mouse models indicate that partial reduction in BER capacity can increase vulnerability to both spontaneous and exposure-dependent pathologies. In humans, measurement of BER variation has been imperfect to this point, yet tools to assess BER in epidemiological surveys are steadily evolving. We provide herein an overview of the BER pathway and discuss the current efforts toward defining the relationship of BER defects with disease susceptibility. PMID:25355293

  5. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

    PubMed

    Gazy, Inbal; Zeevi, David A; Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51

  6. Impaired 53BP1/RIF1 DSB mediated end-protection stimulates CtIP-dependent end resection and switches the repair to PARP1-dependent end joining in G1

    PubMed Central

    Bakr, Ali; Köcher, Sabrina; Volquardsen, Jennifer; Petersen, Cordula; Borgmann, Kerstin; Dikomey, Ekkehard; Rothkamm, Kai; Mansour, Wael Y.

    2016-01-01

    End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway. PMID:27494840

  7. Impaired 53BP1/RIF1 DSB mediated end-protection stimulates CtIP-dependent end resection and switches the repair to PARP1-dependent end joining in G1.

    PubMed

    Bakr, Ali; Köcher, Sabrina; Volquardsen, Jennifer; Petersen, Cordula; Borgmann, Kerstin; Dikomey, Ekkehard; Rothkamm, Kai; Mansour, Wael Y

    2016-09-06

    End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

  8. A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

    PubMed

    Tilgner, K; Neganova, I; Moreno-Gimeno, I; Al-Aama, J Y; Burks, D; Yung, S; Singhapol, C; Saretzki, G; Evans, J; Gorbunova, V; Gennery, A; Przyborski, S; Stojkovic, M; Armstrong, L; Jeggo, P; Lako, M

    2013-08-01

    DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

  9. RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

    PubMed

    Gigi, Vered; Lewis, Susanna; Shestova, Olga; Mijušković, Martina; Deriano, Ludovic; Meng, Wenzhao; Luning Prak, Eline T; Roth, David B

    2014-06-01

    DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

  10. DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

    SciTech Connect

    Ruebe, Claudia E.

    2010-10-01

    Purpose: To evaluate, in a pilot study, the phosphorylated H2AX ({gamma}H2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy. Methods and Materials: The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting {gamma}H2AX foci in blood lymphocytes at defined time points after irradiation of blood samples. Results: Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities, and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function. Conclusions: Double-strand break repair alteration in children may predispose to cancer formation and may affect children's susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.

  11. Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

    PubMed Central

    Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

    2014-01-01

    Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

  12. Acute Normal Tissue Reactions in Head-and-Neck Cancer Patients Treated With IMRT: Influence of Dose and Association With Genetic Polymorphisms in DNA DSB Repair Genes

    SciTech Connect

    Werbrouck, Joke Ruyck, Kim de; Duprez, Frederic; Veldeman, Liv; Claes, Kathleen; Eijkeren, Marc van; Boterberg, Tom; Willems, Petra; Vral, Anne; Neve, Wilfried de; Thierens, Hubert

    2009-03-15

    Purpose: To investigate the association between dose-related parameters and polymorphisms in DNA DSB repair genes XRCC3 (c.-1843A>G, c.562-14A>G, c.722C>T), Rad51 (c.-3429G>C, c.-3392G>T), Lig4 (c.26C>T, c.1704T>C), Ku70 (c.-1310C>G), and Ku80 (c.2110-2408G>A) and the occurrence of acute reactions after radiotherapy. Materials and Methods: The study population consisted of 88 intensity-modulated radiation therapy (IMRT)-treated head-and-neck cancer patients. Mucositis, dermatitis, and dysphagia were scored using the Common Terminology Criteria (CTC) for Adverse Events v.3.0 scale. The population was divided into a CTC0-2 and CTC3+ group for the analysis of each acute effect. The influence of the dose on critical structures was analyzed using dose-volume histograms. Genotypes were determined by polymerase chain reaction (PCR) combined with restriction fragment length polymorphism or PCR-single base extension assays. Results: The mean dose (D{sub mean}) to the oral cavity and constrictor pharyngeus (PC) muscles was significantly associated with the development of mucositis and dysphagia, respectively. These parameters were considered confounding factors in the radiogenomics analyses. The XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes were significantly associated with the development of severe dysphagia (CTC3+). No association was found between the investigated polymorphisms and the development of mucositis or dermatitis. A risk analysis model for severe dysphagia, which was developed based on the XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes and the PC dose, showed a sensitivity of 78.6% and a specificity of 77.6%. Conclusions: The XRCC3c.722C>T and Ku70c.-1310C>G polymorphisms as well as the D{sub mean} to the PC muscles were highly associated with the development of severe dysphagia after IMRT. The prediction model developed using these parameters showed a high sensitivity and specificity.

  13. SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis.

    PubMed

    Pan, Xiaofen; Mou, Jingjing; Liu, Sha; Sun, Ziyi; Meng, Rui; Zhou, Zhenwei; Wu, Gang; Peng, Gang

    2015-06-01

    The present study aimed to investigate the influence of SHP-1 on the radioresistance of the nasopharyngeal carcinoma (NPC) cell line CNE-2 and the relevant underlying mechanisms. The human NPC cell line CNE-2 was transfected with a lentivirus that contained the SHP-1 gene or a nonsense sequence (referred to as LP-H1802Lv201 and LP-NegLv201 cells, respectively). Cells were irradiated with different ionizing radiation (IR) doses. Cell survival, DNA double-strand breaks (DSBs), apoptosis, cell cycle distribution, and the expression of related proteins were assessed using colony formation assay, immunofluorescent assays (IFAs), flow cytometry (FCM) and western blot analyses, respectively. Compared with the control (CNE-2 cells) and LP-NegLv201 cells, LP-H1802Lv201 cells were more resistant to IR. IFAs showed that IR caused less histone H2AX phosphorylation (γH2AX) and RAD51 foci in the LP-H1802Lv201 cells. Compared with the control and LP-NegLv201 cells, LP-H1802Lv201 cells showed increased S phase arrest. After IR, the apoptotic rate of the LP-H1802Lv201 cells was lower in contrast to the control and LP-NegLv201 cells. Western blot analyses showed that IR increased the phosphorylation of ataxia telangiectasia mutated (ATM) kinase, checkpoint kinase 2 (CHK2), ataxia telangiectasia and Rad3-related (ATR) protein, checkpoint kinase 1 (CHK1) and p53. In LP-H1802Lv201 cells, the phosphorylation levels of ATM and CHK2 were significantly increased while the p53 phosphorylation level was decreased compared to these levels in the control and LP-NegLv201 cells. Phosphorylation of ATR and CHK1 did not show significant differences in the three cell groups. Overexpression of SHP-1 in the CNE-2 cells led to radioresistance and the radioresistance was related to enhanced DNA DSB repair, increased S phase arrest and decreased cell apoptosis.

  14. Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

    PubMed Central

    El-Awady, R. A.; Dikomey, E.; Dahm-Daphi, J.

    2001-01-01

    After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G0 fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular. PMID:11328880

  15. The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance.

    PubMed

    Rosu, Simona; Zawadzki, Karl A; Stamper, Ericca L; Libuda, Diana E; Reese, Angela L; Dernburg, Abby F; Villeneuve, Anne M

    2013-01-01

    For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious

  16. DNA damage repair and genetic polymorphisms: Assessment of individual sensitivity and repair capacity

    SciTech Connect

    Cornetta, Tommaso; Festa, Fabiola; Testa, Antonella; Cozzi, Renata Prof. . E-mail: cozzi@uniroma3.it

    2006-10-01

    Purpose: To study the repair capacity after X-ray irradiation in human peripheral blood cells of healthy subjects, in relation to their genotypes. Methods and Materials: The peripheral blood of 50 healthy subjects was irradiated in vitro with 2 Gy of X rays and the induced DNA damage was measured by Comet assay immediately after irradiation. DNA repair was detected by analyzing the cells at defined time intervals after the exposure. Furthermore, all subjects were genotyped for XRCC1, OGG1, and XPC genes. Results: After X-ray irradiation, persons bearing XRCC1 homozygous variant (codon 399) genotype exhibited significantly lower Tail DNA values than those bearing wild-type and heterozygous genotypes. These results are also confirmed at 30 and 60 min after irradiation. Furthermore, XPC heterozygous subjects (variant codon 939) showed lower residual DNA damage 60 min after irradiation compared with wild-type and homozygous genotypes. Conclusion: The results of the present study show that polymorphisms in DNA repair genes could influence individual DNA repair capacity.

  17. Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

    PubMed Central

    Leu, Jyh-Der; Chiu, Yu-Wen; Lo, Chia-Chien; Chiang, Pei-Hsun; Chiu, Su-Jun; Tsai, Cheng-Han; Hwang, Jeng-Jong; Chen, Ran-Chou; Gorbunova, Vera; Lee, Yi-Jang

    2013-01-01

    Purpose A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores, the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and Methods Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation induced double strand break (DSB) repair, systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion CFL-1 over-expression enhances radiosensitivity and this

  18. Becoming a crossover-competent DSB.

    PubMed

    Lake, Cathleen M; Hawley, R Scott

    2016-06-01

    The proper execution of meiotic recombination (or crossing over) is essential for chromosome segregation during the first meiotic division, and thus this process is regulated by multiple, and often elaborate, mechanisms. Meiotic recombination begins with the programmed induction of DNA double-strand breaks (DSBs), of which only a subset are selected to be repaired into crossovers. This crossover selection process is carried out by a number of pro-crossover proteins that regulate the fashion in which DSBs are repaired. Here, we highlight recent studies regarding the process of DSB fate selection by a family of pro-crossover proteins known as the Zip-3 homologs.

  19. XRCC1 deficiency increased the DNA damage induced by γ-ray in HepG2 cell: Involvement of DSB repair and cell cycle arrest.

    PubMed

    Niu, Yujie; Zhang, Xing; Zheng, Yuxin; Zhang, Rong

    2013-09-01

    γ-ray irradiation can induce DNA damages which include base damages, single-strand breaks and double-strand breaks in various type cells. The DNA repair protein XRCC1, as a part of the BER pathway, forms complexes with DNA polymerase beta, DNA ligase III and poly-ADP-ribose polymerase (PARP) in the repair of DNA single strand breaks and also affects the repair of double strand breaks. However, it is still not known well whether XRCC1 contributes to affect the irradiation sensitivity and DNA damage in HepG2 cell and the potential mechanism. Hence, the purpose of this study was to explore whether abrogation of XRCC1 gene expression by shRNA could reduce DNA repair and thus sensitize HepG2 cells to γ-ray. Cell viability was measured by Trypan blue staining and cloning efficiency assay. The DNA damage was detected by Comet assay. Apoptosis and cell cycle were detected by flow cytometry. The DNA-PKcs and gadd153 mRNA expression were determined by Real-time PCR. Our results showed that abrogation of XRCC 1 could sensitize HepG2 cells to γ-ray. This enhanced sensitivity could be attributed to the increased DNA damage and increased cell cycle arrest, which might be related with the increasing of DNA-PKcs and gadd153 mRNA expression. Therefore, our results suggested that the γ-ray irradiation sensitivity could be increased by targeting inhibition of XRCC1 in HepG2 cell.

  20. miR-155 Over-expression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-prone DSB Repair

    PubMed Central

    Czochor, Jennifer R.; Sulkowski, Parker; Glazer, Peter M.

    2016-01-01

    miR-155 is an oncogenic microRNA (miR) that is often over-expressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors including RAD51, MLH1, and MSH6, and its over-expression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that over-expression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 over-expression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone non-homologous end-joining (NHEJ) pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 over-expression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication and repair polymerase, are down-regulated at the mRNA level in the context of miR-155 over-expression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. PMID:26850462

  1. Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans.

    PubMed

    Hyun, Moonjung; Lee, Jihyun; Lee, Kyungjin; May, Alfred; Bohr, Vilhelm A; Ahn, Byungchan

    2008-03-01

    DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.

  2. Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

    PubMed Central

    Hyun, Moonjung; Lee, Jihyun; Lee, Kyungjin; May, Alfred; Bohr, Vilhelm A.; Ahn, Byungchan

    2008-01-01

    DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans. PMID:18203746

  3. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes

    PubMed Central

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  4. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

  5. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK – dependent processing of DNA termini in NHEJ catalyzed DSB repair

    PubMed Central

    Pawelczak, Katherine S.; Turchi, John J.

    2010-01-01

    Artemis is a member of the β–CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5’ to 3’ exonuclease activity and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]6–Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activity, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activity on a Ni-Agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. PMID:20347402

  6. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair.

    PubMed

    Pawelczak, Katherine S; Turchi, John J

    2010-06-04

    Artemis is a member of the beta-CASP family of nucleases in the metallo-beta-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His](6)-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni-agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Biomarkers for DNA DSB inhibitors and radiotherapy clinical trials.

    PubMed

    Liu, Stanley K; Olive, Peggy L; Bristow, Robert G

    2008-09-01

    Major technical advances in radiotherapy, including IMRT and image-guided radiotherapy, have allowed for improved physical precision and increased dose delivery to the tumor, with better sparing of surrounding normal tissue. The development of inhibitors of the sensing and repair of DNA double-strand breaks (DSBs) is exciting and could be combined with precise radiotherapy targeting to improve local control following radiotherapy. However, caution must be exercised in order that DSB inhibitors are combined with radiotherapy in such a manner as to preserve the therapeutic ratio by exploiting repair deficiencies in malignant cells over that of normal cells. In this review, we discuss the rationale and current approaches to targeting DSB sensing and repair pathways in combined modality with radiotherapy. We also describe potential biomarkers that could be useful in detecting functional inhibition of DSB repair in a patient's tissues during clinical radiotherapy trials. Finally, we examine a number of issues relating to the use of DSB-inhibiting molecular agents and radiotherapy in the context of the tumor microenvironment, effects on normal tissues and the optimal timing and duration of the agent in relation to fractionated radiotherapy.

  8. Pulsed-field gel electrophoresis analysis of multicellular DNA double-strand break damage and repair.

    PubMed

    Joshi, Nina; Grant, Stephen G

    2014-01-01

    This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

  9. 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.

  10. miR-3940-5p enhances homologous recombination after DSB in Cr(VI) exposed 16HBE cell.

    PubMed

    Li, Yang; Hu, Guiping; Li, Ping; Tang, Shichuan; Zhang, Ji; Jia, Guang

    2016-02-17

    Hexavalent chromium (Cr(VI)) is a well-recognized human carcinogen, yet the molecular mechanisms by which cause human cancer are still not well understood. MicroRNAs (miRNAs), which are small non-coding RNAs, are involved in carcinogenesis and DNA damage repair. Previous occupational population study showed that hexavalent chromium (Cr(VI)) downregulated plasma miR-3940-5p level, and a low miR-3940-5p level was associated with high XRCC2 expression in lymphocytes, indicating that miR-3940-5p maybe play a protective effect in Cr(VI) induced DNA damage. Here we investigated miR-3940-5p expression and its roles in DNA repair in Cr(VI)-treated 16HBE cells. miR-3940-5p change was detected by qRT-PCR. Rad51 foci formation and double strand break (DSB) were investigated to assess homologous recombination repair (HR) capacity by Immunofluorescent assay and Neutral Comet assay. XRCC2 expression was also evaluated after miRNA oligonucleotides transfection using Western blot. Cr(VI) treatment suppressed miR-3940-5p level in 16HBE cells. miR-3904-5p mimic downregulated XRCC2 expression. As a result, the formation of Rad51-foci was inhibited and DSB repair was prolonged. The results indicate that miR-3940-5p plays a protective effect in Cr(VI) induced DNA damage.

  11. [Study on repair capacity of DNA damage associated with chronic benzene poisoning].

    PubMed

    Xing, Cai-hong; Ji, Zhi-ying; Li, Gui-lan; Yin, Song-nian

    2006-07-01

    To explore the repair capacity of DNA damage associated with chronic benzene poisonings. 63 workers suffered from chronic benzene poisonings and 45 workers exposed to benzene, who were engaged in the same job title, were investigated. Comet assay and cytokinesis-block micronucleus (CBMN) detection were used to evaluate gamma-radiation-induced DNA and chromosomal damage and repair capacity in peripheral blood lymphocyte. The comet tail length difference of the benzene poisoning group (4.64 +/- 1.57 microm) was significantly higher than that of the control group (3.77 +/- 1.30 microm) (P = 0.0029). There was no significant difference of the 3AB index between the poisoning group and the control group. The relative risk of benzene poisoning in the subject with comet tail length difference > 3.81 was significantly higher than that in the subject with comet tail length difference < or = 3.81 microm (OR = 2.490, 95% CI:1.068 - 5.806, P = 0.0346). The relative risk increased along with the comet tail length difference, and the trend was significant (P = 0.0024). There was no significant difference between the relative risk of benzene poisoning in the subject with 3AB index < 0.20 and that in the subject with 3AB index > or = 0.20. DNA repair capacity on DNA-strand level might tightly associate with chronic benzene poisoning. The DNA repair capacity on DNA-strand level would be worse, and the benzene poisoning risk could be higher. There was no clear relation between the DNA repair capacity on chromosome level and the benzene poisoning risk.

  12. Impaired endothelial repair capacity of early endothelial progenitor cells in prehypertension: relation to endothelial dysfunction.

    PubMed

    Giannotti, Giovanna; Doerries, Carola; Mocharla, Pavani S; Mueller, Maja F; Bahlmann, Ferdinand H; Horvàth, Tibor; Jiang, Hong; Sorrentino, Sajoscha A; Steenken, Nora; Manes, Costantina; Marzilli, Mario; Rudolph, K Lenhard; Lüscher, Thomas F; Drexler, Helmut; Landmesser, Ulf

    2010-06-01

    Prehypertension is a highly frequent condition associated with an increased cardiovascular risk. Endothelial dysfunction is thought to promote the development of hypertension and vascular disease; however, underlying mechanisms remain to be further determined. The present study characterizes for the first time the in vivo endothelial repair capacity of early endothelial progenitor cells (EPCs) in patients with prehypertension/hypertension and examines its relation with endothelial function. Early EPCs were isolated from healthy subjects and newly diagnosed prehypertensive and hypertensive patients (n=52). In vivo endothelial repair capacity of EPCs was examined by transplantation into a nude mouse carotid injury model. EPC senescence was determined (RT-PCR of telomere length). NO and superoxide production of EPCs were measured using electron spin resonance spectroscopy analysis. CD34(+)/KDR(+) mononuclear cells and circulating endothelial microparticles were examined by fluorescence-activated cell sorter analysis. Endothelium-dependent and -independent vasodilations were determined by high-resolution ultrasound. In vivo endothelial repair capacity of EPCs was substantially impaired in prehypertensive/hypertensive patients as compared with healthy subjects (re-endothelialized area: 15+/-3%/13+/-2% versus 28+/-3%; P<0.05 versus healthy subjects). Senescence of EPCs in prehypertension/hypertension was substantially increased, and NO production was markedly reduced. Moreover, reduced endothelial repair capacity of early EPCs was significantly related to an accelerated senescence of early EPCs and impaired endothelial function. The present study demonstrates for the first time that in vivo endothelial repair capacity of early EPCs is reduced in patients with prehypertension and hypertension, is related to EPC senescence and impaired endothelial function, and likely represents an early event in the development of hypertension.

  13. Enhanced nucleotide excision repair capacity in lung cancer cells by preconditioning with DNA-damaging agents.

    PubMed

    Choi, Ji Ye; Park, Jeong-Min; Yi, Joo Mi; Leem, Sun-Hee; Kang, Tae-Hong

    2015-09-08

    The capacity of tumor cells for nucleotide excision repair (NER) is a major determinant of the efficacy of and resistance to DNA-damaging chemotherapeutics, such as cisplatin. Here, we demonstrate that using lesion-specific monoclonal antibodies, NER capacity is enhanced in human lung cancer cells after preconditioning with DNA-damaging agents. Preconditioning of cells with a nonlethal dose of UV radiation facilitated the kinetics of subsequent cisplatin repair and vice versa. Dual-incision assay confirmed that the enhanced NER capacity was sustained for 2 days. Checkpoint activation by ATR kinase and expression of NER factors were not altered significantly by the preconditioning, whereas association of XPA, the rate-limiting factor in NER, with chromatin was accelerated. In preconditioned cells, SIRT1 expression was increased, and this resulted in a decrease in acetylated XPA. Inhibition of SIRT1 abrogated the preconditioning-induced predominant XPA binding to DNA lesions. Taking these data together, we conclude that upregulated NER capacity in preconditioned lung cancer cells is caused partly by an increased level of SIRT1, which modulates XPA sensitivity to DNA damage. This study provides some insights into the molecular mechanism of chemoresistance through acquisition of enhanced DNA repair capacity in cancer cells.

  14. Effect of a rapid repair mechanism for nitrification capacity in the load impact wastewater treatment

    NASA Astrophysics Data System (ADS)

    Hong, Wei; Zhang, Bing; Sun, Changdong; Tan, Xin; Liu, Bo; Zou, Xiaofeng

    2017-05-01

    The nitrification capacity in the wastewater treatment is very important, and is particularly vulnerable to impacts. In this study, a rapid repair mechanism for nitrification was built and the result showing that, with the addition of exogenous nitrifying bacteria and organic nutrient, the nitrification capacity in the pilot scale equipment was restored in 20h, the concentration of NH3-N in discharge conforms to the one-class A permitted criterion (GB 18918-2002) and remain stable for long time, while the nitrification capacity in control group would not be fixed by itself in 196h. The repaired experimental group has the advantage of strong shock resistance and stable operation, and under the second high impact load, the concentration of NH3-N in effluent remain stable.

  15. Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment

    SciTech Connect

    Protic, M.; Roilides, E.; Levine, A.S.; Dixon, K.

    1988-07-01

    To determine whether DNA excision repair is enhanced in mammalian cells in response to DNA damage, as it is in bacteria as part of the SOS response, we used an expression vector-host cell reactivation assay to measure cellular DNA repair capacity. When UV-damaged chloramphenicol acetyltransferase (CAT) vector DNA was introduced into monkey cells (CV-1), the level of CAT activity was inversely related to the UV fluence due to inhibition of CAT gene expression by UV photoproducts. When CV-1 cells were treated with either UV radiation or mitomycin C, 24-48 h before transfection, CAT expression from the UV-irradiated plasmid was increased. This increase also occurred in a line of normal human cells, but not in repair-deficient human xeroderma pigmentosum cells. We confirmed that this increase in CAT expression was due to repair, and not to production of damage-free templates by recombination; the frequency of generation of supF+ recombinants after transfection with UV-irradiated pZ189 vectors carrying different point mutations in the supF gene did not significantly increase in carcinogen-treated CV-1 cells. From these results we conclude that carcinogen treatment enhances the excision-repair capacity of normal mammalian cells.

  16. PHF11 promotes DSB resection, ATR signaling, and HR

    PubMed Central

    Gong, Yi; Handa, Naofumi; Kowalczykowski, Stephen C.; de Lange, Titia

    2017-01-01

    Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3′ ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5′ end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5′ dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5′ end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair. PMID:28115467

  17. PHF11 promotes DSB resection, ATR signaling, and HR.

    PubMed

    Gong, Yi; Handa, Naofumi; Kowalczykowski, Stephen C; de Lange, Titia

    2017-01-01

    Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3' ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5' end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5' dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5' end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair.

  18. The Impact of Individual In Vivo Repair of DNA Double-Strand Breaks on Oral Mucositis in Adjuvant Radiotherapy of Head-and-Neck Cancer

    SciTech Connect

    Fleckenstein, Jochen; Kuehne, Martin; Seegmueller, Katharina; Derschang, Sarah; Melchior, Patrick; Graeber, Stefan; Fricke, Andreas; Ruebe, Claudia E.; Ruebe, Christian

    2011-12-01

    Purpose: To evaluate the impact of individual in vivo DNA double-strand break (DSB) repair capacity on the incidence of severe oral mucositis in patients with head-and-neck cancer undergoing adjuvant radiotherapy (RT) or radiochemotherapy (RCT). Patients and Methods: Thirty-one patients with resected head-and-neck cancer undergoing adjuvant RT or RCT were examined. Patients underwent RT of the primary tumor site and locoregional lymph nodes with a total dose of 60-66 Gy (single dose 2 Gy, five fractions per week). Chemotherapy consisted of two cycles of cisplatin and 5-fluorouracil. To assess DSB repair, {gamma}-H2AX foci in blood lymphocytes were quantified before and 0.5 h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and correlated with DSB repair. Results: Sixteen patients received RT alone; 15 patients received RCT. In patients who developed Grade {>=} 3 mucositis (n = 18) the amount of unrepaired DSBs 24 h after radiation exposure and DSB repair half-times did not differ significantly from patients with Grade {<=}2 mucositis (n = 13). Patients with a proportion of unrepaired DSBs after 24 h higher than the mean value + one standard deviation had an increased incidence of severe oral mucositis. Conclusions: Evaluation of in vivo DSB repair by determination of {gamma}-H2AX foci loss is feasible in clinical practice and allows identification of patients with impaired DSB repair. The incidence of oral mucositis is not closely correlated with DSB repair under the evaluated conditions.

  19. Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

    PubMed

    Wagner, Waldemar; Kania, Katarzyna D; Ciszewski, Wojciech M

    2017-04-01

    Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

    2000-01-01

    PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

  1. Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

    2000-01-01

    PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

  2. Maintenance of xylem Network Transport Capacity: A Review of Embolism Repair in Vascular Plants.

    PubMed

    Brodersen, Craig R; McElrone, Andrew J

    2013-01-01

    Maintenance of long distance water transport in xylem is essential to plant health and productivity. Both biotic and abiotic environmental conditions lead to embolism formation within the xylem resulting in lost transport capacity and ultimately death. Plants exhibit a variety of strategies to either prevent or restore hydraulic capacity through cavitation resistance with specialized anatomy, replacement of compromised conduits with new growth, and a metabolically active embolism repair mechanism. In recent years, mounting evidence suggests that metabolically active cells surrounding the xylem conduits in some, but not all, species are capable of restoring hydraulic conductivity. This review summarizes our current understanding of the osmotically driven embolism repair mechanism, the known genetic and anatomical components related to embolism repair, rehydration pathways through the xylem, and the role of capacitance. Anatomical differences between functional plant groups may be one of the limiting factors that allow some plants to refill while others do not, but further investigations are necessary to fully understand this dynamic process. Finally, xylem networks should no longer be considered an assemblage of dead, empty conduits, but instead a metabolically active tissue finely tuned to respond to ever changing environmental cues.

  3. Maintenance of xylem Network Transport Capacity: A Review of Embolism Repair in Vascular Plants

    PubMed Central

    Brodersen, Craig R.; McElrone, Andrew J.

    2013-01-01

    Maintenance of long distance water transport in xylem is essential to plant health and productivity. Both biotic and abiotic environmental conditions lead to embolism formation within the xylem resulting in lost transport capacity and ultimately death. Plants exhibit a variety of strategies to either prevent or restore hydraulic capacity through cavitation resistance with specialized anatomy, replacement of compromised conduits with new growth, and a metabolically active embolism repair mechanism. In recent years, mounting evidence suggests that metabolically active cells surrounding the xylem conduits in some, but not all, species are capable of restoring hydraulic conductivity. This review summarizes our current understanding of the osmotically driven embolism repair mechanism, the known genetic and anatomical components related to embolism repair, rehydration pathways through the xylem, and the role of capacitance. Anatomical differences between functional plant groups may be one of the limiting factors that allow some plants to refill while others do not, but further investigations are necessary to fully understand this dynamic process. Finally, xylem networks should no longer be considered an assemblage of dead, empty conduits, but instead a metabolically active tissue finely tuned to respond to ever changing environmental cues. PMID:23630539

  4. Mutagenesis by cytostatic alkylating agents in yeast strains of differing repair capacities.

    PubMed

    Ruhland, A; Brendel, M

    1979-05-01

    Reversion of two nulcear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (radi rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (rad1 rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom.

  5. SDF-1 enhances wound healing of critical-sized calvarial defects beyond self-repair capacity.

    PubMed

    Jin, Qiming; Giannobile, William V

    2014-01-01

    Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.

  6. RAD51 as a potential surrogate marker for DNA repair capacity in solid malignancies.

    PubMed

    Gachechiladze, Mariam; Škarda, Josef; Soltermann, Alex; Joerger, Markus

    2017-10-01

    Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors, and increased DNA repair capacity is a hallmark mechanism of resistance not only to DNA-damaging treatments such as cytotoxic drugs and radiotherapy, but also to small molecule targeted drugs such as inhibitors of poly-ADP ribose polymerase (PARP). Hence, there is substantial medical need for potent and convenient biomarkers of individual response to DNA-targeted treatment in personalized cancer care. RAD51 is a highly conserved protein that catalyzes DNA repair via homologous recombination, a major DNA repair pathway which directly modulates cellular sensitivity to DNA-damaging treatments. The clinical and biological significance of RAD51 protein expression is still under investigation. Pre-clinical studies consistently show the important role of nuclear RAD51 immunoreactivity in chemo- and radioresistance. Validating data from clinical trials however is limited at present, and some clinical studies show controversial results. This review gives a comprehensive overview on the current knowledge about the prognostic and predictive value of RAD51 protein expression and genetic variability in patients with solid malignancies. © 2017 UICC.

  7. Homeostatic regulation of meiotic DSB formation by ATM/ATR

    SciTech Connect

    Cooper, Tim J.; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J.

    2014-11-15

    Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  8. Homeostatic regulation of meiotic DSB formation by ATM/ATR.

    PubMed

    Cooper, Tim J; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J

    2014-11-15

    Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  9. DNA damage and repair capacity in workers exposed to low concentrations of benzene.

    PubMed

    Lovreglio, Piero; Doria, Denise; Fracasso, Maria Enrica; Barbieri, Anna; Sabatini, Laura; Drago, Ignazio; Violante, Francesco S; Soleo, Leonardo

    2016-03-01

    DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling-station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t-muconic acid, S-phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m(3)), attendants (median 13.8 µg/m(3)), and controls (median 4.1 µg/m(3)). There were no differences in TI and 8-oxodG among the three groups, or between smokers and non-smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2 O2 -damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained.

  10. Exercise capacity, physical activity, and obesity in adults with repaired aortic coarctation.

    PubMed

    Buys, Roselien; Budts, Werner; Delecluse, Christophe; Vanhees, Luc

    2013-01-01

    Patients with aortic coarctation (COA) have a reduced exercise capacity and seem to be more prone to adopt a sedentary lifestyle. During clinical practice, we often observe that patients seem to be overweight. However, data on physical activity and weight status in this population are currently not available. This study aimed to describe weight status, self-reported physical activity levels, and self-perceived health status in adults with repaired COA in comparison with healthy counterparts and to investigate the relationships among exercise capacity, physical activity, weight status, and perceived health status. We studied 103 COA patients who underwent cardiopulmonary exercise testing and who completed the Flemish Physical Activity Computerized Questionnaire and the short-form 36 health survey questionnaire. Patients with COA show a significantly lower exercise tolerance than what would be expected in healthy persons (P < .0001). Weight status was similar to the overall Belgian population, but a tendency toward higher body mass index was present. Patients with COA report a lower level of habitual physical activity (P < .05) as well as reductions in perceived vitality, general health, and mental health (P < .05). Parameters of habitual physical activity are related to exercise capacity (total energy expenditure: r = 0.278, P = .0015). The more active COA patients report higher scores of perceived physical functioning, general health, and mental health. Overweight patients did not perform significantly different than patients with a healthy weight did. Adults with repaired COA have a reduced exercise tolerance, which is related to low physical activity levels. Up to one-third of the COA patients we studied are overweight. As a poor exercise capacity identifies patients at risk for hospitalization and death and obesity might adversely influence the development of cardiovascular disease, it is important to improve the exercise capacity in these patients. Guiding

  11. 8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation.

    PubMed

    Kuboyama, Ayumi; Tanaka, Shoichi; Kawai, Kazuaki; Kasai, Hiroshi; Morii, Haruki; Tamae, Kazuyoshi; Nakashima, Tamiji; Hirano, Takeshi

    2011-05-01

    To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.

  12. Exposure assessment of benzene in Thai workers, DNA-repair capacity and influence of genetic polymorphisms.

    PubMed

    Chanvaivit, Sirirat; Navasumrit, Panida; Hunsonti, Potchanee; Autrup, Herman; Ruchirawat, Mathuros

    2007-01-10

    Exposure to benzene can cause DNA damage and the subsequent development of cancer. In this study, study subjects were 31 laboratory workers at a petrochemical factory and 31 gasoline service attendants. Control subjects were 34 workers from a mail sorting service center. Occupational exposures to benzene were assessed using biomarkers of exposure in blood and urine. Induction of DNA-repair capacity was assessed as a biomarker of early effect. The effects of polymorphisms in a metabolizing gene (CYP2E1), in detoxification genes (NQO1 and GSTT1), and in a DNA-repair gene (XRCC1, codon 399) on biomarker levels were evaluated. The mean individual benzene exposure of laboratory workers (24.40+/-5.82 ppb) and that of gasoline service attendants (112.41+/-13.92 ppb) were significantly higher than in controls (1.39+/-0.17 ppb, p<0.001). Blood benzene levels of laboratory workers (169.12+/-30.60 ppt) and gasoline service attendants (483.46+/-59.62 ppt) were significantly higher than those of the controls (43.30+/-4.89 ppt, p<0.001). Trans,trans-muconic acid levels in post-shift urine samples collected from laboratory workers (0.14+/-0.02 mg/g creatinine) and gasoline service attendants (0.20+/-0.02 mg/g creatinine) were significantly higher than in urine samples of controls (0.04+/-0.01 mg/g creatinine, p<0.001). The level of benzene exposure was correlated with blood benzene levels (R2=0.65, p<0.01) and post-shift urinary trans,trans-muconic acid concentrations (R2=0.49, p<0.01). As a biomarker of early effect, DNA-repair capacity was assessed by use of the cytogenetic challenge assay, i.e., chromosomal aberrations in peripheral lymphocytes were assessed after challenging blood cultures with 1 Gy gamma radiation. A significantly lower DNA-repair capacity--determined as dicentrics in laboratory workers (0.17 per metaphase cell) and in gasoline service attendants (0.19 per metaphase cell) compared with controls (0.12 per metaphase cell, p<0.001)--was observed. The frequency

  13. Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions

    PubMed Central

    Tsai, Ju-Ying; Chen, Fang-Hsin; Hsieh, Tsung-Yu; Hsiao, Ya-Yun

    2015-01-01

    Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to 60Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1–4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing. PMID:25902742

  14. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    SciTech Connect

    Nishiyama, Y.; Rapp, F.

    1981-04-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants.

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

    PubMed

    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

    2009-09-15

    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 H(2)O(2)-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H(2)O(2)-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 H(2)O(2), 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 beta, 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.

  16. DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells

    PubMed Central

    Song, Lipu; Fan, Anran; Zhang, Sheng; Wang, Jianyu; Fan, Nana; Liu, Na; Ye, Xiaoying; Fu, Haifeng; Zhou, Zhongcheng; Wang, Yong; Wei, Hong; Liu, Zhonghua; Li, Ziyi; Lai, Liangxue; Wang, Xumin; Liu, Lin

    2017-01-01

    Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFβ pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies. PMID:28253351

  17. Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells.

    PubMed

    Liu, Chi; Liu, Hui-jun; Xiang, Yang; Tan, Yu-rong; Zhu, Xiao-lin; Qin, Xiao-qun

    2010-08-01

    Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.

  18. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    PubMed

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  19. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    PubMed

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-05

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.

  20. Development of a ratio of emergent to total hernia repairs as a surgical capacity metric.

    PubMed

    Samuel, Jonathan C; Tyson, Anna F; Mabedi, Charles; Mulima, Gift; Cairns, Bruce A; Varela, Carlos; Charles, Anthony G

    2014-01-01

    Non-communicable diseases including surgical conditions are gaining attention in developing countries. Despite this there are few metrics for surgical capacity. We hypothesized that (a) the ratio of emergent to total hernia repairs (E/TH) would correlate with per capita health care expenditures for any given country, and (b) the E/TH is easy to obtain in resource-poor settings. We performed a systematic review to identify the E/TH for as many countries as possible (Prospero registry CRD42013004645). We screened 1285 English language publications since 1990; 23 met inclusion criteria. Primary data was also collected from Kamuzu Central Hospital (KCH) in Lilongwe, Malawi. A total of 13 countries were represented. Regression analysis was used to determine the correlation between per capita health care spending and the E/TH. There is a strong correlation between the log values of the ratio emergent to total groin hernias and the per capita health care spending that is robust across country income levels (R(2) = 0.823). Primary data from KCH was easily obtained and demonstrated a similar correlation. The ratio of emergent to total groin hernias is a potential measure of surgical capacity using data that is easily attainable. Further studies should validate this metric against other accepted health care capacity indicators. Systematic review registered with Prospero (CRD42013004645). Copyright © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

  1. Oxidative damage to RPA limits the nucleotide excision repair capacity of human cells

    PubMed Central

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-01-01

    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 (ROS) that diminish NER capacity by causing protein damage. The RPA DNA binding protein plays 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. PMID:26134950

  2. DNA DSB measurements and modelling approaches based on gamma-H2AX foci time evolution

    NASA Astrophysics Data System (ADS)

    Esposito, Giuseppe; Campa, Alessandro; Antonelli, Francesca; Mariotti, Luca; Belli, Mauro; Giardullo, Paola; Simone, Giustina; Antonella Tabocchini, Maria; Ottolenghi, Andrea

    DNA double strand breaks (DSBs) induced by ionising radiation are considered the main dam-age related to the deleterious consequences in the cells. Unrepaired or mis-repaired DSBs can cause mutations or loss of chromosome regions which can eventually lead to cell death or neo-plastic transformation. Quantification of the number and complexity of DSBs induced by low doses of radiation remains a complex problem. About ten years ago Rogakou et al. proposed an immunofluorescent technique able to detect even a single DSB per cell. This approach is based on the serine 139 phosphorylation of many molecules (up to 2000) of histone H2AX (γg-H2AX) following the induction of a DSB in the DNA. DSB can be visualized as foci by immunofluores-cence by using phospho-specific antibodies, so that enumeration of foci can be used to measure DSB induction and processing. It is still not completely clear how γ-H2AX dephosphorylation takes place; however it has been related with DSB repair, in particular with the efficiency of DSB repair. In this work we analyse the H2AX phosphorylation-dephosphorylation kinetics after irradiation of primary human fibroblasts (AG1522 cell line) with radiation of differing quality, that is γ-rays and α-particles (125 keV/µm), with the aim of comparing the time evolution of γ-H2AX foci. Our results show that, after a dose of 0.5 Gy, both γ-rays and α-particles induce the maximum number of γ-H2AX foci within 30 minutes from irradiation, that this number depends on the radiation type and is consistent with the number of track traversal in α-irradiated nuclei, that the dephosphorylation kinetics are very different, being the α-induced foci rate of disappearence slower than that of γ-induced foci. In this work a modellistic approach to estimate the number of DSB induced by γ-rays detectable by using the γ-H2AX assay is presented. The competing processes of appearance and disappearance of visible foci will be modeled taking into account the

  3. Exercise training improves in vivo endothelial repair capacity of early endothelial progenitor cells in subjects with metabolic syndrome.

    PubMed

    Sonnenschein, Kristina; Horváth, Tibor; Mueller, Maja; Markowski, Andrea; Siegmund, Tina; Jacob, Christian; Drexler, Helmut; Landmesser, Ulf

    2011-06-01

    Endothelial dysfunction and injury are considered to contribute considerably to the development and progression of atherosclerosis. It has been suggested that intense exercise training can increase the number and angiogenic properties of early endothelial progenitor cells (EPCs). However, whether exercise training stimulates the capacity of early EPCs to promote repair of endothelial damage and potential underlying mechanisms remain to be determined. The present study was designed to evaluate the effects of moderate exercise training on in vivo endothelial repair capacity of early EPCs, and their nitric oxide and superoxide production as characterized by electron spin resonance spectroscopy analysis in subjects with metabolic syndrome. Twenty-four subjects with metabolic syndrome were randomized to an 8 weeks exercise training or a control group. Superoxide production and nitric oxide (NO) availability of early EPCs were characterized by using electron spin resonance (ESR) spectroscopy analysis. In vivo endothelial repair capacity of EPCs was examined by transplantation into nude mice with defined carotid endothelial injury. Endothelium-dependent, flow-mediated vasodilation was analysed using high-resolution ultrasound. Importantly, exercise training resulted in a substantially improved in vivo endothelial repair capacity of early EPCs (24.0 vs 12.7%; p < 0.05) and improved endothelium-dependent vasodilation. Nitric oxide production of EPCs was substantially increased after exercise training, but not in the control group. Moreover, exercise training reduced superoxide production of EPCs, which was not observed in the control group. The present study suggests for the first time that moderate exercise training increases nitric oxide production of early endothelial progenitor cells and reduces their superoxide production. Importantly, this is associated with a marked beneficial effect on the in vivo endothelial repair capacity of early EPCs in subjects with

  4. Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase.

    PubMed

    Yasuhira, Shinji; Saito, Takeshi; Maesawa, Chihaya; Masuda, Tomoyuki

    2012-08-01

    Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Heavy ion induced DNA-DSB in yeast and mammalian cells

    NASA Technical Reports Server (NTRS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

  6. OsSDS is essential for DSB formation in rice meiosis.

    PubMed

    Wu, Zhigang; Ji, Jianhui; Tang, Ding; Wang, Hongjun; Shen, Yi; Shi, Wenqing; Li, Yafei; Tan, Xuelin; Cheng, Zhukuan; Luo, Qiong

    2015-01-01

    SDS is a meiosis specific cyclin-like protein and required for DMC1 mediated double-strand break (DSB) repairing in Arabidopsis. Here, we found its rice homolog, OsSDS, is essential for meiotic DSB formation. The Ossds mutant is normal in vegetative growth but both male and female gametes are inviable. The Ossds meiocytes exhibit severe defects in homologous pairing and synapsis. No γH2AX immunosignals in Ossds meiocytes together with the suppression of chromosome fragmentation in Ossds-1 Osrad51c, both provide strong evidences that OsSDS is essential for meiotic DSB formation. Immunostaining investigations revealed that meiotic chromosome axes are normally formed but both SC installation and localization of recombination elements are failed in Ossds. We suspected that this cyclin protein has been differentiated pretty much between monocots and dicots on its function in meiosis.

  7. The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

    PubMed

    Serrentino, Maria-Elisabetta; Borde, Valérie

    2012-07-15

    A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots.

  8. Women with endometriosis have a higher DNA repair capacity and diminished breast cancer risk

    PubMed Central

    Matta, Jaime L.; Flores, Idhaliz; Morales, Luisa M.; Monteiro, Janice; Alvarez-Garriga, Carolina; Bayona, Manuel

    2014-01-01

    Introduction Breast cancer (BC) and endometriosis are important reproductive health diseases for women. Although endometriosis is not a malignant condition, some of its characteristics mimic that of a malignancy. Endometriosis is associated with increased risk of certain cancers; however, whether it alters BC risk is unclear. This study evaluates the association of endometriosis and BC and explores whether DNA repair capacity (DRC) plays a role in such a relationship. Materials and Methods A case-control study of 991 women (385 with BC and 606 controls, all recruited over 5 years) was undertaken in Puerto Rico. Eighty participants with self-reported surgically diagnosed endometriosis were identified, 20 of whom also had a diagnosis of BC. Data from a structured questionnaire and DRC measurements were assessed to determine the association between BC, DRC, and endometriosis. Results Participants with BC cases were 50% less likely to have history of endometriosis (OR = 0.5 95%CI: 0.3, 0.9, p = 0.038) than women without BC controls. Findings that did not reach statistical significance included the following: women with history of endometriosis had a slightly higher DRC level than those without it; BC cases and history of endometriosis were less likely to have had endometriosis diagnosis before age 38 as compared to controls with endometriosis. Discussion Here we report an inverse association between endometriosis and BC, the former possibly conferring a protective effect on the latter. Although the mechanisms involved are unknown they may include protection provided by higher DRC and or hormonal treatments for endometriosis. A larger sample of endometriosis cases is necessary to confirm these results and answer the question of whether a higher DRC capacity may contribute to this potential protection, and to identify other factors at play. PMID:25473592

  9. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    NASA Astrophysics Data System (ADS)

    Amundson, S. A.; Chen, D. J.

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-inducedtk - mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  10. White matter involvement after TBI: Clues to axon and myelin repair capacity.

    PubMed

    Armstrong, Regina C; Mierzwa, Amanda J; Marion, Christina M; Sullivan, Genevieve M

    2016-01-01

    Impact-acceleration forces to the head cause traumatic brain injury (TBI) with damage in white matter tracts comprised of long axons traversing the brain. White matter injury after TBI involves both traumatic axonal injury (TAI) and myelin pathology that evolves throughout the post-injury time course. The axon response to initial mechanical forces and secondary insults follows the process of Wallerian degeneration, which initiates as a potentially reversible phase of intra-axonal damage and proceeds to an irreversible phase of axon fragmentation. Distal to sites of axon disconnection, myelin sheaths remain for prolonged periods, which may activate neuroinflammation and inhibit axon regeneration. In addition to TAI, TBI can cause demyelination of intact axons. These evolving features of axon and myelin pathology also represent opportunities for repair. In experimental TBI, demyelinated axons exhibit remyelination, which can serve to both protect axons and facilitate recovery of function. Myelin remodeling may also contribute to neuroplasticity. Efficient clearance of myelin debris is a potential target to attenuate the progression of chronic pathology. During the early phase of Wallerian degeneration, interventions that prevent the transition from reversible damage to axon disconnection warrant the highest priority, based on the poor regenerative capacity of axons in the CNS. Clinical evaluation of TBI will need to address the challenge of accurately detecting the extent and stage of axon damage. Distinguishing the complex white matter changes associated with axons and myelin is necessary for interpreting advanced neuroimaging approaches and for identifying a broader range of therapeutic opportunities to improve outcome after TBI.

  11. Somatic mosaicism for DNA repair capacity in fibroblasts derived from a group A xeroderma pigmentosum patient

    SciTech Connect

    Chang, H.R.; Ishizaki, K.; Sasaki, M.S.; Toguchida, J.; Kato, M.; Nakamura, Y.; Kawamura, S.; Moriguchi, T.; Ikenaga, M. )

    1989-10-01

    A female Japanese xeroderma pigmentosum (XP) patient with severe skin lesions and various neurologic abnormalities was assigned to complementation group A by conventional cell fusion studies. Ultraviolet (UV)-irradiated skin fibroblasts showed a biphasic survival curve, as measured by colony-forming ability. The surviving fraction decreased rapidly up to 2 J/m2 of UV, with a steep slope of D(O) (mean lethal dose) = 0.95 J/m2. At much higher doses it decreased more slowly, with D(O) = 3.5 J/m2. To elucidate the cause of this unique survival response, we isolated a large number of independent clones from single colonies and measured their responses to UV. Of 81 clones analyzed, ten showed a marked resistance to killing by UV, which was only slightly more sensitive than normal cells, and these clones had a rate of unscheduled DNA synthesis (UDS) that was about 45% of normal cells. By contrast, the remaining 71 clones were extremely sensitive to UV, typical of XP group A strains, and had a UDS level 1%-3% of normals. Analysis of restriction fragment length polymorphism using seven polymorphic DNA probes indicated that the UV-resistant clones were derived from the same individual as the UV-sensitive clones. These results clearly demonstrate that this patient's fibroblast cells consist of two types with differing responses to UV, and provide direct evidence of somatic mosaicism for DNA repair capacity in an XP patient.

  12. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    SciTech Connect

    Amundson, S.A.; Chen, D.J.

    1994-12-31

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by {alpha}-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and {alpha}-particles. WTK1 is also more mutable by {alpha}-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while {alpha}-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by {alpha}-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  13. Radiobiological basis of total body irradiation with different dose rate and fractionation: repair capacity of hemopoietic cells

    SciTech Connect

    Song, C.W.; Kim, T.H.; Khan, F.M.; Kersey, J.H.; Levitt, S.H.

    1981-12-01

    Total body irradiation (TBI) followed by bone marrow transplantation is being used in the treatment of malignant or non-malignant hemopoietic disorders. It has been believed that the ability of hemopoietic cells to repair sublethal radiation damage is negligible. Therefore, several schools of investigators suggested that TBI in a single exposure at extremely low dose rate (5 rad/min) over several hours, or in several fractions in 2-3 days, should yield a higher therapeutic gain, as compared with a single exposure at a high dose rate (25 rad/min). We reviewed the existing data in the literature, in particular, the response of hemopoietic cells to fractionated doses of irradiation and found that the repair capacity of both malignant and non-malignant hemopoietic cells might be greater than has been thought. It is concluded that we should not underestimate the ability of hemopoietic cells to repair sublethal radiation damage in using TBI.

  14. Radiobiological basis of total body irradiation with different dose rate and fractionation: repair capacity of hemopoietic cells

    SciTech Connect

    Song, C.W.; Kim, T.H.; Khan, F.M.; Kersey, J.H.; Levitt, S.H.

    1981-12-01

    Total body irradiation (TBI) followed by bone marrow transplantation is being used in the treatment of malignant or non-malignant hemopoietic disorders. It has been believed that the ability of hemopoietic cells to repair sublethal radiation damage is negligible. Therefore, several schools of investigators suggested that TBI in a single exposure at extremely low dose rate (5 rad/min) over several hours, or in several fractions in 2-3 days, should yield a higher therapeutic gain, as compared with a single exposure at a high dose rate (26 rad/min). We reviewed the existing data in the literature, in particular, the response of hemopoietic cells to fractionated doses of irradiation and found that the repair capacity of both malignant and non-malignant hemopoietic cells might be greater than has been thought. It is concluded that we should not underestimate the ability of hemopoietic cells to repair sublethal radiation damage in using TBI.

  15. HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis.

    PubMed

    Goodyer, William; Kaitna, Susanne; Couteau, Florence; Ward, Jordan D; Boulton, Simon J; Zetka, Monique

    2008-02-01

    Repair of the programmed meiotic double-strand breaks (DSBs) that initiate recombination must be coordinated with homolog pairing to generate crossovers capable of directing chromosome segregation. Chromosome pairing and synapsis proceed independently of recombination in worms and flies, suggesting a paradoxical lack of coregulation. Here, we find that the meiotic axis component HTP-3 links DSB formation with homolog pairing and synapsis. HTP-3 forms complexes with the DSB repair components MRE-11/RAD-50 and the meiosis-specific axis component HIM-3. Loss of htp-3 or mre-11 recapitulates meiotic phenotypes consistent with a failure to generate DSBs, suggesting that HTP-3 associates with MRE-11/RAD-50 in a complex required for meiotic DSB formation. Loss of HTP-3 eliminates HIM-3 localization to axes and HIM-3-dependent homolog alignment, synapsis, and crossing over. Our study reveals a mechanism for coupling meiotic DSB formation with homolog pairing through the essential participation of an axis component with complexes mediating both processes.

  16. Aerobic endurance capacity affects spatial memory and SIRT1 is a potent modulator of 8-oxoguanine repair

    PubMed Central

    Sarga, Linda; Hart, Nikolett; Koch, Lauren; Britton, Steve; Hajas, Gyorgy; Boldogh, Istvan; Ba, Xuequing; Radak, Zsolt

    2013-01-01

    Regular exercise promotes brain function via a wide range of adaptive responses, including the increased expression of antioxidant and oxidative DNA damage-repairing systems. Accumulation of oxidized DNA base lesions and strand breaks is etiologically linked to for example aging processes and age-associated diseases. Here we tested whether exercise training has an impact on brain function, extent of neurogenesis, and expression of 8-oxoguanine DNA glycosylase-1 (Ogg1) and SIRT1 (silent mating type information regulation 2 homolog). To do so, we utilized strains of rats with low- and high- running capacity (LCR and HCR) and examined learning and memory, DNA synthesis, expression, and posttranslational modification of Ogg1 hippocampal cells. Our results showed that rats with higher aerobic/running capacity had better spatial memory, and expressed less Ogg1, when compared to LCR rats. Furthermore, exercise increased SIRT1 expression and decreased acetylated Ogg1 (AcOgg1) levels, a post-translational modification important for efficient repair of 8-oxoG. Our data on cell cultures revealed that nicotinamide, a SIRT1-specific inhibitor, caused the greatest increase in the acetylation of Ogg1, a finding further supported by our other observations that silencing SIRT1 also markedly increased the levels of AcOgg1. These findings imply that high-running capacity is associated with increased hippocampal function, and SIRT1 level/activity and inversely correlates with AcOgg1 levels and thereby the repair of genomic 8-oxoG. PMID:23973402

  17. Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines.

    PubMed

    Parfitt, S L; Milner, R J; Salute, M E; Hintenlang, D E; Farese, J P; Bacon, N J; Bova, F J; Rajon, D A; Lurie, D M

    2011-09-01

    Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.

  18. Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity

    PubMed Central

    Doddrell, Robin D.S.; Edwards, Philip; Morrison, Helen

    2017-01-01

    Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS. PMID:28137778

  19. Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions.

    PubMed

    Tsai, Ju-Ying; Chen, Fang-Hsin; Hsieh, Tsung-Yu; Hsiao, Ya-Yun

    2015-07-01

    Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  20. Regenerative Capacity of Old Muscle Stem Cells Declines without Significant Accumulation of DNA Damage

    PubMed Central

    Cousin, Wendy; Ho, Michelle Liane; Desai, Rajiv; Tham, Andrea; Chen, Robert Yuzen; Kung, Sunny; Elabd, Christian; Conboy, Irina M.

    2013-01-01

    The performance of adult stem cells is crucial for tissue homeostasis but their regenerative capacity declines with age, leading to failure of multiple organs. In skeletal muscle this failure is manifested by the loss of functional tissue, the accumulation of fibrosis, and reduced satellite cell-mediated myogenesis in response to injury. While recent studies have shown that changes in the composition of the satellite cell niche are at least in part responsible for the impaired function observed with aging, little is known about the effects of aging on the intrinsic properties of satellite cells. For instance, their ability to repair DNA damage and the effects of a potential accumulation of DNA double strand breaks (DSBs) on their regenerative performance remain unclear. This work demonstrates that old muscle stem cells display no significant accumulation of DNA DSBs when compared to those of young, as assayed after cell isolation and in tissue sections, either in uninjured muscle or at multiple time points after injury. Additionally, there is no significant difference in the expression of DNA DSB repair proteins or globally assayed DNA damage response genes, suggesting that not only DNA DSBs, but also other types of DNA damage, do not significantly mark aged muscle stem cells. Satellite cells from DNA DSB-repair-deficient SCID mice do have an unsurprisingly higher level of innate DNA DSBs and a weakened recovery from gamma-radiation-induced DNA damage. Interestingly, they are as myogenic in vitro and in vivo as satellite cells from young wild type mice, suggesting that the inefficiency in DNA DSB repair does not directly correlate with the ability to regenerate muscle after injury. Overall, our findings suggest that a DNA DSB-repair deficiency is unlikely to be a key factor in the decline in muscle regeneration observed upon aging. PMID:23704914

  1. Roles of chromatin remodellers in DNA double strand break repair.

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

    Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

  2. Dimethyloxaloylglycine increases bone repair capacity of adipose-derived stem cells in the treatment of osteonecrosis of the femoral head

    PubMed Central

    Zhu, Zhen-Hong; Song, Wen-Qi; Zhang, Chang-Qing; Yin, Ji-Min

    2016-01-01

    Mesenchymal stem cells have been widely studied to promote local bone regeneration of osteonecrosis of the femoral head (ONFH). Previous studies observed that dimethyloxaloylglycine (DMOG) enhanced the angiogenic and osteogenic activity of mesenchymal stem cells by activating the expression of hypoxia inducible factor-1α (HIF-1α), thereby improving the bone repair capacity of mesenchymal stem cells. In the present study, it was investigated whether DMOG could increase the bone repair capacity of adipose-derived stem cells (ASCs) in the treatment of ONFH. Western blot analysis was performed to detect HIF-1α protein expression in ASCs treated with different concentrations of DMOG. The results showed DMOG enhanced HIF-1α expression in ASCs in a dose-dependent manner at least for 7 days. Furthermore, DMOG-treated ASCs were transplanted into the necrotic area of a rabbit model of ONFH to treat the disease. Four weeks later, micro-computed tomography (CT) quantitative analysis showed that 58.8±7.4% of the necrotic area was regenerated in the DMOG-treated ASCs transplantation group, 45.5±3.4% in normal ASCs transplantation group, 25.2±2.8% in only core decompression group and 10.6±2.6% in the untreated group. Histological analysis showed that transplantation of DMOG-treated ASCs clearly improved the bone regeneration of the necrotic area compared with the other three groups. Micro-CT and immunohistochemical analysis demonstrated the revasculation of the necrotic area were also increased significantly in the DMOG-treated ASC group compared with the control groups. Thus, it is hypothesized that DMOG could increase the bone repair capacity of ASCs through enhancing HIF-1α expression in the treatment of ONFH. PMID:27882083

  3. DNA double-strand break repair pathway choice and cancer.

    PubMed

    Aparicio, Tomas; Baer, Richard; Gautier, Jean

    2014-07-01

    Since DNA double-strand breaks (DSBs) contribute to the genomic instability that drives cancer development, DSB repair pathways serve as important mechanisms for tumor suppression. Thus, genetic lesions, such as BRCA1 and BRCA2 mutations, that disrupt DSB repair are often associated with cancer susceptibility. In addition, recent evidence suggests that DSB "mis-repair", in which DSBs are resolved by an inappropriate repair pathway, can also promote genomic instability and presumably tumorigenesis. This notion has gained currency from recent cancer genome sequencing studies which have uncovered numerous chromosomal rearrangements harboring pathological DNA repair signatures. In this perspective, we discuss the factors that regulate DSB repair pathway choice and their consequences for genome stability and cancer.

  4. Early age decline in DNA repair capacity in the liver: in depth profile of differential gene expression

    PubMed Central

    Guedj, Avital; Geiger-Maor, Anat; Galun, Eithan; Amsalem, Hagai; Rachmilewitz, Jacob

    2016-01-01

    Aging is associated with progressive decline in cell function and with increased damage to macromolecular components. DNA damage, in the form of double-strand breaks (DSBs), increases with age and in turn, contributes to the aging process and age-related diseases. DNA strand breaks triggers a set of highly orchestrated signaling events known as the DNA damage response (DDR), which coordinates DNA repair. However, whether the accumulation of DNA damage with age is a result of decreased repair capacity, remains to be determined. In our study we showed that with age there is a decline in the resolution of foci containing γH2AX and pKAP-1 in diethylnitrosamine (DEN)-treated mouse livers, already evident at a remarkably early age of 6-months. Considerable age-dependent differences in global gene expression profiles in mice livers after exposure to DEN, further affirmed these age related differences in the response to DNA damage. Functional analysis identified p53 as the most overrepresented pathway that is specifically enhanced and prolonged in 6-month-old mice. Collectively, our results demonstrated an early decline in DNA damage repair that precedes ‘old age’, suggesting this may be a driving force contributing to the aging process rather than a phenotypic consequence of old age. PMID:27922819

  5. Modulation of DNA-Induced Damage and Repair Capacity in Humans after Dietary Intervention with Lutein-Enriched Fermented Milk

    PubMed Central

    Herrero-Barbudo, Carmen; Soldevilla, Beatriz; Pérez-Sacristán, Belén; Blanco-Navarro, Inmaculada; Herrera, Mercedes; Granado-Lorencio, Fernando; Domínguez, Gemma

    2013-01-01

    Dietary factors provide protection against several forms of DNA damage. Additionally, consumer demand for natural products favours the development of bioactive food ingredients with health benefits. Lutein is a promising biologically active component in the food industry. The EFSA Panel on Dietetic Products, Nutrition and Allergies considers that protection from oxidative damage may be a beneficial physiological effect but that a cause and effect relationship has not been established. Thus, our aim was to evaluate the safety and potential functional effect of a lutein-enriched milk product using the Comet Assay in order to analyze the baseline, the induced DNA-damage and the repair capacity in the lymphocytes of 10 healthy donors before and after the intake of the mentioned product. Our data suggest that the regular consumption of lutein-enriched fermented milk results in a significant increase in serum lutein levels and this change is associated with an improvement in the resistance of DNA to damage and the capacity of DNA repair in lymphocytes. Our results also support the lack of a genotoxic effect at the doses supplied as well as the absence of interactions and side effects on other nutritional and biochemicals markers. PMID:24040187

  6. Modulation of DNA-induced damage and repair capacity in humans after dietary intervention with lutein-enriched fermented milk.

    PubMed

    Herrero-Barbudo, Carmen; Soldevilla, Beatriz; Pérez-Sacristán, Belén; Blanco-Navarro, Inmaculada; Herrera, Mercedes; Granado-Lorencio, Fernando; Domínguez, Gemma

    2013-01-01

    Dietary factors provide protection against several forms of DNA damage. Additionally, consumer demand for natural products favours the development of bioactive food ingredients with health benefits. Lutein is a promising biologically active component in the food industry. The EFSA Panel on Dietetic Products, Nutrition and Allergies considers that protection from oxidative damage may be a beneficial physiological effect but that a cause and effect relationship has not been established. Thus, our aim was to evaluate the safety and potential functional effect of a lutein-enriched milk product using the Comet Assay in order to analyze the baseline, the induced DNA-damage and the repair capacity in the lymphocytes of 10 healthy donors before and after the intake of the mentioned product. Our data suggest that the regular consumption of lutein-enriched fermented milk results in a significant increase in serum lutein levels and this change is associated with an improvement in the resistance of DNA to damage and the capacity of DNA repair in lymphocytes. Our results also support the lack of a genotoxic effect at the doses supplied as well as the absence of interactions and side effects on other nutritional and biochemicals markers.

  7. Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes

    SciTech Connect

    Jones, I M; Thomas, C B; Xi, T; Mohrenweiser, H W; Nelson, D O

    2006-07-03

    Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by Random Forests Regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach 6 SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate

  8. Evaluation of kidney repair capacity using 99mTc-DMSA in ischemia/reperfusion injury models.

    PubMed

    Kwak, Wonjung; Jang, Hee-Seong; Belay, Takele; Kim, Jinu; Ha, Yeong Su; Lee, Sang Woo; Ahn, Byeong-Cheol; Lee, Jaetae; Park, Kwon Moo; Yoo, Jeongsoo

    2011-03-04

    Quantitative (99m)Tc-DMSA renal uptake was studied in different renal ischemia/reperfusion (I/R) mice models for the assessment of renal repair capacity. Mice models of nephrectomy, uni- and bi-lateral I/R together with sham-operated mice were established. At 1h, 1d, 4d, 1, 2 and 3 wk after I/R, (99m)Tc-DMSA (27.7 ± 1.3 MBq) was injected via tail vein and after 3h post-injection, the mice were scanned for 30 min with pinhole equipped gamma camera. Higher uptake of (99m)Tc-DMSA was measured in normal kidneys of uni-lateral I/R model and nephrectomized kidney I/R model at 3 wk post-surgery. Comparing the restoration capacities of the affected kidneys of nephrectomy, uni- and bi-lateral I/R models, higher repair capacity was observed in the nephrectomized model followed by bi-lateral then uni-lateral models. The normal kidney may retard the restoration of damaged kidney in uni-lateral I/R model. Moreover, 3 wk after Uni-I/R, the size of injured kidney was significantly smaller than non-ischemic contralateral and sham operated kidneys, while nephrectomy I/R kidneys were significantly enlarged compared to all others at 3 wk post-surgery. Very strong correlation between (99m)Tc-DMSA uptake and weight of dissected kidneys in I/R models was observed. Consistent with (99m)Tc-DMSA uptake results, all histological results indicate that kidney recovery after injury is correlated with the amount of intact tubules and kidney sizes. In summary, our study showed good potentials of (99m)Tc-DMSA scan as a promising non-invasive method for evaluation of kidney restoration after I/R injuries. Interestingly, mice with Bi-I/R injury showed faster repair capacity than those with uni-I/R.

  9. On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

    PubMed

    Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

    2011-01-02

    The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.

  10. Re-establishment of nucleosome occupancy during double-strand break repair in budding yeast.

    PubMed

    Tsabar, Michael; Hicks, Wade M; Tsaponina, Olga; Haber, James E

    2016-11-01

    Homologous recombination (HR) is an evolutionarily conserved pathway in eukaryotes that repairs a double-strand break (DSB) by copying homologous sequences from a sister chromatid, a homologous chromosome or an ectopic location. Recombination is challenged by the packaging of DNA into nucleosomes, which may impair the process at many steps, from resection of the DSB ends to the re-establishement of nucleosomes after repair. However, nucleosome dynamics during DSB repair have not been well described, primarily because of a lack of well-ordered nucleosomes around a DSB. We designed a system in budding yeast Saccharomyces cerevisiae to monitor nucleosome dynamics during repair of an HO endonuclease-induced DSB. Nucleosome occupancy around the break is lost following DSB formation, by 5'-3' resection of the DSB end. Soon after repair is complete, nucleosome occupancy is partially restored in a repair-dependent but cell cycle-independent manner. Full re-establishment of nucleosome protection back to the level prior to DSB induction is achieved when the cell cycle resumes following repair. These findings may have implications to the mechanisms by which cells sense the completion of repair.

  11. Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems.

    PubMed

    Bocian-Ostrzycka, Katarzyna M; Grzeszczuk, Magdalena J; Dziewit, Lukasz; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    The bacterial proteins of the Dsb family-important components of the post-translational protein modification system-catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp.

  12. Structure of a DsbF homologue from Corynebacterium diphtheriae

    PubMed Central

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-01-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein. PMID:25195886

  13. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    PubMed

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  14. Limited integrative repair capacity of native cartilage autografts within cartilage defects in a sheep model.

    PubMed

    Gelse, Kolja; Riedel, Dominic; Pachowsky, Milena; Hennig, Friedrich F; Trattnig, Siegfried; Welsch, Götz H

    2015-03-01

    The purpose of this study was to investigate integration and cellular outgrowth of native cartilage autografts transplanted into articular cartilage defects. Native cartilage autografts were applied into chondral defects in the femoral condyle of adult sheep. Within the defects, the calcified cartilage layer was either left intact or perforated to induce bone marrow stimulation. Empty defects served as controls. The joints were analyzed after 6 and 26 weeks by macroscopic and histological analysis using the ICRS II Score and Modified O'Driscoll Scores. Non-treated defects did not show any endogenous regenerative response and bone marrow stimulation induced fibrous repair tissue. Transplanted native cartilage grafts only insufficiently integrated with the defect borders. Cell death and loss of proteoglycans were present at the margins of the grafts at 6 weeks, which was only partially restored at 26 weeks. Significant cellular outgrowth from the grafts or defect borders could not be observed. Bonding of the grafts could be improved by additional bone marrow stimulation providing ingrowing cells that formed a fibrous interface predominantly composed of type I collagen. Transplanted native cartilage grafts remain as inert structures within cartilage defects and fail to induce integrative cartilage repair which rather demands additional cells provided by additional bone marrow stimulation. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  15. Maintenance of xylem network transport capacity: a review of embolism repair in vascular plants

    USDA-ARS?s Scientific Manuscript database

    Maintenance of long distance water transport in xylem is essential to plant health and productivity. Both biotic and abiotic environmental conditions lead to embolism formation within the xylem resulting in lost transport capacity and ultimately death following systemic spread. Plants exhibit a vari...

  16. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    PubMed

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  17. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability

    PubMed Central

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-01-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. PMID:25287622

  18. Exploring the potential interference of estuarine sediment contaminants with the DNA repair capacity of human hepatoma cells.

    PubMed

    Pinto, Miguel Ferreira; Louro, Henriqueta; Costa, Pedro M; Caeiro, Sandra; Silva, Maria João

    2015-01-01

    Estuaries may be reservoirs of a wide variety of pollutants, including mutagenic and carcinogenic substances that may impact on the ecosystem and human health. A previous study showed that exposure of human hepatoma (HepG2) cells to extracts from sediment samples collected in two areas (urban/industrial and riverine/agricultural) of an impacted estuary (Sado, Portugal), produced differential cytotoxic and genotoxic effects. Those effects were found to be consistent with levels and nature of sediment contamination. The present study aimed at evaluating whether the mixtures of contaminants contained in those extracts were able to modulate DNA repair capacity of HepG2 cells. The residual level of DNA damage was measured by the comet assay in cells exposed for 24 or 48 h to different extracts, after a short preexposure to a challenging concentration range of ethyl methanesulfonate (EMS), as a model alkylating agent. The results suggested that the mixture of contaminants present in the tested samples, besides a potential direct effect on the DNA molecule, may also interfere with DNA repair mechanisms in HepG2 cells, thus impairing their ability to deal with genotoxic stress and, possibly, facilitating accumulation of mutations. Humans are environmentally/occupationally exposed to mixtures rather than to single chemicals. Thus, the observation that estuarine contaminants induce direct and indirect DNA strand breakage in human cells, the latter through the impairment of DNA repair, raises additional concerns regarding potential hazards from exposure and the need to further explore these endpoints in the context of environmental risk assessment.

  19. A novel strategy to enhance mesenchymal stem cell migration capacity and promote tissue repair in an injury specific fashion.

    PubMed

    Xinaris, C; Morigi, M; Benedetti, V; Imberti, B; Fabricio, A S; Squarcina, E; Benigni, A; Gagliardini, E; Remuzzi, G

    2013-01-01

    Mesenchymal stem cells (MSCs) of bone marrow origin appear to be an attractive candidate for cell-based therapies. However, the major barrier to the effective implementation of MSC-based therapies is the lack of specific homing of exogenously infused cells and overall the inability to drive them to the diseased or damaged tissue. In order to circumvent these limitations, we developed a preconditioning strategy to optimize MSC migration efficiency and potentiate their beneficial effect at the site of injury. Initially, we screened different molecules by using an in vitro injury-migration setting, and subsequently, we evaluated the effectiveness of the different strategies in mice with acute kidney injury (AKI). Our results showed that preconditioning of MSCs with IGF-1 before infusion improved cell migration capacity and restored normal renal function after AKI. The present study demonstrates that promoting migration of MSCs could increase their therapeutic potential and indicates a new therapeutic paradigm for organ repair.

  20. Expression and crystallization of DsbA from Staphylococcus aureus

    SciTech Connect

    Heras, B. Kurz, M.; Jarrott, R.; Byriel, K. A.; Jones, A.; Thöny-Meyer, L.; Martin, J. L.

    2007-11-01

    Free-interface diffusion crystallization chips were used to identify crystallization conditions for S. aureus DsbA, representing the first Gram-positive DsbA to be crystallized. Native and selenomethionine-derivative crystals diffracted to 2.1 and 2.4 Å resolution, respectively. Bacterial Dsb proteins catalyse the in vivo formation of disulfide bonds, a critical step in the stability and activity of many proteins. Most studies on Dsb proteins have focused on Gram-negative bacteria and thus the process of oxidative folding in Gram-positive bacteria is poorly understood. To help elucidate this process in Gram-positive bacteria, DsbA from Staphylococcus aureus (SaDsbA) has been focused on. Here, the expression, purification, crystallization and preliminary diffraction analysis of SaDsbA are reported. SaDsbA crystals diffract to a resolution limit of 2.1 Å and belong to the hexagonal space group P6{sub 5} or P6{sub 1}, with unit-cell parameters a = b = 72.1, c = 92.1 Å and one molecule in the asymmetric unit (64% solvent content)

  1. Preferential binding of an unfolded protein to DsbA.

    PubMed Central

    Frech, C; Wunderlich, M; Glockshuber, R; Schmid, F X

    1996-01-01

    The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. PMID:8617214

  2. Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

    PubMed Central

    Headey, Stephen J.; Vazirani, Mansha; Shouldice, Stephen R.; Coinçon, Mathieu; Tay, Stephanie; Morton, Craig J.; Simpson, Jamie S.; Martin, Jennifer L.

    2017-01-01

    At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors. PMID:28346540

  3. Chromosome position determines the success of double-strand break repair.

    PubMed

    Lee, Cheng-Sheng; Wang, Ruoxi W; Chang, Hsiao-Han; Capurso, Daniel; Segal, Mark R; Haber, James E

    2016-01-12

    Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5' to 3' resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor.

  4. 53BP1 fosters fidelity of homology-directed DNA repair.

    PubMed

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Jiri; Lukas, Claudia

    2016-08-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.

  5. Meiotic DSB patterning: A multifaceted process

    PubMed Central

    Cooper, Tim J.; Garcia, Valerie; Neale, Matthew J.

    2016-01-01

    Abstract Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control—spatial regulation—detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed. PMID:26730703

  6. Meiotic DSB patterning: A multifaceted process.

    PubMed

    Cooper, Tim J; Garcia, Valerie; Neale, Matthew J

    2016-01-01

    Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control--spatial regulation--detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed.

  7. On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA.

    PubMed

    Luo, Man; Guan, Yi-Xin; Yao, Shan-Jing

    2011-10-15

    DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.

  8. Sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts.

    PubMed

    Koyanagi, Masamichi; Iwasaki, Masayoshi; Rupp, Stefan; Tedesco, Francesco Saverio; Yoon, Chang-Hwan; Boeckel, Jes-Niels; Trauth, Janina; Schütz, Corina; Ohtani, Kisho; Goetz, Rebekka; Iekushi, Kazuma; Bushoven, Philipp; Momma, Stefan; Mummery, Christine; Passier, Robert; Henschler, Reinhard; Akintuerk, Hakan; Schranz, Dietmar; Urbich, Carmen; Galvez, Beatriz G; Cossu, Giulio; Zeiher, Andreas M; Dimmeler, Stefanie

    2010-04-16

    Complementation of pluripotency genes may improve adult stem cell functions. Here we show that clonally expandable, telomerase expressing progenitor cells can be isolated from peripheral blood of children. The surface marker profile of the clonally expanded cells is distinct from hematopoietic or mesenchymal stromal cells, and resembles that of embryonic multipotent mesoangioblasts. Cell numbers and proliferative capacity correlated with donor age. Isolated circulating mesoangioblasts (cMABs) express the pluripotency markers Klf4, c-Myc, as well as low levels of Oct3/4, but lack Sox2. Therefore, we tested whether overexpression of Sox2 enhances pluripotency and facilitates differentiation of cMABs in cardiovascular lineages. Lentiviral transduction of Sox2 (Sox-MABs) enhanced the capacity of cMABs to differentiate into endothelial cells and cardiomyocytes in vitro. Furthermore, the number of smooth muscle actin positive cells was higher in Sox-MABs. In addition, pluripotency of Sox-MABs was shown by demonstrating the generation of endodermal and ectodermal progenies. To test whether Sox-MABs may exhibit improved therapeutic potential, we injected Sox-MABs into nude mice after acute myocardial infarction. Four weeks after cell therapy with Sox-MABs, cardiac function was significantly improved compared to mice treated with control cMABs. Furthermore, cell therapy with Sox-MABs resulted in increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. The complementation of Sox2 in Oct3/4-, Klf4-, and c-Myc-expressing cMABs enhanced the differentiation into all 3 cardiovascular lineages and improved the functional recovery after acute myocardial infarction.

  9. Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

    SciTech Connect

    Vivian, Julian P.; Scoullar, Jessica; Robertson, Amy L.; Bottomley, Stephen P.; Horne, James; Chin, Yanni; Wielens, Jerome; Thompson, Philip E.; Velkov, Tony; Piek, Susannah; Byres, Emma; Beddoe, Travis; Wilce, Matthew C.J.; Kahler, Charlene M.; Rossjohn, Jamie; Scanlon, Martin J.

    2009-09-02

    DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-{angstrom} resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

  10. Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters

    SciTech Connect

    Fertil, B.; Deschavanne, P.J.; Debieu, D.; Malaise, E.P.

    1988-10-01

    Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.

  11. The repair capacity of lung cancer cell lines A549 and H1299 depends on HMGB1 expression level and the p53 status.

    PubMed

    Yusein-Myashkova, Shazie; Stoykov, Ivan; Gospodinov, Anastas; Ugrinova, Iva; Pasheva, Evdokia

    2016-07-01

    Elucidation of the cellular components responsive to chemotherapeutic agents as cisplatin rationalizes the strategy for anticancer chemotherapy. The removal of the cisplatin/DNA lesions gives the chance to the cancer cells to survive and compromises the chemotherapeutical treatment. Therefore, the cell repair efficiency is substantial for the clinical outcome. High mobility group box 1 (HMGB1) protein is considered to be involved in the removal of the lesions as it binds with high affinity to cisplatin/DNA adducts. We demonstrated that overexpression of HMGB1 protein inhibited cis-platinated DNA repair in vivo and the effect strongly depended on its C-terminus. We registered increased levels of DNA repair after HMGB1 silencing only in p53 defective H1299 lung cancer cells. Next, introduction of functional p53 resulted in DNA repair inhibition. H1299 cells overexpressing HMGB1 were significantly sensitized to treatment with cisplatin demonstrating the close relation between the role of HMGB1 in repair of cis-platinated DNA and the efficiency of the anticancer drug, the process being modulated by the C-terminus. In A549 cells with functional p53, the repair of cisplatin/DNA adducts is determined by а complex action of HMGB1 and p53 as an increase of DNA repair capacity was registered only after silencing of both proteins.

  12. Recombinational DNA repair in a cellular context: a search for the homology search.

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

    Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.

  13. 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.

  14. 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.

  15. Suboptimal DNA repair capacity predisposes coke-oven workers to accumulate more chromosomal damages in peripheral lymphocytes.

    PubMed

    Cheng, Juan; Leng, Shuguang; Li, Haishan; Huang, Chuanfeng; Niu, Yong; Zhang, Linyuan; Liang, Xuemiao; Lin, Han; Zheng, Yuxin

    2009-03-01

    DNA repair is an essential mechanism for cells to maintain their genomic integrity under endogenous or exogenous assault. Reduced DNA repair capacity (DRC) is associated with increased risk for several environmentally related cancers. The micronucleus in peripheral lymphocytes has been validated as a biomarker of chromosomal damage, increasing cancer risk in human populations. We hypothesized that suboptimal DRC is associated with the increase in chromosomal damage among 94 coke-oven workers and 64 noncoke-oven controls. DRC was evaluated in isolated lymphocytes by comet assay. Chromosomal damage in peripheral lymphocytes was detected by cytokinesis-block micronucleus assay. Four common coding single nucleotide polymorphisms in the XRCC1 gene were genotyped. Coke-oven workers have significantly increased urinary 1-hydroxypyrene (9.0; 6.8-11.7 microg/L versus 1.5, 1.3-1.7 microg/L; P<0.01) and micronucleus frequency (7.4 per thousand+/-4.3 per thousand versus 3.0 per thousand+/-3.0 per thousand; P<0.01), and decreased DRC (55.9%+/-16.4% versus 63.6%+/-18.5%; P<0.01) compared with controls. Significant correlations between DRC and micronucleus frequency were found in coke-oven workers (r=-0.32; P<0.01; n = 94) and all study subjects (r=-0.32; P<0.001; n=158) but not in controls (r=-0.21; P=0.11; n=64). Variants of the Arg399Gln polymorphism were associated with a decreased DRC in both coke-oven workers (51.6%+/-16.1% versus 60.6%+/-15.7%; P<0.01) and controls (59.1%+/-18.5% versus 68.4%+/-17.5%; P=0.04). The complicated interrelationship of these multiple biomarkers was also identified by path analysis. These findings should facilitate developing a biomarker-based risk assessment model for lung cancer in this occupational population.

  16. Crossover Invariance Determined by Partner Choice for Meiotic DNA Break Repair

    PubMed Central

    Hyppa, Randy W.; Smith, Gerald R.

    2010-01-01

    SUMMARY Crossovers between meiotic homologs are crucial for their proper segregation, and crossover number and position are carefully controlled. Crossover homeostasis in budding yeast maintains crossovers at the expense of non-crossovers when double-strand DNA break (DSB) frequency is reduced. The mechanism of maintaining constant crossover levels in other species has been unknown. Here we investigate in fission yeast a different aspect of crossover control – the near invariance of crossover frequency per kb of DNA despite large variations in DSB intensity across the genome. Crossover invariance involves the choice of sister chromatid vs. homolog for DSB repair. At strong DSB hotspots, intersister repair outnumbers interhomolog repair ~3:1, but our genetic and physical data indicate the converse in DSB-cold regions. This unanticipated mechanism of crossover control may operate in many species and explain, for example, the large excess of DSBs over crossovers and the repair of DSBs on unpaired chromosomes in diverse species. PMID:20655467

  17. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    PubMed

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  18. The role of the Mre11-Rad50-Nbs1 complex in double-strand break repair-facts and myths.

    PubMed

    Takeda, Shunichi; Hoa, Nguyen Ngoc; Sasanuma, Hiroyuki

    2016-08-01

    Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5'-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51-DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation-induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution.

  19. Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocytes during 36 weeks in rabbit model.

    PubMed

    Kazemnejad, Somaieh; Khanmohammadi, Manijeh; Mobini, Sahba; Taghizadeh-Jahed, Masoud; Khanjani, Sayeh; Arasteh, Shaghayegh; Golshahi, Hannaneh; Torkaman, Giti; Ravanbod, Roya; Heidari-Vala, Hamed; Moshiri, Ali; Tahmasebi, Mohammad-Naghi; Akhondi, Mohammad-Mehdi

    2016-06-01

    The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies.

  20. Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena.

    PubMed

    Lukaszewicz, Agnieszka; Shodhan, Anura; Loidl, Josef

    2015-11-01

    The resection of 5'-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3' single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3'-5' direction and Exo1 in the opposite 5'-3' direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line.

    PubMed

    Borràs-Fresneda, Mireia; Barquinero, Joan-Francesc; Gomolka, Maria; Hornhardt, Sabine; Rössler, Ute; Armengol, Gemma; Barrios, Leonardo

    2016-06-01

    Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

  2. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line

    PubMed Central

    Borràs-Fresneda, Mireia; Barquinero, Joan-Francesc; Gomolka, Maria; Hornhardt, Sabine; Rössler, Ute; Armengol, Gemma; Barrios, Leonardo

    2016-01-01

    Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols. PMID:27245205

  3. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

    SciTech Connect

    Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

    2010-11-01

    Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m{sup 3}) and high (above 50 mg/m{sup 3}) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 {+-} 1.00 SSB/10{sup 9} Da), followed by high exposure group (0.72 {+-} 0.81 SSB/10{sup 9} Da) and controls (0.65 {+-} 0.82 SSB/10{sup 9} Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

  4. Non-DSB clustered DNA lesions. Does theory colocalize with the experiment?

    NASA Astrophysics Data System (ADS)

    Nikitaki, Zacharenia; Nikolov, Vladimir; Mavragani, Ifigeneia V.; Plante, Ianik; Emfietzoglou, Dimitris; Iliakis, George; Georgakilas, Alexandros G.

    2016-11-01

    Ionizing radiation results in various kinds of DNA lesions such as double strand breaks (DSBs) and other non-DSB base lesions. These lesions may be formed in close proximity (i.e., within a few nanometers) resulting in clustered types of DNA lesions. These damage clusters are considered the fingerprint of ionizing radiation, notably charged particles of high linear energy transfer (LET). Accumulating theoretical and experimental evidence suggests that the induction of these clustered lesions appears under various irradiation conditions but also as a result of high levels of oxidative stress. The biological significance of these clustered DNA lesions pertains to the inability of cells to process them efficiently compared to isolated DNA lesions. The results in the case of unsuccessful or erroneous repair can vary from mutations up to chromosomal instability. In this mini review, we discuss of several Monte Carlo simulations codes and experimental evidence regarding the induction and repair of radiation-induced non-DSB complex DNA lesions. We also critically present the most widely used methodologies (i.e., gel electrophoresis and fluorescence microscopy [in situ colocalization assays]). Based on the comparison of different approaches, we provide examples and suggestions for the improved detection of these lesions in situ. Based on the current status of knowledge, we conclude that there is a great need for improvement of the detection techniques at the cellular or tissue level, which will provide valuable information for understanding the mechanisms used by the cell to process clustered DNA lesions.

  5. The GH/IGF-1 axis in a critical period early in life determines cellular DNA repair capacity by altering transcriptional regulation of DNA repair-related genes: implications for the developmental origins of cancer.

    PubMed

    Podlutsky, Andrej; Valcarcel-Ares, Marta Noa; Yancey, Krysta; Podlutskaya, Viktorija; Nagykaldi, Eszter; Gautam, Tripti; Miller, Richard A; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

    2017-02-23

    Experimental, clinical, and epidemiological findings support the concept of developmental origins of health and disease (DOHAD), suggesting that early-life hormonal influences during a sensitive period around adolescence have a powerful impact on cancer morbidity later in life. The endocrine changes that occur during puberty are highly conserved across mammalian species and include dramatic increases in circulating GH and IGF-1 levels. Importantly, patients with developmental IGF-1 deficiency due to GH insensitivity (Laron syndrome) do not develop cancer during aging. Rodents with developmental GH/IGF-1 deficiency also exhibit significantly decreased cancer incidence at old age, marked resistance to chemically induced carcinogenesis, and cellular resistance to genotoxic stressors. Early-life treatment of GH/IGF-1-deficient mice and rats with GH reverses the cancer resistance phenotype; however, the underlying molecular mechanisms remain elusive. The present study was designed to test the hypothesis that developmental GH/IGF-1 status impacts cellular DNA repair mechanisms. To achieve that goal, we assessed repair of γ-irradiation-induced DNA damage (single-cell gel electrophoresis/comet assay) and basal and post-irradiation expression of DNA repair-related genes (qPCR) in primary fibroblasts derived from control rats, Lewis dwarf rats (a model of developmental GH/IGF-1 deficiency), and GH-replete dwarf rats (GH administered beginning at 5 weeks of age, for 30 days). We found that developmental GH/IGF-1 deficiency resulted in persisting increases in cellular DNA repair capacity and upregulation of several DNA repair-related genes (e.g., Gadd45a, Bbc3). Peripubertal GH treatment reversed the radiation resistance phenotype. Fibroblasts of GH/IGF-1-deficient Snell dwarf mice also exhibited improved DNA repair capacity, showing that the persisting influence of peripubertal GH/IGF-1 status is not species-dependent. Collectively, GH/IGF-1 levels during a critical period

  6. Simulation of DSB yield for high LET radiation.

    PubMed

    Friedrich, T; Durante, M; Scholz, M

    2015-09-01

    A simulation approach for the calculation of the LET-dependent yield of double-strand breaks (DSB) is presented. The model considers DSB formed as two close-lying single-strand breaks (SSB), whose formation is mediated by both intra-track processes (single electrons) or at local doses larger than about 1000 Gy in particle tracks also by electron inter-track processes (two independent electron tracks). A Monte Carlo algorithm and an analytical formula for the DSB yield are presented. The approach predicts that the DSB yield is enhanced after charged particle irradiation of high LET compared with X-ray or gamma radiation. It is used as an inherent part of the local effect model, which is applied to estimate the relative biological effectiveness of high LET radiation.

  7. DNA double strand break (DSB) induction and cell survival in iodine-enhanced computed tomography (CT)

    NASA Astrophysics Data System (ADS)

    Streitmatter, Seth W.; Stewart, Robert D.; Jenkins, Peter A.; Jevremovic, Tatjana

    2017-08-01

    A multi-scale Monte Carlo model is proposed to assess the dosimetric and biological impact of iodine-based contrast agents commonly used in computed tomography. As presented, the model integrates the general purpose MCNP6 code system for larger-scale radiation transport and dose assessment with the Monte Carlo damage simulation to determine the sub-cellular characteristics and spatial distribution of initial DNA damage. The repair-misrepair-fixation model is then used to relate DNA double strand break (DSB) induction to reproductive cell death. Comparisons of measured and modeled changes in reproductive cell survival for ultrasoft characteristic k-shell x-rays (0.25-4.55 keV) up to orthovoltage (200-500 kVp) x-rays indicate that the relative biological effectiveness (RBE) for DSB induction is within a few percent of the RBE for cell survival. Because of the very short range of secondary electrons produced by low energy x-ray interactions with contrast agents, the concentration and subcellular distribution of iodine within and near cellular targets have a significant impact on the estimated absorbed dose and number of DSB produced in the cell nucleus. For some plausible models of the cell-level distribution of contrast agent, the model predicts an increase in RBE-weighted dose (RWD) for the endpoint of DSB induction of 1.22-1.40 for a 5-10 mg ml-1 iodine concentration in blood compared to an RWD increase of 1.07  ±  0.19 from a recent clinical trial. The modeled RWD of 2.58  ±  0.03 is also in good agreement with the measured RWD of 2.3  ±  0.5 for an iodine concentration of 50 mg ml-1 relative to no iodine. The good agreement between modeled and measured DSB and cell survival estimates provides some confidence that the presented model can be used to accurately assess biological dose for other concentrations of the same or different contrast agents.

  8. The dipeptidyl peptidase-4 inhibitor vildagliptin has the capacity to repair β-cell dysfunction and insulin resistance.

    PubMed

    Horie, A; Tokuyama, Y; Ishizuka, T; Suzuki, Y; Marumo, K; Oshikiri, K; Ide, K; Sunaga, M; Kanatsuka, A

    2014-10-01

    The aim of the present study was to determine whether the dipeptidyl peptidase (DPP)-4 inhibitor could repair pancreatic β-cell dysfunction and insulin resistance. Ten subjects with type 2 diabetes who had never received DPP-4 inhibitor treatment were enrolled in the study. Just before and 3 months after twice-daily administration of vildagliptin (50 mg tablets), insulin secretion and insulin sensitivity were estimated using 2-compartment model analysis of C-peptide kinetics and insulin-modified minimal model parameters, respectively. The first-phase insulin secretion (CS1) was determined as the sum of the C-peptide secretion rate (CSR) from 0 to 5 min (normal range 6.8-18.5 ng/ml/min). The whole-body insulin sensitivity index (SI) was calculated using a minimal model software program (normal range 2.6-7.6×10(-4)/min/μU/ml). After vildagliptin treatment, reductions in mean (± SE) HbA1c were noted (43.28±1.53 vs. 40.98±1.77 mmol/mol; p=0.019). Vildagliptin treatment increased the area under the curve for the C peptide reactivity (CPR) (AUCCPR; 26.66±5.15 vs. 33.02±6.12 ng/ml · 20 min; p=0.003) and CS1 (0.80±0.20 vs. 1.35±0.38 ng/ml/min; p=0.037) in response to an intravenous glucose load. -Vildagliptin treatment significantly increased SI (0.46±0.27 vs. 1.21±0.48×10(-4)/min/μU/ml; p=0.037). The long-term administration of vildagliptin improved CS1 and Si suggesting that this drug has the capacity to repair impairments in pancreatic β-cell function and insulin resistance in type 2 diabetes.

  9. Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways.

    PubMed

    Sears, Catherine R; Turchi, John J

    2012-07-13

    The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.

  10. Differential promoter methylation of kinesin family member 1a in plasma is associated with breast cancer and DNA repair capacity

    PubMed Central

    GUERRERO-PRESTON, RAFAEL; HADAR, TAL; OSTROW, KIMBERLY LASKIE; SOUDRY, ETHAN; ECHENIQUE, MIGUEL; ILI-GANGAS, CARMEN; PÉREZ, GABRIELA; PEREZ, JIMENA; BREBI-MIEVILLE, PRISCILLA; DESCHAMPS, JOSÉ; MORALES, LUISA; BAYONA, MANUEL; SIDRANSKY, DAVID; MATTA, JAIME

    2014-01-01

    Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts. PMID:24927296

  11. Differential promoter methylation of kinesin family member 1a in plasma is associated with breast cancer and DNA repair capacity.

    PubMed

    Guerrero-Preston, Rafael; Hadar, Tal; Ostrow, Kimberly Laskie; Soudry, Ethan; Echenique, Miguel; Ili-Gangas, Carmen; Pérez, Gabriela; Perez, Jimena; Brebi-Mieville, Priscilla; Deschamps, José; Morales, Luisa; Bayona, Manuel; Sidransky, David; Matta, Jaime

    2014-08-01

    Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥ 0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts.

  12. Effects of a new antibacterial adhesive on the repair capacity of the pulp-dentine complex in infected teeth.

    PubMed

    Tziafas, D; Koliniotou-Koumpia, E; Tziafa, C; Papadimitriou, S

    2007-01-01

    To evaluate the effects of a self-etching/priming adhesive system, containing the antibacterial monomer 12-methacryloyloxy-dodecylpyridinium bromide (MDPB), on the repair capacity of the pulp-dentine complex in infected cavities in dog's teeth. Class V cavities with a residual dentine thickness ranging from 0.3-0.8 mm were prepared on the buccal surface of permanent teeth in four dogs. Pulpal exposures were performed in half of the cavities. Millipore filters that had been incubated for 3 h in a 10(5) milky suspension of a-streptococci were placed in the cavities, which were then filled temporarily. After 24 h, the filters were removed and both the exposed and non-exposed cavities were washed with sterile saline and assigned to four groups which were treated with either the experimental antibacterial adhesive system, or Clearfil SE bond, Dycal and Teflon discs. Stereotype connective tissue reactions (inflammatory cell response and/or tissue necrosis) and pulp-specific reparative tissue responses (reduction of odontoblasts and tertiary dentine formation) were assessed at post-operative periods of 4 and 8 weeks. Neither severe inflammation nor tissue necrosis was observed, either in the dentinal cavities or pulpal exposures treated with the self-etch adhesive containing MDPB. Rates of tertiary dentine formation in infected dentinal cavities treated with this system were comparable with those observed after dentine treatment with the Ca(OH)2-based material. Dentinal bridging was not seen in pulpal exposures treated with the experimental adhesive. The new antibacterial adhesive system maintained pulp vitality and primary odontoblastic function in infected nonexposed and exposed cavities but interfered with reparative dentine formation in infected pulpal exposures.

  13. Spectrum of Radiation-Induced Clustered Non-DSB Damage - A Monte Carlo Track Structure Modeling and Calculations.

    PubMed

    Watanabe, Ritsuko; Rahmanian, Shirin; Nikjoo, Hooshang

    2015-05-01

    The aim of this report is to present the spectrum of initial radiation-induced cellular DNA damage [with particular focus on non-double-strand break (DSB) damage] generated by computer simulations. The radiation types modeled in this study were monoenergetic electrons (100 eV-1.5 keV), ultrasoft X-ray photons Ck, AlK and TiK, as well as some selected ions including 3.2 MeV/u proton; 0.74 and 2.4 MeV/u helium ions; 29 MeV/u nitrogen ions and 950 MeV/u iron ions. Monte Carlo track structure methods were used to simulate damage induction by these radiation types in a cell-mimetic condition from a single-track action. The simulations took into account the action of direct energy deposition events and the reaction of hydroxyl radicals on atomistic linear B-DNA segments of a few helical turns including the water of hydration. Our results permitted the following conclusions: a. The absolute levels of different types of damage [base damage, simple and complex single-strand breaks (SSBs) and DSBs] vary depending on the radiation type; b. Within each damage class, the relative proportions of simple and complex damage vary with radiation type, the latter being higher with high-LET radiations; c. Overall, for both low- and high-LET radiations, the ratios of the yields of base damage to SSBs are similar, being about 3.0 ± 0.2; d. Base damage contributes more to the complexity of both SSBs and DSBs, than additional SSB damage and this is true for both low- and high-LET radiations; and e. The average SSB/DSB ratio for low-LET radiations is about 18, which is about 5 times higher than that for high-LET radiations. The hypothesis that clustered DNA damage is more difficult for cells to repair has gained currency among radiobiologists. However, as yet, there is no direct in vivo experimental method to validate the dependence of kinetics of DNA repair on DNA damage complexity (both DSB and non-DSB types). The data on the detailed spectrum of DNA damage presented here, in particular

  14. β-Cyclodextrin-Linked Polyethylenimine Nanoparticles Facilitate Gene Transfer and Enhance the Angiogenic Capacity of Mesenchymal Stem Cells for Wound Repair and Regeneration.

    PubMed

    Peng, Li-Hua; Wei, Wei; Shan, Ying-Hui; Zhang, Tian-Yuan; Zhang, Chen-Zhen; Wu, Jia-He; Yu, Lian; Lin, Jun; Liang, Wen-Quan; Khang, Gilson; Gao, Jian-Qing

    2015-04-01

    Repair of deep wounds by cell transplantation strongly depends on angiogenesis and on the regeneration of skin and appendages. In this study, plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165) was transduced into bone-marrow mesenchymal stem cells (MSCs) using a nonviral vector, β-cyclodextrin-linked polyethylenimine, to enhance angiogenic capacity. The effects of MSCs administered by intradermal injection or transplantation on wound closure were compared in a full-thickness excision wound model. The results showed that the MSC-seeded sponge had significantly stronger acceleration in wound closure than the MSC injection. The effects on wound repair and regeneration of transplanted MSCs and pDNA-VEGF1 65-transfected MSCs (TMSCs) with gelatin/β-tricalcium phosphate scaffold were also investigated. Compared with MSC transplantation, TMSC transplantation showed higher efficacy in stimulating wound closure, promoting dermal collagen synthesis and regulating the deposition of newly formed collagen. In addition, the angiogenic capacity of the TMSCs was higher than that of the MSCs. The results indicate that the nonviral genetic engineering of the MSCs is a promising strategy to enhance the angiogenic capacity of MSCs for wound repair and angiogenesis. Functional gene-activated MSCs may be used as cost-effective and accessible seed cells for skin tissue engineering and as novel carriers for wound gene therapy.

  15. DNA-DSB in CHO-K1 cells induced by heavy-ions: Break rejoining and residual damage (GSI)

    NASA Technical Reports Server (NTRS)

    Taucher-Scholz, G.; Heilmann, J.; Becher, G.; Kraft, G.

    1994-01-01

    DNA double strand breaks (DSB's) are the critical lesions involved in cellular effects of ionizing radiation. Therefore, the evaluation of DSB induction in mammalian cells after heavy ion irradiation is an essential task for the assessment of high-LET radiation risk in space. Of particular interest has been the question of how the biological efficiency for the cellular inactivation endpoint relates to the initial lesions (DSBs) at varying LETs. For cell killing, an increased Relative Biological Efficiency (RBE) has been determined for highLET radiation around 100-200 keV/mu m. At higher LET, the RBE's decrease again to values below one for the very heavy particles. At GSI, DSB-induction was measured in CHO-K1 cells following irradiation with accelerated particles covering a wide LET range. The electrophoretic elution of fragmented DNA out of agarose plugs in a constant electrical field was applied for the detection of DSB's. The fraction of DNA retained was determined considering the relative intensities of ethidium bromide fluorescence in the well and in the gel lane. Dose-effect curves were established, from which the RBE for DSB induction was calculated at a fraction of 0.7 of DNA retained In summary, these rejoining studies are in line with an enhanced severity of the DNA DSB's at higher LET's, resulting in a decreased repairability of the induced lesions. However, no information concerning the fidelity of strand breaks rejoining is provided in these studies. To assess correct rejoining of DNA fragments an experimental system involving individual DNA hybridization bands has been set up. In preliminary experiments Sal I generated DNA fragments of 0.9 Mbp were irradiated with xrays and incubated for repair However, restitution of the original signals was not observed, probably due to the high radiation dose necessary for breakage of a fragment of this size. A banding pattern with NotI hybridization signals in a higher MW range (3Mbp) has been obtained by varying

  16. Radiosensitisation by pharmacological ascorbate in glioblastoma multiforme cells, human glial cells, and HUVECs depends on their antioxidant and DNA repair capabilities and is not cancer specific.

    PubMed

    Castro, M Leticia; McConnell, Melanie J; Herst, Patries M

    2014-09-01

    We previously showed that 5 mM ascorbate radiosensitized early passage radioresistant glioblastoma multiforme (GBM) cells derived from one patient tumor. Here we investigate the sensitivity of a panel of cell lines to 5 mM ascorbate and 6 Gy ionizing radiation, made up of three primary human GBM cells, three GBM cell lines, a human glial cell line, and primary human vascular endothelial cells. The response of different cells lines to ascorbate and/or radiation was determined by measuring viability, colony-forming ability, generation and repair of double-stranded DNA breaks (DSBs), cell cycle progression, antioxidant capacity and generation of reactive oxygen species. Individually, radiation and ascorbate both decreased viability and clonogenicity by inducing DNA damage, but had differential effects on cell cycle progression. Radiation led to G2/M arrest in most cells whereas ascorbate caused accumulation in S phase, which was moderately associated with poor DSB repair. While high dose ascorbate radiosensitized all cell lines in clonogenic assays, the sensitivity to radiation, high dose ascorbate, and combined treatment varied between cell lines. Normal glial cells were similar to GBM cells with respect to free radical scavenging potential and effect of treatment on DNA damage and repair, viability, and clonogenicity. Both GBM cells and normal cells coped equally poorly with oxidative stress caused by radiation and/or high dose ascorbate, dependent primarily on their antioxidant and DSB repair capacity.

  17. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  18. DNA double-strand break repair pathway choice in Dictyostelium.

    PubMed

    Hsu, Duen-Wei; Kiely, Rhian; Couto, C Anne-Marie; Wang, Hong-Yu; Hudson, Jessica J R; Borer, Christine; Pears, Catherine J; Lakin, Nicholas D

    2011-05-15

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

  19. How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability.

    PubMed

    Jeggo, Penny A; Löbrich, Markus

    2015-10-01

    DNA DSBs (double-strand breaks) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that, in normal cells, DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilization of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. In the present review, we evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilization of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We consider the ways by which cancers modify and hijack these processes to gain genomic instability.

  20. A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair

    PubMed Central

    Jain, Suvi; Sugawara, Neal; Lydeard, John; Vaze, Moreshwar; Tanguy Le Gac, Nicolas; Haber, James E.

    2009-01-01

    A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a “recombination execution checkpoint” (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence. PMID:19204116

  1. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

    PubMed Central

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body. PMID:25565522

  2. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

    PubMed

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

  3. 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

  4. Chromatin remodeling in DNA double-strand break repair.

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

    ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.

  5. Impaired Endothelial Repair Capacity of Early Endothelial Progenitor Cells in Hypertensive Patients With Primary Hyperaldosteronemia: Role of 5,6,7,8-Tetrahydrobiopterin Oxidation and Endothelial Nitric Oxide Synthase Uncoupling.

    PubMed

    Chen, Long; Ding, Mei-Lin; Wu, Fang; He, Wen; Li, Jin; Zhang, Xiao-Yu; Xie, Wen-Li; Duan, Sheng-Zhong; Xia, Wen-Hao; Tao, Jun

    2016-02-01

    Although hyperaldosteronemia exerts detrimental impacts on vascular endothelium in addition to elevating blood pressure, the effects and molecular mechanisms of hyperaldosteronemia on early endothelial progenitor cell (EPC)-mediated endothelial repair after arterial damage are yet to be determined. The aim of this study was to investigate the endothelial repair capacity of early EPCs from hypertensive patients with primary hyperaldosteronemia (PHA). In vivo endothelial repair capacity of early EPCs from PHAs (n=20), age- and blood pressure-matched essential hypertension patients (n=20), and age-matched healthy subjects (n=20) was evaluated by transplantation into a nude mouse carotid endothelial denudation model. Endothelial function was evaluated by flow-mediated dilation of brachial artery in human subjects. In vivo endothelial repair capacity of early EPCs and flow-mediated dilation were impaired both in PHAs and in essential hypertension patients when compared with age-matched healthy subjects; however, the early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs were impaired more severely than essential hypertension patients. Oral spironolactone improved early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs. Increased oxidative stress, oxidative 5,6,7,8-tetrahydrobiopterin degradation, endothelial nitric oxide synthase uncoupling and decreased nitric oxide production were found in early EPCs from PHAs. Nicotinamide adenine dinucleotide phosphate oxidase subunit p47(phox) knockdown or 5,6,7,8-tetrahydrobiopterin supplementation attenuated endothelial nitric oxide synthase uncoupling and enhanced in vivo endothelial repair capacity of early EPCs from PHAs. In conclusion, PHAs exhibited more impaired endothelial repair capacity of early EPCs than did essential hypertension patients independent of blood pressure, which was associated with mineralocorticoid receptor-dependent oxidative stress and subsequently 5

  6. Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining.

    PubMed

    Grabarz, Anastazja; Barascu, Aurélia; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2012-01-01

    A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.

  7. A new light on the meiotic DSB catalytic complex.

    PubMed

    Robert, Thomas; Vrielynck, Nathalie; Mézard, Christine; de Massy, Bernard; Grelon, Mathilde

    2016-06-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

  8. Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene. Results Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF. Conclusion These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis. PMID:24134223

  9. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-01-24

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

  10. Association between common genetic variation in Cockayne syndrome A and B genes and nucleotide excision repair capacity among smokers.

    PubMed

    Leng, Shuguang; Bernauer, Amanda; Stidley, Christine A; Picchi, Maria A; Sheng, Xin; Frasco, Melissa A; Van Den Berg, David; Gilliland, Frank D; Crowell, Richard E; Belinsky, Steven A

    2008-08-01

    Mutagen sensitivity in in vitro cultured lymphocytes challenged by benzo[a]pyrene diolepoxide (BPDE) has been validated as an intrinsic susceptibility factor for several cancers. Bulky BPDE-DNA adducts are repaired via either transcription-coupled repair or global genome nucleotide excision repair depending on the location of lesions. Cockayne syndrome A (CSA) and B (CSB) play essential roles in integrating the recognition of damage, chromatin remodeling, and the core nucleotide excision repair proteins. This study evaluated the hypothesis that common genetic variation in CSA and CSB is associated with mutagen sensitivity induced by BPDE in 276 cancer-free smokers. Tag single nucleotide polymorphisms (SNP; n = 37) selected across the entire coding and putative regulatory regions of CSA and CSB based on a high-density SNP database were genotyped by the Illumina Golden Gate assay. Major principal components of CSA and CSB that captured the linkage disequilibrium from multiple SNPs were globally associated with the number of breaks per cell at the threshold of 80% (P < or = 0.02 for both genes). Haplotype H125 in CSA and H97 in CSB as well as SNPs in high linkage disequilibrium with these two haplotypes were significantly associated with a 13% to 15% reduction in the mean number of chromatid breaks per cell (P < 0.05). A resampling-based omnibus test supported the significant association between SNPs and haplotypes in CSA and mutagen sensitivity induced by BPDE (P = 0.035). This study implicates transcription-coupled repair in protecting the cell from BPDE-induced DNA damage.

  11. The COP9 signalosome is vital for timely repair of DNA double-strand breaks

    PubMed Central

    Meir, Michal; Galanty, Yaron; Kashani, Lior; Blank, Michael; Khosravi, Rami; Fernández-Ávila, María Jesús; Cruz-García, Andrés; Star, Ayelet; Shochot, Lea; Thomas, Yann; Garrett, Lisa J.; Chamovitz, Daniel A.; Bodine, David M.; Kurz, Thimo; Huertas, Pablo; Ziv, Yael; Shiloh, Yosef

    2015-01-01

    The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability. PMID:25855810

  12. Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

    PubMed

    Page, Melissa M; Salmon, Adam B; Leiser, Scott F; Robb, Ellen L; Brown, Melanie F; Miller, Richard A; Stuart, Jeffrey A

    2009-04-15

    Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.

  13. Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

    PubMed

    Carballo, Jesús A; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S

    2013-06-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  14. Exercise capacity, quality of life, and resilience after repair of tetralogy of Fallot: a cross-sectional study of patients operated between 1964 and 2009.

    PubMed

    Frigiola, Alessandra; Bull, Catherine; Wray, Jo

    2014-02-01

    Patients with repaired tetralogy of Fallot have good long-term survival but less is known about the subjectively assessed quality of life or objectively measured functional status of those who have not required subsequent pulmonary valve replacement. We assessed these parameters in a group of children and adults free from pulmonary valve replacement after tetralogy of Fallot repair. A random sample of 50 subjects--16 children and 34 adults, aged 4.1-56.7 years---who had undergone tetralogy of Fallot repair and were free from subsequent pulmonary valve replacement underwent cardiopulmonary exercise testing and completed standardised questionnaires assessing health-related quality of life and resilience. Patients were generally asymptomatic (median New York Heart Association class = I). Exercise capacity was within two standard deviations of normal for most children and adults (mean z VO2max: 0.20 ± 1.5; mean z VE/VCO2: -0.9 ± 1.3). Children reported a total health-related quality of life score similar to healthy norms (78 ± 10 versus 84 ± 1, p = 0.73). Adult survivors also reported quality of life scores comparable to healthy norms. Resilience was highly correlated with all domains of health-related quality of life (r = 0.713, p < 0.0001). Patients who have undergone tetralogy of Fallot repair in childhood and have not required pulmonary valve replacement have a good long-term health-related quality of life. The finding that patients with greater resilience had better health-related quality of life suggests that it may be beneficial to implement interventions to foster resilience.

  15. Decreased cell survival and DNA repair capacity after UVC irradiation in association with down-regulation of GRP78/BiP in human RSa cells

    SciTech Connect

    Zhai Ling; Kita, Kazuko . E-mail: kita@faculty.chiba-u.jp; Wano, Chieko; Wu Yuping; Sugaya, Shigeru; Suzuki, Nobuo

    2005-05-01

    In contrast to extensive studies on the roles of molecular chaperones, such as heat shock proteins, there are only a few reports about the roles of GRP78/BiP, an endoplasmic reticulum (ER) stress-induced molecular chaperone, in mammalian cell responses to DNA-damaging stresses. To investigate whether GRP78/BiP is involved in resistance to a DNA-damaging agent, UVC (principally 254 nm in wavelength), we established human cells with down-regulation of GRP78/BiP by transfection of human RSa cells with antisense cDNA for GRP78/BiP. We found that the transfected cells showed higher sensitivity to UVC-induced cell death than control cells transfected with the vector alone. In the antisense-cDNA transfected cells, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4) photoproducts) in vivo and DNA synthesis activity of whole cell extracts to repair UVC-irradiated plasmids in vitro were remarkably decreased compared with those in the control cells. Furthermore, the antisense-cDNA transfected cells also showed slightly higher sensitivity to cisplatin-induced cell death than the control cells. Cisplatin-induced DNA damage is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. The present results suggest that GRP78/BiP plays a protective role against UVC-induced cell death possibly via nucleotide excision repair, at least in the human RSa cells tested.

  16. Transcript-RNA-templated DNA recombination and repair.

    PubMed

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  17. Human resident CD34+ stromal cells/telocytes have progenitor capacity and are a source of αSMA+ cells during repair.

    PubMed

    Díaz-Flores, L; Gutiérrez, R; García, M P; González, M; Sáez, F J; Aparicio, F; Díaz-Flores, L; Madrid, J F

    2015-05-01

    We studied the progenitor capacity of human resident CD34+ stromal cells/telocytes (SC/TCs) in the enteric wall affected by inflammatory/repair processes (appendicitis, diverticulitis of large bowel and Crohn's disease of the terminal ileum) at different stages of evolution (inflammatory, proliferative and remodelling). In these conditions, CD34+ SC/TCs are activated, showing changes, which include the following overlapping events: 1) separation from adjacent structures (e.g., from vascular walls) and location in oedematous spaces, 2) morphological modifications (in cell shape and size) with presence of transitional cell forms between quiescent and activated CD34+ SC/TCs, 3) rapid proliferation and 4) loss of CD34 expression and gain of αSMA expression. These events mainly occur in the inflammatory and proliferative stages. During the loss of CD34 expression, the following findings are observed: a) irregular cell labelling intensity for anti-CD34, b) co-localization of CD34 and actin, c) concurrent irregular labelling intensity for αSMA and d) αSMA expression in all stromal cells, with total loss of CD34 expression. While CD34 expression was conserved, a high proliferative capacity (Ki-67 expression) was observed and vice versa. In the segments of the ileum affected by Crohn's disease, the stromal cells around fissures were αSMA+ and, in the transitional zones with normal enteric wall, activated CD34+ SC/TCs were observed. In conclusion, human resident CD34+ SC/TCs in the enteric wall have progenitor capacity and are activated with or without differentiation into αSMA+ stromal cells during inflammatory/repair processes.

  18. ATM specifically mediates repair of double-strand breaks with blocked DNA ends

    PubMed Central

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Ariel Lieberman, Jenna; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M.; Cortés-Ledesma, Felipe

    2014-01-01

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders. PMID:24572510

  19. ATM specifically mediates repair of double-strand breaks with blocked DNA ends.

    PubMed

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Lieberman, Jenna Ariel; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M; Cortés-Ledesma, Felipe

    2014-02-27

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.

  20. XRCC1 Arg399Gln was associated with repair capacity for DNA damage induced by occupational chromium exposure

    PubMed Central

    2012-01-01

    Background Occupational chromium exposure may induce DNA damage and lead to lung cancer and other work-related diseases. DNA repair gene polymorphisms, which may alter the efficiency of DNA repair, thus may contribute to genetic susceptibility of DNA damage. The aim of this study was to test the hypothesis that the genetic variations of 9 major DNA repair genes could modulate the hexavalent chromium (Cr (VI))-induced DNA damage. Findings The median (P25-P75) of Olive tail moment was 0.93 (0.58–1.79) for individuals carrying GG genotype of XRCC1 Arg399Gln (G/A), 0.73 (0.46–1.35) for GA heterozygote and 0.50 (0.43–0.93) for AA genotype. Significant difference was found among the subjects with three different genotypes (P = 0.048) after adjusting the confounding factors. The median of Olive tail moment of the subjects carrying A allele (the genotypes of AA and GA) was 0.66 (0.44–1.31), which was significantly lower than that of subjects with GG genotype (P = 0.043). The A allele conferred a significantly reduced risk of DNA damage with the OR of 0.39 (95% CI: 0.15–0.99, P = 0.048). No significant association was found between the XRCC1Arg194Trp, ERCC1 C8092A, ERCC5 His1104Asp, ERCC6 Gly399Asp, GSTP1 Ile105Val, OGG1 Ser326Cys, XPC Lys939Gln, XPD Lys751Gln and DNA damage. Conclusion The polymorphism of Arg399Gln in XRCC1 was associated with the Cr (VI)- induced DNA damage. XRCC1 Arg399Gln may serve as a genetic biomarker of susceptibility for Cr (VI)- induced DNA damage. PMID:22642904

  1. Short-Term Change of Exercise Capacity in Patients with Pulmonary Valve Replacement after Tetralogy of Fallot Repair

    PubMed Central

    Hwang, Tae Woong; Kim, Sung Ook; Kim, Moon Sun; Kim, Seong Ho; Lee, Sang Yun; Choi, Eun Young; Park, Su Jin; Kwon, Hye Won; Lim, Hyo Bin

    2017-01-01

    Background and Objectives The aim of this study was to investigate the effect of pulmonary valve replacement (PVR) on exercise capacity and determine cardiopulmonary exercise (CPEX) parameters associated with improvement in right ventricle (RV) function. Subjects and Methods We retrospectively analyzed CPEX and magnetic resonance imaging parameters in a total of 245 patients who underwent PVR from January 1998 to October 2015. In addition, we analyzed the characteristics of the patients who showed improved exercise capacity after PVR. Results Twenty-eight patients met the inclusion criteria for the study. CPEX parameters after PVR showed no significant changes in all patients. However, baseline predicted peak oxygen uptake (VO2peak) (%) value was significantly lower in patients with significant improvement in exercise capacity after PVR, as compared to patients who showed decreased exercise capacity after PVR (60.83±10.28 vs. 75.81±13.83) (p=0.003). In addition, patients with improved exercise capacity showed a positive correlation between the change of right ventricular ejection fraction (RVEF) (%) and the change of anaerobic threshold (r=0.733, p=0.007); whereas, patients with decreased exercise capacity showed a negative correlation between the change of RVEF (%) and the change of predicted VO2peak (%) (r=−0.575, p=0.020). Conclusion The importance of predicted VO2peak (%) in evaluating exercise capacity differentiated from other CPEX variables. The change of anaerobic threshold and predicted VO2peak (%) might be a useful predictor of the change in RV function after PVR. PMID:28382082

  2. Impaired left ventricular myocardial mechanics and their relation to pulmonary regurgitation, right ventricular enlargement and exercise capacity in asymptomatic children after repair of tetralogy of Fallot.

    PubMed

    Fernandes, Fernanda P; Manlhiot, Cedric; Roche, Susan L; Grosse-Wortmann, Lars; Slorach, Cameron; McCrindle, Brian W; Mertens, Luc; Kantor, Paul F; Friedberg, Mark K

    2012-05-01

    Left ventricular (LV) dysfunction is common in adults late after repair of tetralogy of Fallot (TOF). The early detection of myocardial dysfunction may be important, but LV myocardial strain and dyssynchrony are not well studied in children with TOF. The objective of this study was to investigate LV strain and dyssynchrony in asymptomatic children and adolescents after contemporary repair of TOF. The hypothesis was that impaired LV myocardial mechanics are related to pulmonary regurgitation, right ventricular (RV) enlargement, and exercise capacity. Children and adolescents were prospectively studied after TOF repair. LV regional strain and dyssynchrony were assessed using two-dimensional speckle-tracking echocardiography. Ventricular volumes, ejection fraction, and pulmonary regurgitation were assessed using magnetic resonance imaging. Exercise capacity was determined using metabolic exercise testing. One hundred twenty-four subjects (53 patients with TOF and 71 controls) were studied. Regional circumferential (e.g., basal lateral wall, -15.0 ± 7.0% vs -19.0 ± 7.0%, P = .02) and radial (e.g., basal posterior wall, 32.0 ± 18.0% vs 48.0 ± 21.0%, P < .001) LV strain and longitudinal septal strain (-18.5 ± 3.5% vs -20.2 ± 2.8%, P = .01) were significantly reduced in patients with TOF compared with controls. LV mechanical dyssynchrony indices were not significantly different between groups (e.g., standard deviation of time to peak circumferential strain, 52.5 ± 40.4 vs 50.5 ± 27.1 msec, P = .81). Higher pulmonary regurgitation volume and larger RV end-diastolic volume were associated with decreased LV radial strain (P = .09). There was no association between LV longitudinal, radial, or circumferential dyssynchrony and indexed pulmonary regurgitation flow volume, RV end-diastolic volume, or RV ejection fraction. LV circumferential and radial strain are significantly reduced in children and adolescents after TOF repair and are associated with pulmonary

  3. Gene Targeting Without DSB Induction Is Inefficient in Barley.

    PubMed

    Horvath, Mihaly; Steinbiss, Hans-Henning; Reiss, Bernd

    2016-01-01

    Double strand-break (DSB) induction allowed efficient gene targeting in barley (Hordeum vulgare), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the acetolactate synthase (ALS) gene was established, a target gene which had been used previously in rice and Arabidopsis thaliana. Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in A. thaliana and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines. In addition, P-N selection was tested. In contrast to the high gene targeting efficiencies obtained in the absence of DSB induction in A. thaliana or rice, not one single gene targeting event was obtained in barley. These data suggest that gene targeting efficiencies are very low in barley and can substantially differ between different plants, even at the same target locus. They also suggest that the amount of labour and time would become unreasonably high to use these methods as a tool in routine applications. This is particularly true since DSB induction offers efficient alternatives. Barley, unlike rice and A. thaliana has a large, complex genome, suggesting that genome size or complexity could be the reason for the low efficiencies. We discuss to what extent transformation methods, genome size or genome complexity could contribute to the striking differences in the gene targeting efficiencies between barley, rice and A. thaliana.

  4. Gene Targeting Without DSB Induction Is Inefficient in Barley

    PubMed Central

    Horvath, Mihaly; Steinbiss, Hans-Henning; Reiss, Bernd

    2017-01-01

    Double strand-break (DSB) induction allowed efficient gene targeting in barley (Hordeum vulgare), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the acetolactate synthase (ALS) gene was established, a target gene which had been used previously in rice and Arabidopsis thaliana. Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in A. thaliana and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines. In addition, P-N selection was tested. In contrast to the high gene targeting efficiencies obtained in the absence of DSB induction in A. thaliana or rice, not one single gene targeting event was obtained in barley. These data suggest that gene targeting efficiencies are very low in barley and can substantially differ between different plants, even at the same target locus. They also suggest that the amount of labour and time would become unreasonably high to use these methods as a tool in routine applications. This is particularly true since DSB induction offers efficient alternatives. Barley, unlike rice and A. thaliana has a large, complex genome, suggesting that genome size or complexity could be the reason for the low efficiencies. We discuss to what extent transformation methods, genome size or genome complexity could contribute to the striking differences in the gene targeting efficiencies between barley, rice and A. thaliana. PMID:28105032

  5. Position effects influencing intrachromosomal repair of a double-strand break in budding yeast.

    PubMed

    Wang, Ruoxi W; Lee, Cheng-Sheng; Haber, James E

    2017-01-01

    Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238 kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.

  6. 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.

  7. Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity

    PubMed Central

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta

    2013-01-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2–8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16INK4a), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier). PMID:23689827

  8. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity.

    PubMed

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta; Tucsek, Zsuzsanna; Toth, Peter; Deak, Ferenc; Gautam, Tripti; Csiszar, Anna; Sonntag, William E

    2013-12-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2-8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16 (INK4a) ), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

  9. Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

    PubMed

    Cesare, Anthony J

    2014-11-01

    Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression.

  10. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    PubMed Central

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  11. Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51.

    PubMed

    Downing, Brandon; Morgan, Rachel; VanHulle, Kelly; Deem, Angela; Malkova, Anna

    2008-10-14

    DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.

  12. Preferential repair of DNA double-strand break at the active gene in vivo.

    PubMed

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  13. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

    PubMed

    Shen, Hexi; Strunks, Gary D; Klemann, Bart J P M; Hooykaas, Paul J J; de Pater, Sylvia

    2017-01-05

    Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. Copyright © 2017 Shen et al.

  14. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

    PubMed Central

    Shen, Hexi; Strunks, Gary D.; Klemann, Bart J. P. M.; Hooykaas, Paul J. J.; de Pater, Sylvia

    2016-01-01

    Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. PMID:27866150

  15. Cadmium Induced Cell Apoptosis, DNA Damage, Decreased DNA Repair Capacity, and Genomic Instability during Malignant Transformation of Human Bronchial Epithelial Cells

    PubMed Central

    Zhou, Zhiheng; Wang, Caixia; Liu, Haibai; Huang, Qinhai; Wang, Min; Lei, Yixiong

    2013-01-01

    Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells in the fifth, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice using flow cytometry, Hoechst 33258 staining, comet assay, quantitative real-time polymerase chain reaction (PCR), Western blot analysis, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis. We observed a progressive increase in cell population of the G0/G1 phase of the cell cycle and the rate of apoptosis, DNA damage, and cadmium-induced apoptotic morphological changes in cerebral cortical neurons during malignant transformation. Gene expression analysis revealed increased expression of cell proliferation (PCNA), cell cycle (CyclinD1), pro-apoptotic activity (Bax), and DNA damage of the checkpoint genes ATM, ATR, Chk1, Chk2, Cdc25A. Decreased expression of the anti-apoptotic gene Bcl-2 and the DNA repair genes hMSH2, hMLH1, ERCC1, ERCC2, and hOGG1 was observed. RAPD-PCR revealed genomic instability in cadmium-exposed cells, and sequence analysis showed mutation of exons in hMSH2, ERCC1, XRCC1, and hOGG1 in tumorigenic cells. This study suggests that Cadmium can increase cell apoptosis and DNA damage, decrease DNA repair capacity, and cause mutations, and genomic instability leading to malignant transformation. This process could be a viable mechanism for cadmium-induced cancers. PMID:24046522

  16. Mechanism of RAD51-dependent DNA interstrand cross-link repair.

    PubMed

    Long, David T; Räschle, Markus; Joukov, Vladimir; Walter, Johannes C

    2011-07-01

    DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.

  17. Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair.

    PubMed

    Wang, Zai; Zhou, Zhong-Jun; Liu, De-Pei; Huang, Jian-Dong

    2006-11-24

    Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.

  18. Structural and Functional Characterization of Three DsbA Paralogues from Salmonella enterica Serovar Typhimurium*

    PubMed Central

    Heras, Begoña; Totsika, Makrina; Jarrott, Russell; Shouldice, Stephen R.; Gunčar, Gregor; Achard, Maud E. S.; Wells, Timothy J.; Argente, M. Pilar; McEwan, Alastair G.; Schembri, Mark A.

    2010-01-01

    In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence. PMID:20233716

  19. Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice.

    PubMed

    Kwon, Yong-Ik; Abe, Kiyomi; Osakabe, Keishi; Endo, Masaki; Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Shimada, Hiroaki; Toki, Seiichi

    2012-12-01

    During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.

  20. Transcript RNA supports precise repair of its own DNA gene.

    PubMed

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  1. Differential mutagenicity of N-methyl-N-nitrosocarbamate insectides in Escherichia coli strains having different DNA repair capacities.

    PubMed

    Yoshikawa, K; Uchino, H; Kurata, H

    1978-12-01

    Four isogenic strains of Escherichia coli with the same auxotrophic marker (arg Fam--namely wild-type, uvrA-, polA- and recA-) were used for testing the lethalities and mutagenicities of 1-naphthyl N-methyl-N-nitrosocarbamate (nitroso-NAC), 3-methylphenyl N-methyl-N-nitrosocarbamate (nitroso-MTMC), and 3,4-dimethylphenyl N-methyl-N-nitrosocarbamate (nitroso-MPMC). The strains recA- and polA- showed a similarly higher sensitivity to killing than wild-type and uvrA- after treatments with each of the three chemicals, whereas the strains wild-type, uvrA-, and polA- were equally mutable by these compounds at equal doses. The strain recA- was hardly mutable by nitroso-NAC, but significant levels of Arg+ mutations were observed after treatments with nitroso-MTMC and nitroso-MPMC. These and previous results suggest that both nitroso-MTMC and nitroso-MPMC are similar in their mutagenicity pattern to N-methyl-N'-nitro-N-nitrosoguanidine whereas nitroso-NAC is similar to methyl methanesulfonate or X-rays, and that the major damage to DNA of the three agents is not excisable by the uvrA+-dependent excision repair, probably methylation in DNA.

  2. Control of gene editing by manipulation of DNA repair mechanisms.

    PubMed

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  3. The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

    SciTech Connect

    Paxman, Jason J.; Borg, Natalie A.; Horne, James; Thompson, Philip E.; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S.; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M.; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P.; Rossjohn, Jamie; Scanlon, Martin J.

    2010-09-07

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  4. The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes.

    PubMed

    Paxman, Jason J; Borg, Natalie A; Horne, James; Thompson, Philip E; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P; Rossjohn, Jamie; Scanlon, Martin J

    2009-06-26

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  5. Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria.

    PubMed

    Raczko, Anna M; Bujnicki, Janusz M; Pawlowski, Marcin; Godlewska, Renata; Lewandowska, Magdalena; Jagusztyn-Krynicka, Elzbieta K

    2005-01-01

    In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a beta-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba (dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.

  6. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair**

    PubMed Central

    Coates, Julia; Jhujh, Satpal; Mehmood, Shahid; Tamura, Naoka; Travers, Jon; Wu, Qian; Draviam, Viji M.; Robinson, Carol V.; Blundell, Tom L.; Jackson, Stephen P.

    2014-01-01

    XRCC4 and XLF are two structurally-related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF; also called C9orf142) as a new XRCC4-superfamily member, and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX−/− cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro, and assembly of core non-homologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery. PMID:25574025

  7. Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.

    PubMed

    Pfeiffer, Petra; Odersky, Andrea; Goedecke, Wolfgang; Kuhfittig-Kulle, Steffi

    2014-01-01

    Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

  8. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    PubMed

    Stamper, Ericca L; Rodenbusch, Stacia E; Rosu, Simona; Ahringer, Julie; Villeneuve, Anne M; Dernburg, Abby F

    2013-01-01

    Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  9. 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

  10. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    PubMed

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  11. RecA bundles mediate homology pairing between distant sisters during DNA break repair.

    PubMed

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J

    2014-02-13

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  12. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    NASA Astrophysics Data System (ADS)

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2014-02-01

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  13. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    PubMed Central

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recombination (HR), which is mostly error-free3,4. Consequently, the relative contribution of these different pathways to DSB repair in the cell cycle has a profound influence on the maintenance of genetic integrity. How then are DSB directed for repair by different, potentially competing, repair pathways? Here we identify a role for CtIP in this process in DT40. We establish that CtIP is not only required for repair of DSB by HR in S/G2 phase, but also for MMEJ in G1. The function of CtIP in HR, but not MMEJ, is dependent on the phosphorylation of serine residue 327 and recruitment of BRCA1. Cells expressing CtIP protein that cannot be phosphorylated at serine 327 are specifically defective in HR and exhibit decreased level of single-stranded DNA (ssDNA) after DNA damage, while MMEJ remains unaffected. Our data support a model in which phosphorylation of serine 327 of CtIP as cells enter S-phase and the recruitment of BRCA1 functions as a molecular switch to shift the balance of DSB repair from error-prone DNA end-joining to error-free homologous recombination (Supplementary Fig. 1). PMID:19357644

  14. Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms.

    PubMed

    Bezine, Elisabeth; Malaisé, Yann; Loeuillet, Aurore; Chevalier, Marianne; Boutet-Robinet, Elisa; Salles, Bernard; Mirey, Gladys; Vignard, Julien

    2016-10-24

    The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity.

  15. Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms

    PubMed Central

    Bezine, Elisabeth; Malaisé, Yann; Loeuillet, Aurore; Chevalier, Marianne; Boutet-Robinet, Elisa; Salles, Bernard; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity. PMID:27775089

  16. Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity

    PubMed Central

    Nickson, Catherine M.; Moori, Parisa; Carter, Rachel J.; Rubbi, Carlos P.; Parsons, Jason L.

    2017-01-01

    Patients with human papillomavirus type 16 (HPV)-associated oropharyngeal squamous cell carcinomas (OPSCC) display increased sensitivity to radiotherapy and improved survival rates in comparison to HPV-negative forms of the disease. However the cellular mechanisms responsible for this characteristic difference are unclear. Here, we have investigated the contribution of DNA damage repair pathways to the in vitro radiosensitivity of OPSCC cell lines. We demonstrate that two HPV-positive OPSCC cells are indeed more radiosensitive than two HPV-negative OPSCC cells, which correlates with reduced efficiency for the repair of ionising radiation (IR)-induced DNA double strand breaks (DSB). Interestingly, we show that HPV-positive OPSCC cells consequently have upregulated levels of the proteins XRCC1, DNA polymerase β, PNKP and PARP-1 which are involved in base excision repair (BER) and single strand break (SSB) repair. This translates to an increased capacity and efficiency for the repair of DNA base damage and SSBs in these cells. In addition, we demonstrate that HPV-positive but interestingly more so HPV-negative OPSCC display increased radiosensitivity in combination with the PARP inhibitor olaparib. This suggests that PARP inhibition in combination with radiotherapy may be an effective treatment for both forms of OPSCC, particularly for HPV-negative OPSCC which is relatively radioresistant. PMID:28415784

  17. Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase

    PubMed Central

    Byriel, Karl; Huston, Wilhelmina M.; Furlong, Emily; Heras, Begoña; Martin, Jennifer L.

    2016-01-01

    The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins. PMID:28030602

  18. 'Relax and Repair' to restrain aging.

    PubMed

    Krishnan, Vaidehi; Liu, Baohua; Zhou, Zhongjun

    2011-10-01

    The maintenance of genomic integrity requires the precise identification and repair of DNA damage. Since DNA is packaged and condensed into higher order chromatin, the events associated with DNA damage recognition and repair are orchestrated within the layers of chromatin. Very similar to transcription, during DNA repair, chromatin remodelling events and histone modifications act in concert to 'open' and relax chromatin structure so that repair proteins can gain access to DNA damage sites. One such histone mark critical for maintaining chromatin structure is acetylated lysine 16 of histone H4 (AcH4K16), a modification that can disrupt higher order chromatin organization and convert it into a more 'relaxed' configuration. We have recently shown that impaired H4K16 acetylation delays the accumulation of repair proteins to double strand break (DSB) sites which results in defective genome maintenance and accelerated aging in a laminopathy-based premature aging mouse model. These results support the idea that epigenetic factors may directly contribute to genomic instability and aging by regulating the efficiency of DSB repair. In this article, the interplay between epigenetic misregulation, defective DNA repair and aging is discussed.

  19. The non-homologous end-joining (NHEJ) mathematical model for the repair of double-strand breaks: II. Application to damage induced by ultrasoft X rays and low-energy electrons.

    PubMed

    Taleei, Reza; Girard, Peter M; Sankaranarayanan, Krishnaswami; Nikjoo, Hooshang

    2013-05-01

    We investigated the kinetics of simple and complex types of double-strand breaks (DSB) using our newly proposed mechanistic mathematical model for NHEJ DSB repair. For this purpose the simulated initial spectrum of DNA DSB, induced in an atomistic canonical model of B-DNA by low-energy single electron tracks, 100 eV to 4.55 keV, and the electrons generated by ultrasoft X rays (CK, AlK and TiK), were subjected to NHEJ repair processes. The activity elapsed time of sequentially independent steps of repair performed by proteins involved in NHEJ repair process were calculated for separate DSB. The repair kinetics of DSBs were computed and compared with published data on repair kinetics obtained by pulsed-field gel electrophoresis method. The comparison shows good agreement for V79-4 cells irradiated with ultrasoft X rays. The average times for the repair of simple and complex DSB confirm that double-strand break complexity is a potential explanation for the slow component of DSB repair observed in V79-4 cells irradiated by ultrasoft X rays.

  20. Can a mixed damage interfere with DNA-protein cross-links repair?

    PubMed

    Marzano, C; Severin, E; Bordin, F

    2001-01-01

    Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

  1. Exposure of nondividing populations of primary human fibroblasts to UV (254 nm) radiation induces a transient enhancement in capacity to repair potentially lethal cellular damage

    SciTech Connect

    Tyrrell, R.M.

    1984-02-01

    Nondividing (arrested) populations of primary human fibroblasts from normal individuals exposed to an intial dose (1.5 or 3 Jm/sup -2/) of far-UV (254 nm) radiation and then incubated in medium containing low (0.5%) serum develop enhanced resistance to inactivation of cloning efficiency by a second (challenge) dose of UV. The resistance develops within 2-4 days, after which there is a decline. Resistance develops to a higher degree and more rapidly (1-2 days) in cells derived from patients with the variant form of xeroderma pigmentosum. Excision-deficient cells from xeroderma pigmentosum complementation group A individuals also develop UV resistance after a lower (0.2 Jm/sup -2/) exposure to UV. Enhanced UV resistance does not develop in UV-irradiated cell populations incubated with the protein synthesis inhibitor cycloheximide (5 ..mu..M). These observations are consistent with the interpretation that exposure of human fibroblasts to low doses of UV induces synthesis of a protein involved in a metabolic pathway that transiently enhances the capacity of cells to repair potentially lethal damage resulting from a subsequent dose of UV.

  2. Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes.

    PubMed

    Park, Eun-Jung; Hur, Shin-Kyoung; Kwon, Jongbum

    2010-10-15

    Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

  3. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

    PubMed

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

    2017-02-08

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52(+/+) counterparts, which display normal CSR, Rad52(-/-) B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52(-/-) versus Rad52(+/+) B cells on Ku86 knockdown.

  4. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

    PubMed Central

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

    2017-01-01

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

  5. Comparing Benign and Malignant Neoplasia and DSB Induction for Low-and High-LET Radiation

    NASA Astrophysics Data System (ADS)

    Burns, Fredric; (Eric) Tang, Moon-Shong; Wu, Feng

    One-and 2-stage models based on DNA double strand breaks (DSBs) have been developed to describe the dose and LET dependence of cancer induction in rat skin exposed to the Bragg plateau of several ion beams or electron radiation. Data are presented showing that carcinomas (malignant) and fibromas (benign) are induced differently by low and high LET radiation. DSBs are subject to complex repair processes, including homologous and non-homologous end joining, that slowly eliminate broken chromosome ends but at the expense of elevating genomic instability that increases the risk of neoplasia. In this formulation the initial molecular lesion in radiation carcinogenesis is assumed to be a DNA double strand break (DSB). The 2-event model assumes that pairs of DSBs join to create cellular genomic instability that eventually progresses to malignancy. The 1-event model assumes that joining is insignificant but that unrepaired DSBs remain and are sufficiently destabilizing to produce low-grade neoplasias. The respective expected relationships between neoplasia yield (Y), radiation dose (D) and LET (L) are: Y(D) = CLD + BD2 (A) for 2-events and Y(D) = CLD (B) for 1-event. Respective B and C values have been evaluated empirically for carcinomas, fibromas and DSBs, the latter via the -H2Ax technique in surrogate keratinocytes, for several types of radiations, including, 40Ar ions, 56Fe ions, 20Ne ions, protons, electrons and x-rays. Fibromas outnumber carcinomas by about 6:1 but are more sensitive than carcinomas to the cytolethal effect of the radiations. The 2-event model agrees well with carcinoma yields in rat skin but fails to model fibromas correctly. Instead the fibroma yields best fitted with the 1-event model for the high LET ion radiations, but at very low LET (electron radiation), an empirical D3 component becomes apparent which is not currently incorporated into the theoretical model. At higher LET values, the D3 component was not detected. The overall results are

  6. The accumulation of un-repairable DNA damage in laminopathy progeria fibroblasts is caused by ROS generation and is prevented by treatment with N-acetyl cysteine.

    PubMed

    Richards, Shane A; Muter, Joanne; Ritchie, Pamela; Lattanzi, Giovanna; Hutchison, Christopher J

    2011-10-15

    Fibroblasts from patients with the severe laminopathy diseases, restrictive dermopathy (RD) and Hutchinson Gilford progeria syndrome (HGPS), are characterized by poor growth in culture, the presence of abnormally shaped nuclei and the accumulation of DNA double-strand breaks (DSB). Here we show that the accumulation of DSB and poor growth of the fibroblasts but not the presence of abnormally shaped nuclei are caused by elevated levels of reactive oxygen species (ROS) and greater sensitivity to oxidative stress. Basal levels of ROS and sensitivity to H(2)O(2) were compared in fibroblasts from normal, RD and HGPS individuals using fluorescence activated cell sorting-based assays. Basal levels of ROS and stimulated levels of ROS were both 5-fold higher in the progeria fibroblasts. Elevated levels of ROS were correlated with lower proliferation indices but not with the presence of abnormally shaped nuclei. DSB induced by etoposide were repaired efficiently in normal, RD and HGPS fibroblasts. In contrast, DSB induced by ROS were repaired efficiently in normal fibroblasts, but in RD and HGPS fibroblasts many ROS-induced DSB were un-repairable. The accumulation of ROS-induced DSB appeared to cause the poor growth of RD and HGPS fibroblasts, since culture in the presence of the ROS scavenger N-acetyl cysteine (NAC) reduced the basal levels of DSB, eliminated un-repairable ROS-induced DSB and greatly improved population-doubling times. Our findings suggest that un-repaired ROS-induced DSB contribute significantly to the RD and HGPS phenotypes and that inclusion of NAC in a combinatorial therapy might prove beneficial to HGPS patients.

  7. Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks.

    PubMed

    Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D

    2016-05-08

    The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.

  8. Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

    PubMed Central

    Alexander, Jessica L.; Beagan, Kelly; Orr-Weaver, Terry L.; McVey, Mitch

    2016-01-01

    Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context. PMID:27849606

  9. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

  10. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair

    PubMed Central

    Tsabar, Michael; Waterman, David P.; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V.; Memisoglu, Gonen; Haber, James E.

    2016-01-01

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

  11. Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination.

    PubMed

    Kim, Jung-Ae; Hicks, Wade M; Li, Jin; Tay, Sue Yen; Haber, James E

    2011-02-01

    In response to a DNA double-strand break (DSB), cells undergo a transient cell cycle arrest prior to mitosis until the break is repaired. In budding yeast (Saccharomyces cerevisiae), the DNA damage checkpoint is regulated by a signaling cascade of protein kinases, including Mec1 and Rad53. When DSB repair is complete, cells resume cell cycle progression (a process called "recovery") by turning off the checkpoint. Recovery involves two members of the protein phosphatase 2C (PP2C) family, Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4) enzyme, Pph3. Here, we demonstrate a new function of these three phosphatases in DSB repair. Cells lacking all three phosphatases Pph3, Ptc2, and Ptc3 exhibit synergistic sensitivities to the DNA-damaging agents camptothecin and methyl methanesulfonate, as well as hydroxyurea but not to UV light. Moreover, the simultaneous absence of Pph3, Ptc2, and Ptc3 results in defects in completing DSB repair, whereas neither single nor double deletion of the phosphatases causes a repair defect. Specifically, cells lacking all three phosphatases are defective in the repair-mediated DNA synthesis. Interestingly, the repair defect caused by the triple deletion of Pph3, Ptc2, and Ptc3 is most prominent when a DSB is slowly repaired and the DNA damage checkpoint is fully activated.

  12. Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development.

    PubMed

    Orii, Kenji E; Lee, Youngsoo; Kondo, Naomi; McKinnon, Peter J

    2006-06-27

    The repair of DNA double-strand breaks (DSBs) occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity and organismal survival. Although inactivation of either pathway leads to embryonic lethality, here we show selective requirements for each DNA DSB repair pathway at different stages of mammalian nervous system development. DNA damage-induced apoptosis resulting from inactivation of HR (Xrcc2 deficiency) only occurred in proliferating neural precursor cells, whereas disruption of NHEJ (DNA ligase IV deficiency) mainly affected differentiating cells at later developmental stages. Therefore, these data suggest that NHEJ is dispensable for a substantial portion of early development because DSB repair during this period utilizes HR. Moreover, DNA damage-induced apoptosis required the ataxia telangiectasia mutated (Atm) kinase after disruption of NHEJ, but not HR, during neurogenesis. However, embryonic lethality arising from disruption of either repair pathway was rescued by loss of p53 and resulted in specific tumor types reflective of the particular DSB repair pathway inactivated. Thus, these data reveal distinct tissue- and cell-type requirements for each DNA DSB repair pathway during neural development and provide insights for understanding the contributions of DNA DSB responses to disease.

  13. Preliminary crystallographic data of the three homologues of the thiol–disulfide oxidoreductase DsbA in Neisseria meningitidis

    SciTech Connect

    Lafaye, Céline; Griat, Mickael; Serre, Laurence

    2008-02-01

    The Neisseria meningitidis genome possesses three genes encoding active DsbAs. To throw light on the reason for this genetic multiplicity, the three enzymes have been purified and crystallized. Bacterial virulence depends on the correct folding of surface-exposed proteins, a process that is catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. Uniquely among bacteria, the Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host-interactive biology, while the function of DsbA3 remains unknown. In an attempt to shed light on the reason for this multiplicity of dsbA genes, the three enzymes from N. meningitidis have been purified and crystallized in the presence of high concentrations of ammonium sulfate. The best crystals were obtained using DsbA1 and DsbA3; they belong to the orthorhombic and tetragonal systems and diffract to 1.5 and 2.7 Å resolution, respectively.

  14. Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity.

    PubMed

    Dumoulin, Alexis; Grauschopf, Ulla; Bischoff, Markus; Thöny-Meyer, Linda; Berger-Bächi, Brigitte

    2005-11-01

    DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.

  15. Meningocele repair

    MedlinePlus

    ... Myelodysplasia repair; Spinal dysraphism repair; Meningomyelocele repair; Neural tube defect repair; Spina bifida repair ... If your child has hydrocephalus, a shunt (plastic tube) will be put in the child's brain to ...

  16. Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

    PubMed Central

    Fattah, Farjana; Lee, Eu Han; Weisensel, Natalie; Wang, Yongbao; Lichter, Natalie; Hendrickson, Eric A.

    2010-01-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice. PMID:20195511

  17. Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

    PubMed Central

    Grabowska, Anna D.; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M.; Wösten, Marc M. S. M.; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M.; van Putten, Jos P. M.; Jagusztyn-Krynicka, E. Katarzyna

    2014-01-01

    Background Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Methods and Results Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Conclusions Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether

  18. DNA Repair Capacity in Peripheral Lymphocytes Predicts Survival of Patients With Non–Small-Cell Lung Cancer Treated With First-Line Platinum-Based Chemotherapy

    PubMed Central

    Wang, Li-E; Yin, Ming; Dong, Qiong; Stewart, David J.; Merriman, Kelly W.; Amos, Christopher I.; Spitz, Margaret R.; Wei, Qingyi

    2011-01-01

    Purpose Platinum-based regimens are the standard chemotherapy for patients with advanced non–small-cell lung cancer (NSCLC). DNA repair capacity (DRC) in tumor cells plays an important role in resistance to platinum-based drugs. We have previously reported that efficient DRC, as assessed by an in vitro lymphocyte-based assay, was a determinant of poor survival in patients with NSCLC in a relatively small data set. In this larger independent study of 591 patients with NSCLC, we further evaluated whether DRC in peripheral lymphocytes predicts survival of patients with NSCLC who receive platinum-based chemotherapy. Patients and Methods All patients were recruited at The University of Texas MD Anderson Cancer Center and donated blood samples before the start of any chemotherapy. We measured DRC in cultured T lymphocytes by using the host-cell reactivation assay, and we assessed associations between DRC in peripheral lymphocytes and survival of patients with NSCLC who were treated with first-line platinum-based chemotherapy. Results We found an inverse association between DRC in peripheral lymphocytes and patient survival. Compared with patients in the low tertile of DRC, patients with NSCLC in the high tertile of DRC had significantly worse overall and 3-year survival (adjusted hazard ratio [HR], 1.33; 95% CI, 1.04 to 1.71; P = .023; and HR, 1.35; 95% CI, 1.04 to 1.76; P = .025, respectively). This trend was more pronounced in patients with early-stage tumors, adenocarcinoma, or squamous cell carcinoma. Conclusion We confirmed that DRC in peripheral lymphocytes is an independent predictor of survival for patients with NSCLC treated with platinum-based chemotherapy. PMID:21947825

  19. The role of DNA repair on cell killing by charged particles

    NASA Astrophysics Data System (ADS)

    Eguchi-Kasai, K.; Murakami, M.; Itsukaichi, H.; Fukutsu, K.; Kanai, T.; Furusawa, Y.; Sato, K.; Ohara, H.; Yatagai, F.

    It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderate RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.

  20. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair.

    PubMed

    Kato, Akihiro; Komatsu, Kenshi

    2015-07-14

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair.

  1. Poorer right ventricular systolic function and exercise capacity in women after repair of tetralogy of fallot: a sex comparison of standard deviation scores based on sex-specific reference values in healthy control subjects.

    PubMed

    Sarikouch, Samir; Boethig, Dietmar; Peters, Brigitte; Kropf, Siegfried; Dubowy, Karl-Otto; Lange, Peter; Kuehne, Titus; Haverich, Axel; Beerbaum, Philipp

    2013-11-01

    In repaired congenital heart disease, there is increasing evidence of sex differences in cardiac remodeling, but there is a lack of comparable data for specific congenital heart defects such as in repaired tetralogy of Fallot. In a prospective multicenter study, a cohort of 272 contemporary patients (158 men; mean age, 14.3±3.3 years [range, 8-20 years]) with repaired tetralogy of Fallot underwent cardiac magnetic resonance for ventricular function and metabolic exercise testing. All data were transformed to standard deviation scores according to the Lambda-Mu-Sigma method by relating individual values to their respective 50th percentile (standard deviation score, 0) in sex-specific healthy control subjects. No sex differences were observed in age at repair, type of repair conducted, or overall hemodynamic results. Relative to sex-specific controls, repaired tetralogy of Fallot in women had larger right ventricular end-systolic volumes (standard deviation scores: women, 4.35; men, 3.25; P=0.001), lower right ventricular ejection fraction (women, -2.83; men, -2.12; P=0.011), lower right ventricular muscle mass (women, 1.58; men 2.45; P=0.001), poorer peak oxygen uptake (women, -1.65; men, -1.14; P<0.001), higher VE/VCO2 (ventilation per unit of carbon dioxide production) slopes (women, 0.88; men 0.58; P=0.012), and reduced peak heart rate (women, -2.16; men -1.74; P=0.017). Left ventricular parameters did not differ between sexes. Relative to their respective sex-specific healthy control subjects, derived standard deviation scores in repaired tetralogy of Fallot suggest that women perform poorer than men in terms of right ventricular systolic function as tested by cardiac magnetic resonance and exercise capacity. This effect cannot be explained by selection bias. Further outcome data are required from longitudinal cohort studies.

  2. Disarming Burkholderia pseudomallei: Structural and Functional Characterization of a Disulfide Oxidoreductase (DsbA) Required for Virulence In Vivo

    PubMed Central

    McMahon, Róisín M.; Marshall, Laura E.; Halili, Maria; Furlong, Emily; Tay, Stephanie; Sarkar-Tyson, Mitali

    2014-01-01

    Abstract Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei. Conclusion: These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism. Antioxid. Redox Signal. 20, 606–617. PMID:23901809

  3. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-04-01

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  4. RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

    PubMed Central

    Chen, Su; Wang, Chen; Sun, Luxi; Wang, Da-Liang; Chen, Lu; Huang, Zhuan; Yang, Qi; Gao, Jie; Yang, Xi-Bin; Chang, Jian-Feng; Chen, Ping; Lan, Li

    2014-01-01

    Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients. PMID:25384975

  5. DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7.

    PubMed

    Gkotzamanidou, Maria; Terpos, Evangelos; Bamia, Christina; Munshi, Nikhil C; Dimopoulos, Meletios A; Souliotis, Vassilis L

    2016-09-01

    DNA repair activity of malignant cells seems to influence therapeutic outcome and patients' survival. Herein, we investigated the mechanistic basis for the link between DNA repair efficiency and response to antimyeloma therapy. Nucleotide excision repair (NER), interstrand cross-links repair (ICL/R), double-strand breaks repair (DSB/R), and chromatin structure were evaluated in multiple myeloma (MM) cell lines (melphalan-sensitive RPMI8226; melphalan-resistant LR5) and bone marrow plasma cells (BMPCs) from MM patients who responded (n = 17) or did not respond (n = 9) to subsequent melphalan therapy. The effect of DSB/R inhibition was also evaluated. Responders' BMPCs showed slower rates of NER and DSB/R (P <0022), similar rates of ICL/R, and more condensed chromatin structure compared with nonresponders. Moreover, apoptosis rates of BMPCs were inversely correlated with individual DNA repair efficiency and were higher in responders' cells compared with those of nonresponders (P = .0011). Similarly, RPMI8226 cells showed slower rates of NER and DSB/R, comparable rates of ICL/R, more condensed chromatin structure, and higher sensitivity than LR5 cells. Interestingly, cotreatment of BMPCs or cell lines with DSB/R inhibitors significantly reduced the rates of DSB/R and increased melphalan sensitivity of the cells, with the nonhomologous end-joining inhibitor SCR7 showing the strongest effect. Together, responders' BMPCs are characterized by lower efficiencies of NER and DSB/R mechanisms, resulting in higher accumulation of the extremely cytotoxic ICLs and DSBs lesions, which in turn triggers the induction of the apoptotic pathway. Moreover, the enhancement of melphalan cytotoxicity by DSB/R inhibition offers a promising strategy toward improvement of existing antimyeloma regimens. © 2016 by The American Society of Hematology.

  6. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection

    PubMed Central

    Westmoreland, James W.; Resnick, Michael A.

    2016-01-01

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5′-3′ resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced ‘dirty’ DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. PMID:26503252

  7. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection.

    PubMed

    Westmoreland, James W; Resnick, Michael A

    2016-01-29

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5'-3' resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced 'dirty' DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  8. Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.

    PubMed

    He, Mu-Dan; Zhang, Feng-Hua; Wang, Hua-Lin; Wang, Hou-Peng; Zhu, Zuo-Yan; Sun, Yong-Hua

    2015-10-01

    DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    PubMed

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  10. Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy.

    PubMed

    Xu, Fei; Fang, Yixuan; Yan, Lili; Xu, Lan; Zhang, Suping; Cao, Yan; Xu, Li; Zhang, Xiaoying; Xie, Jialing; Jiang, Gaoyue; Ge, Chaorong; An, Ni; Zhou, Daohong; Yuan, Na; Wang, Jianrong

    2017-03-27

    Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

  11. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

  12. Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

    PubMed Central

    Xu, Fei; Fang, Yixuan; Yan, Lili; Xu, Lan; Zhang, Suping; Cao, Yan; Xu, Li; Zhang, Xiaoying; Xie, Jialing; Jiang, Gaoyue; Ge, Chaorong; An, Ni; Zhou, Daohong; Yuan, Na; Wang, Jianrong

    2017-01-01

    Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult. PMID:28345663

  13. Preliminary crystallographic data of the three homologues of the thiol–disulfide oxidoreductase DsbA in Neisseria meningitidis

    PubMed Central

    Lafaye, Céline; Iwena, Thomas; Ferrer, Jean-Luc; Kroll, J. Simon; Griat, Mickael; Serre, Laurence

    2008-01-01

    Bacterial virulence depends on the correct folding of surface-exposed proteins, a process that is catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. Uniquely among bacteria, the Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host-interactive biology, while the function of DsbA3 remains unknown. In an attempt to shed light on the reason for this multiplicity of dsbA genes, the three enzymes from N. meningitidis have been purified and crystallized in the presence of high concentrations of ammonium sulfate. The best crystals were obtained using DsbA1 and DsbA3; they belong to the orthorhombic and tetragonal systems and diffract to 1.5 and 2.7 Å resolution, respectively. PMID:18259062

  14. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  15. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  16. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

    SciTech Connect

    Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  17. Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

    PubMed Central

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand

    2008-01-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  18. RNA-templated DNA repair

    PubMed Central

    Storici, Francesca; Bebenek, Katarzyna; Kunkel, Thomas A.; Gordenin, Dmitry A.; Resnick, Michael A.

    2007-01-01

    RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons1 and in the elongation of telomeres2. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining3. An indirect role for RNA in DNA repair, following reverse transcription and formation of a complementary DNA, has been observed in the non-homologous joining of DSB ends4,5. In the yeast Saccharomyces cerevisiae, in which homologous recombination is efficient3, RNA was shown to mediate recombination, but only indirectly through a cDNA intermediate6,7 generated by the reverse transcriptase function of Ty retrotransposons in Ty particles in the cytoplasm8. Although pairing between duplex DNA and single-strand (ss)RNA can occur in vitro9,10 and in vivo11, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We show here that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. This and the observation that even yeast replicative DNA polymerases such as α and δ can copy short RNA template tracts in vitro demonstrate that RNA can transfer genetic information in vivo through direct homologous interaction with chromosomal DNA. PMID:17429354

  19. PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks.

    PubMed

    Metzger, Michael J; Stoddard, Barry L; Monnat, Raymond J

    2013-07-01

    Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed 'nickase' variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.

  20. A receiver function study across the Dead Sea Basin (DSB)

    NASA Astrophysics Data System (ADS)

    Mohsen, A.; Asch, G.; Hofstetter, R.; Kind, R.; Weber, M.

    2008-12-01

    Beginning in September 2006, a temporary network of 30 broadband and 45 short-period seismic stations has been set up on both sides of the Dead Sea Basin (DSB). During one and a half year of successful operation, data were continuously recorded in the field at 100 Hz and 200 Hz sample frequency for the broadband and short-period seismic stations, respectively. The raw data were converted to miniseed format and archived as full seed volume in the GEOFON data center of the GFZ. In the present work, the Receiver Function Method has been applied to the three component passive source data to investigate seismic discontinuities from the crust down to the upper mantle. Unusual negative phases at about 1s delay time have been observed at several stations in the Dead Sea region on the top of the assumed salt diapir. First preliminary receiver function analysis reveals a crustal thickness of about 30 -35 km in the investigated area and possibly low-velocity layer beneath the Moho. It also shows a basin which is possibly filled with salt about 10 km thick beneath the Lisan peninsula (Dead Sea).

  1. Blockage of Autophagy in C6 Glioma Cells Enhanced Radiosensitivity Possibly by Attenuating DNA-PK-Dependent DSB Due to Limited Ku Nuclear Translocation and DNA Binding.

    PubMed

    Liu, C; He, W; Jin, M; Li, H; Xu, H; Liu, H; Yang, K; Zhang, T; Wu, G; Ren, J

    2015-01-01

    Glioblastoma multiforme (GBM) is the most lethal brain tumor and notorious for its resistance to ionizing radiation (IR). Recent evidence suggests that one possible mechanism that enables resistance to IR and protects cells against therapeutic stress is cellular autophagy. The molecular basis for this pro-survival function, however, remains elusive. Herein, we report a molecular mechanism by which IR-induced autophagy accelerates the repair of DNA double-strand breaks (DSB). We demonstrate that IR induces the accumulation of autophagosomes, which is accompanied by elevated expression of autophagyrelated genes beclin-1, atg5, atg7, and atg12. Beclin-1 knockdown impaired the induction of IR-mediated autophagy and significantly sensitized glioma cells to radiation therapy in vitro and in vivo. Furthermore, our data is the first to demonstrate that the radiosensitizing effect of beclin-1 knockdown may result from the disruption of nuclear translocation and DNA binding activity of Ku proteins and consequent attenuation of DSB repair. Our findings help advance our understanding of the molecular mechanisms underlying IR-induced autophagy and provide a promising adjunctive therapeutic strategy for the radiosensitization of malignant glioma.

  2. Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

    PubMed Central

    Wilson, Paul F.; Hinz, John M.; Urbin, Salustra S.; Nham, Peter B.; Thompson, Larry H.

    2010-01-01

    The repair of DNA double-strand breaks (DSB) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and nonhomologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after γ-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300 cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction (~20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had ~2-fold higher chromatid-type CAs and a remarkable ~25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al. DNA Repair 4, 782–792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2. PMID:20434408

  3. Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

    PubMed

    Guigueno, A; Belin, P; Boquet, P L

    1997-05-01

    The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.

  4. Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

    PubMed Central

    Guigueno, A; Belin, P; Boquet, P L

    1997-01-01

    The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency. PMID:9150222

  5. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  6. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  7. Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

    PubMed Central

    Lin, Han; Ha, Kyungsoo; Lu, Guojun; Fang, Xiao; Cheng, Ranran; Zuo, Qiuhong

    2015-01-01

    Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair. PMID:26283732

  8. Repair of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    The genetic information of cells continuously undergoes damage induced by intracellular processes including energy metabolism, DNA replication and transcription, and by environmental factors such as mutagenic chemicals and UV and ionizing radiation. This causes numerous DNA lesions, including double strand breaks (DSBs). Since cells cannot escape this damage or normally function with a damaged genome, several DNA repair mechanisms have evolved. Although most "single-stranded" DNA lesions are rapidly removed from DNA without permanent damage, DSBs completely break the DNA molecule, presenting a real challenge for repair mechanisms, with the highest risk among DNA lesions of incorrect repair. Hence, DSBs can have serious consequences for human health. Therefore, in this chapter, we will refer only to this type of DNA damage. In addition to the biochemical aspects of DSB repair, which have been extensively studied over a long period of time, the spatio-temporal organization of DSB induction and repair, the importance of which was recognized only recently, will be considered in terms of current knowledge and remaining questions.

  9. Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.

    PubMed

    Kostyrko, Kaja; Mermod, Nicolas

    2016-04-07

    DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.

  10. Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms

    PubMed Central

    Kostyrko, Kaja; Mermod, Nicolas

    2016-01-01

    DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways. PMID:26657630

  11. Effect of environmental factors and cell physiological state on Pulsed Electric Fields resistance and repair capacity of various strains of Escherichia coli.

    PubMed

    Somolinos, M; García, D; Mañas, P; Condón, S; Pagán, R

    2008-06-10

    The aim was to determine the resistance variation of four strains of Escherichia coli to Pulsed Electric Fields (PEF), the role of the sigma factor RpoS in PEF resistance, as well as the influence of several environmental factors and the cell physiological state on the PEF resistance and repair capacity. The rpoS null mutant, E. coli BJ4L1, exhibited decreased PEF resistance as compared with its wild-type parent, BJ4. W3110 and O157:H7 were the most PEF-resistant strains: whereas 2 and more than 3 Log10 cycles of BJ4 and BJ4L1 cells, respectively, were inactivated after 50 pulses at 35 kV/cm, only 0.5 Log10 cycle of inactivation of W3110 and O157:H7 was attained. A different pattern was observed and the resistance variation among strains was largely reduced, when selective recovery media were used. At exponential growth phase, the resistance of the four strains was lower, and more than 4 Log10 cycles of inactivation of all strains tested were attained at 30 kV/cm. Previous heat and cold shock treatments scarcely influenced cell PEF resistance. PEF survival increased with the reduction in water activity of the treatment medium to 0.94: the occurrence of sublethally injured cells was negligible, and less than 1 Log10 cycle of inactivation was attained at 35 kV/cm. PEF-treated cells were sensitive to a subsequent storage at pH 4.0 or in the presence of sorbic acid, attaining a final inactivation of 4-5 Log10 cycles after 24 hour-incubation. In conclusion, the work confirms the role of rpoS in PEF resistance. E. coli strains exhibit large differences in PEF resistance. These differences were less important when cells were recovered under selective conditions. Both resistance variation among strains and occurrence of sublethal damage were noticeably influenced by the environmental factors tested.

  12. Transcriptional regulation of the assT-dsbL-dsbI gene cluster in Salmonella enterica serovar Typhi IMSS-1 depends on LeuO, H-NS, and specific growth conditions.

    PubMed

    Gallego-Hernández, A L; Hernández-Lucas, I; De la Cruz, M A; Olvera, L; Morett, E; Medina-Aparicio, L; Ramírez-Trujillo, J A; Vázquez, A; Fernández-Mora, M; Calva, E

    2012-05-01

    The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ΔleuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions.

  13. Transcriptional Regulation of the assT-dsbL-dsbI Gene Cluster in Salmonella enterica Serovar Typhi IMSS-1 Depends on LeuO, H-NS, and Specific Growth Conditions

    PubMed Central

    Gallego-Hernández, A. L.; Hernández-Lucas, I.; De la Cruz, M. A.; Olvera, L.; Morett, E.; Medina-Aparicio, L.; Ramírez-Trujillo, J. A.; Vázquez, A.; Fernández-Mora, M.

    2012-01-01

    The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ΔleuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions. PMID:22343301

  14. Radio-over-fiber DSB-to-SSB conversion using semiconductor lasers at stable locking dynamics.

    PubMed

    Hsieh, Kun-Lin; Hung, Yu-Han; Hwang, Sheng-Kwang; Lin, Chien-Chung

    2016-05-02

    In radio-over-fiber systems, optical single-sideband (SSB) modulation signals are preferred to optical double-sideband (DSB) modulation signals for fiber distribution in order to mitigate the microwave power fading effect. However, typically adopted modulation schemes generate DSB signals, making DSB-to-SSB conversion necessary before or after fiber distribution. This study investigates a semiconductor laser at stable locking dynamics for such conversion. The conversion relies solely on the nonlinear dynamical interaction between an input DSB signal and the laser. Only a typical semiconductor laser is therefore required as the key conversion unit, and no pump or probe signal is necessary. The conversion can be achieved for a broad tunable range of microwave frequency up to at least 60 GHz. In addition, the conversion can be carried out even when the microwave frequency, the power of the input DSB signal, or the frequency of the input DSB signal fluctuates over a wide range, leading to high adaptability and stability of the conversion system. After conversion, while the microwave phase quality, such as linewidth and phase noise, is mainly preserved, a bit-error ratio down to 10-9 is achieved for a data rate up to at least 8 Gb/s with a detection sensitivity improvement of more than 1.5 dB.

  15. Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

    SciTech Connect

    Kurz, M.; Iturbe-Ormaetxe, I.; Jarrott, R.; O’Neill, S. L.; Byriel, K. A.; Martin, J. L. Heras, B.

    2008-02-01

    The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.

  16. Linking Cancer Metabolism to DNA Repair and Accelerated Senescence

    PubMed Central

    Efimova, Elena V.; Takahashi, Satoe; Shamsi, Noumaan A.; Wu, Ding; Labay, Edwardine; Ulanovskaya, Olesya A.; Weichselbaum, Ralph R.; Kozmin, Sergey A.; Kron, Stephen J.

    2015-01-01

    Conventional wisdom ascribes metabolic reprogramming in cancer to meeting increased demands for intermediates to support rapid proliferation. Prior models have proposed benefits toward cell survival, immortality and stress resistance while the recent discovery of oncometabolites has shifted attention to chromatin targets affecting gene expression. To explore further effects of cancer metabolism and epigenetic deregulation, DNA repair kinetics were examined in cells treated with metabolic intermediates, oncometabolites and/or metabolic inhibitors by tracking resolution of double strand breaks (DSBs) in irradiated MCF7 breast cancer cells. Disrupting cancer metabolism revealed roles for both glycolysis and glutaminolysis in promoting DSB repair and preventing accelerated senescence after irradiation. Targeting pathways common to glycolysis and glutaminolysis uncovered opposing effects of the hexosamine biosynthetic pathway (HBP) and tricarboxylic acid (TCA) cycle. Treating cells with the HBP metabolite N-acetylglucosamine (GlcNAc) or augmenting protein O-GlcNAcylation with small molecules or RNAi targeting O-GlcNAcase enhanced DSB repair, while targeting O-GlcNAc transferase reversed GlcNAc’s effects. Opposing the HBP, TCA metabolites including α-ketoglutarate blocked DSB resolution. Strikingly, DNA repair could be restored by the oncometabolite 2-hydroxyglutarate (2-HG). Targeting downstream effectors of histone methylation and demethylation implicated the PRC1/2 polycomb complexes as the ultimate targets for metabolic regulation, reflecting known roles for Polycomb group proteins in non-homologous end-joining (NHEJ) DSB repair. Our findings that epigenetic effects of cancer metabolic reprogramming may promote DNA repair provide a molecular mechanism by which deregulation of metabolism may not only support cell growth but also maintain cell immortality, drive therapeutic resistance and promote genomic instability. PMID:26538285

  17. DNA double-strand–break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice

    PubMed Central

    Schipler, Agnes; Iliakis, George

    2013-01-01

    Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice. PMID:23804754

  18. The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells.

    PubMed

    Sevcik, Jan; Falk, Martin; Kleiblova, Petra; Lhota, Filip; Stefancikova, Lenka; Janatova, Marketa; Weiterova, Lenka; Lukasova, Emilie; Kozubek, Stanislav; Pohlreich, Petr; Kleibl, Zdenek

    2012-05-01

    The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.

  19. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    PubMed

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

  20. DSB:Ce3+ scintillation glass for future

    NASA Astrophysics Data System (ADS)

    Auffray, E.; Akchurin, N.; Benaglia, A.; Borisevich, A.; Cowden, C.; Damgov, J.; Dormenev, V.; Dragoiu, C.; Dudero, P.; Korjik, M.; Kozlov, D.; Kunori, S.; Lecoq, P.; Lee, S. W.; Lucchini, M.; Mechinsky, V.; Pauwels, K.

    2015-02-01

    One of the main challenges for detectors at future high-energy collider experiments is the high precision measurement of hadron and jet energy and momentum. One possibility to achieve this is the dual-readout technique, which allows recording simultaneously scintillation and Cherenkov light in an active medium in order to extract the electromagnetic fraction of the total shower energy on an event- by-event basis. Making use of this approach in the high luminosity LHC, however, puts stringent requirements on the active materials in terms of radiation hardness. Consequently, the R&D carried out on suitable scintillating materials focuses on the detector performance as well as on radiation tolerance. Among the different scintillating materials under study, scintillating glasses can be a suitable solution due to their relatively simple and cost effective production. Recently a new type of inorganic scintillating glass: Cerium doped DSB has been developed by Radiation Instruments and New Components LLC in Minsk for oil logging industry. This material can be produced either in form of bulk or fiber shape with diameter 0.3-2mm and length up to 2000 mm. It is obtained by standard glass production technology at temperature 1400°C with successive thermal annealing treatment at relatively low temperature. The production of large quantities is relatively easy and the production costs are significantly lower compared to crystal fibers. Therefore, this material is considered as an alternative and complementary solution to crystal fibers in view of a production at industrial scale, as required for a large dual readout calorimeter. In this paper, the first results on optical, scintillation properties as well as the radiation damage behaviour obtained on different samples made with different raw materials and various cerium concentrations will be presented.

  1. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells.

    PubMed

    Shahar, O D; Raghu Ram, E V S; Shimshoni, E; Hareli, S; Meshorer, E; Goldberg, M

    2012-07-26

    DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ∼10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability.

  2. Alternative end-joining and classical nonhomologous end-joining pathways repair different types of double-strand breaks during class-switch recombination.

    PubMed

    Cortizas, Elena M; Zahn, Astrid; Hajjar, Maurice E; Patenaude, Anne-Marie; Di Noia, Javier M; Verdun, Ramiro E

    2013-12-01

    Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

  3. Relative contribution of four nucleases, CtIP, Dna2, Exo1 and Mre11, to the initial step of DNA double-strand break repair by homologous recombination in both the chicken DT40 and human TK6 cell lines.

    PubMed

    Hoa, Nguyen Ngoc; Akagawa, Remi; Yamasaki, Tomomi; Hirota, Kouji; Sasa, Kentaro; Natsume, Toyoaki; Kobayashi, Junya; Sakuma, Tetsushi; Yamamoto, Takashi; Komatsu, Kenshi; Kanemaki, Masato T; Pommier, Yves; Takeda, Shunichi; Sasanuma, Hiroyuki

    2015-12-01

    Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.

  4. Non-homologous end joining repair in Xenopus egg extract.

    PubMed

    Zhu, Songli; Peng, Aimin

    2016-06-21

    Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair mechanism. We characterized here a series of plasmid-based DSB templates that were repaired in Xenopus egg extracts via the canonical, Ku-dependent NHEJ pathway. We showed that the template with compatible ends was efficiently repaired without end processing, in a manner that required the kinase activity of DNA-PKcs but not ATM. Moreover, non-compatible ends with blunt/3'-overhang, blunt/5'-overhang, and 3'-overhang/5'-overhang were predominantly repaired with fill-in and ligation without the removal of end nucleotides. In contrast, 3'-overhang/3'-overhang and 5'-overhang/5'-overhang templates were processed by resection of 3-5 bases and fill-in of 1-4 bases prior to end ligation. Therefore, the NHEJ machinery exhibited a strong preference for precise repair; the presence of neither non-compatible ends nor protruding single strand DNA sufficiently warranted the action of nucleases. ATM was required for the efficient repair of all non-compatible ends including those repaired without end processing by nucleases, suggesting its role beyond phosphorylation and regulation of Artemis. Finally, dephosphorylation of the 5'-overhang/3'-overhang template reduced the efficiency of DNA repair without increasing the risk of end resection, indicating that end protection via prompt end ligation is not the sole mechanism that suppresses the action of nucleases.

  5. Contribution of phenolic compounds to the UV-B screening capacity of developing barley primary leaves in relation to DNA damage and repair under elevated UV-B levels.

    PubMed

    Schmitz-Hoerner, Rainer; Weissenböck, Gottfried

    2003-09-01

    Epidermally located UV-absorbing hydroxycinnamic acid conjugates and flavonoid glycosides are known to be efficient UV-B protectants in higher plants, although important biological molecules are not always fully protected. However, repair mechanisms also exist, such as repair of damaged DNA by photolyases. To distinguish between the relative importance of the phenolic compounds and of DNA repair, developing primary leaves of two barley lines, mutant ant 30-310, deficient in flavonoids, and its parent line Ca 33787, were grown under relatively high visible light (650-700 micromol m(-2) s(-1) max for 6 h in a 13 h photoperiod) and supplemented with (+ UV-B) or without (-UV-B) 12 kJ m(-2) UV-B(BE) for 6 h daily. UV-B screening capacity of the leaf phenolics was determined at 315 nm during leaf development and compared with thymine dimers (TD) accumulation, as an indicator of UV-B-induced DNA damage and potential subsequent repair. The degree of damage was related to the phenolic contents of the leaves. UV-B screening capacity was increased ca. 4-fold in the parent line (+ UV-B), mainly due to UV-induced flavonoid (saponarin, lutonarin) accumulation in epidermal and subepidermal mesophyll tissue, relative to the flavonoid-deficient mutant. Nevertheless, in the parent line an 8-fold increase in TD levels occurred over the growth period of 18 days, whereas the mutant accumulated additional DNA damage, with 6- to 9-fold higher TD amounts. Surprisingly, under the high UV-B irradiation, growth and development of the primary leaves in both lines were only slightly reduced.

  6. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  7. Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired.

    PubMed

    Morel, Frederic; Renoux, Monique; Lachaume, Philippe; Alziari, Serge

    2008-01-01

    Mitochondrial DNA lesions cause numerous human diseases, and it is therefore important to identify the mechanisms whereby the mitochondrion repairs the damage. We have studied in cultured Drosophila cells the repair of bleomycin-induced double-strand breaks (DSBs) in mitochondrial DNA. Our results show that DSBs are repaired as rapidly and effectively in the mitochondria as in the nucleus. DNA repair is complete within 2h following bleomycin treatment, showing that Drosophila mitochondria have an effective system of DSB repair. The mechanism and mitochondrial proteins involved remain to be identified.

  8. NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR.

    PubMed

    Yin, Kelvin; Chhabra, Yash; Tropée, Romain; Lim, Yi Chieh; Fane, Mitchell; Dray, Eloise; Sturm, Richard A; Smith, Aaron G

    2017-09-01

    Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184-96. ©2017 AACR. ©2017 American Association for Cancer Research.

  9. Engineering of Helicobacter pylori Dimeric Oxidoreductase DsbK (HP0231)

    PubMed Central

    Bocian-Ostrzycka, Katarzyna M.; Grzeszczuk, Magdalena J.; Banaś, Anna M.; Jastrząb, Katarzyna; Pisarczyk, Karolina; Kolarzyk, Anna; Łasica, Anna M.; Collet, Jean-François; Jagusztyn-Krynicka, Elżbieta K.

    2016-01-01

    The formation of disulfide bonds that are catalyzed by proteins of the Dsb (disulfide bond) family is crucial for the correct folding of many extracytoplasmic proteins. Thus, this formation plays an essential, pivotal role in the assembly of many virulence factors. The Helicobacter pylori disulfide bond-forming system is uncomplicated compared to the best-characterized Escherichia coli Dsb pathways. It possesses only two extracytoplasmic Dsb proteins named HP0377 and HP0231. As previously shown, HP0377 is a reductase involved in the process of cytochrome c maturation. Additionally, it also possesses disulfide isomerase activity. HP0231 was the first periplasmic dimeric oxidoreductase involved in disulfide generation to be described. Although HP0231 function is critical for oxidative protein folding, its structure resembles that of dimeric EcDsbG, which does not confer this activity. However, the HP0231 catalytic motifs (CXXC and the so-called cis-Pro loop) are identical to that of monomeric EcDsbA. To understand the functioning of HP0231, we decided to study the relations between its sequence, structure and activity through an extensive analysis of various HP0231 point mutants, using in vivo and in vitro strategies. Our work shows the crucial role of the cis-Pro loop, as changing valine to threonine in this motif completely abolishes the protein function in vivo. Functioning of HP0231 is conditioned by the combination of CXXC and the cis-Pro loop, as replacing the HP0231 CXXC motif by the motif from EcDsbG or EcDsbC results in bifunctional protein, at least in E. coli. We also showed that the dimerization domain of HP0231 ensures contact with its substrates. Moreover, the activity of this oxidase is independent on the structure of the catalytic domain. Finally, we showed that HP0231 chaperone activity is independent of its redox function. PMID:27507968

  10. Crustal structure of the Dead Sea Basin (DSB) from a receiver function analysis

    NASA Astrophysics Data System (ADS)

    Mohsen, A.; Asch, G.; Mechie, J.; Kind, R.; Hofstetter, R.; Weber, M.; Stiller, M.; Abu-Ayyash, K.

    2011-01-01

    The Dead Sea Transform (DST) is a major left-lateral strike-slip fault that accommodates the relative motion between the African and Arabian plates, connecting a region of extension in the Red Sea to the Taurus collision zone in Turkey over a length of about 1100 km. The Dead Sea Basin (DSB) is one of the largest basins along the DST. The DSB is a morphotectonic depression along the DST, divided into a northern and a southern sub-basin, separated by the Lisan salt diapir. We report on a receiver function study of the crust within the multidisciplinary geophysical project, DEad Sea Integrated REsearch (DESIRE), to study the crustal structure of the DSB. A temporary seismic network was operated on both sides of the DSB between 2006 October and 2008 April. The aperture of the network is approximately 60 km in the E-W direction crossing the DSB on the Lisan peninsula and about 100 km in the N-S direction. Analysis of receiver functions from the DESIRE temporary network indicates that Moho depths vary between 30 and 38 km beneath the area. These Moho depth estimates are consistent with results of near-vertical incidence and wide-angle controlled-source techniques. Receiver functions reveal an additional discontinuity in the lower crust, but only in the DSB and west of it. This leads to the conclusion that the internal crustal structure east and west of the DSB is different at the present-day. However, if the 107 km left-lateral movement along the DST is taken into account, then the region beneath the DESIRE array where no lower crustal discontinuity is observed would have lain about 18 Ma ago immediately adjacent to the region under the previous DESERT array west of the DST where no lower crustal discontinuity is recognized.

  11. Crustal structure of the Dead Sea Basin (DSB) from a receiver function analysis

    NASA Astrophysics Data System (ADS)

    Mohsen, A.; Asch, G.; Mechie, J.; Hofstetter, R.; Kind, R.; Weber, M. H.; Stiller, M.; Abu-Ayyash, K.

    2010-12-01

    The Dead Sea Transform (DST) is a major left-lateral strike-slip fault that accommodates the relative motion between the African and Arabian plates, connecting a region of extension in the Red Sea to the Taurus collision zone in Turkey over a length of about 1100 km. The Dead Sea Basin (DSB) is one of the largest basins along the DST. The DSB is a morphotectonic depression along the DST, divided into a northern and a southern sub-basin, separated by the Lisan salt diapir. We report on a receiver function study of the crust within the multidisciplinary geophysical project, DEad Sea Integrated REsearch (DESIRE), to study the crustal structure of the DSB. A temporary seismic network was operated on both sides of the DSB between October 2006 and April 2008. The aperture of the network is approximately 60 km in the E-W direction crossing the DSB on the Lisan peninsula and about 100 km in the N-S direction. Analysis of receiver functions from the DESIRE temporary network indicates that Moho depths vary between 30-38 km beneath the area. These Moho depth estimates are consistent with results of near-vertical incidence and wide-angle controlled-source techniques. Receiver functions reveal an additional discontinuity in the lower crust, but only in the DSB and west of it. This leads to the conclusion that the internal crustal structure east and west of the DSB is different at the present-day. However, if the 107 km left-lateral movement along the DST is taken into account, then the region beneath the DESIRE array where no lower crustal discontinuity is observed would have lain about 18 Ma ago immediately adjacent to the region under the previous DESERT array west of the DST where no lower crustal discontinuity is recognized.

  12. Asymmetrical TWDM-PON with 4 × 25-Gb/s downstream DSB OFDM and 4 × 10-Gb/s upstream OOK modulations

    NASA Astrophysics Data System (ADS)

    Lin, Bangjiang; Li, Yiwei; Zhang, Shihao; Tang, Xuan

    2015-12-01

    We propose and experimentally demonstrate an asymmetrical time wavelength division multiplexing passive optical network (TWDM-PON) scheme with 100-Gb/s downstream and 40-Gb/s upstream capacity using four pairs of wavelengths. Double sideband (DSB) orthogonal frequency division multiplexing (OFDM) with 16-QAM mapping is used for downstream transmission, while on-off keying (OOK) modulation is used for upstream transmission. The experimental results show that the power budget for bidirectional transmission is more than 35.5-dB. This scheme can be a promising solution for 100-Gb/s TWDM-PON with high power budget.

  13. DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncatula.

    PubMed

    Dupuy, Pierre; Gourion, Benjamin; Sauviac, Laurent; Bruand, Claude

    2016-11-23

    The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double strand breaks (DSB). In particular, the reactive oxygen (ROS) and nitrogen (RNS) species produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSB. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, a S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis, or that DSB are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.

  14. Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

    PubMed Central

    Shee, Chandan; Gibson, Janet L.; Darrow, Michele C.; Gonzalez, Caleb; Rosenberg, Susan M.

    2011-01-01

    Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example—a general stress-response–controlled switch to error-prone DNA break (double-strand break, DSB) repair—was suggested to be peculiar to an Escherichia coli F′ conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F− E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response–controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment. PMID:21808005

  15. Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

    PubMed Central

    Checchi, Paula M.; Lawrence, Katherine S.; Van, Mike V.; Larson, Braden J.; Engebrecht, JoAnne

    2014-01-01

    During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes. PMID:24939994

  16. Novel Image Processing Interface to Relate DSB Spatial Distribution from Immunofluorescence Foci Experiments to the State-of-the-Art Models of DNA Breakage

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.

    2004-01-01

    A recently developed software (NASARadiationTrackImage) allows a quick and automatic segmentation of foci that indicate spatial localization of specific proteins that are visualized by immunofluorescence. Of interest are the spatial and temporal distribution of foci such as gammaH2AX, a signal of the phosphorylation of a variant of the histone H2A that has been shown to correspond to DSBs, or proteins involved in DSB processing, such as ATM, Rad51, and p53, following exposures of human cells to high charge and energy (HZE) ion irradiation. Experimental data are recorded as sets of two-dimensional images in color with cells and foci of gammaH2AX, ATM, Rad51 or others shown. Different cells, levels of radiation and timing after radiation were recorded. The software allows us to calculate the number of foci per cell, overall intensity of light in foci and their spatial organization. A simple statistical model allows for testing of foci overlap (eclipse). A more complex statistical model previously known as DNAbreak simulates track structure and random chromosome geometry. It has one adjustable parameter corresponding to an average intensity of DSB creation in cubic micrometers of DNA volume per particle track or unit dose. Its limitation is the low-resolution limit both in physical space and DSB's along DNA. It works adequately on the scale of a cell and provides further insights on how the geometry of tracks and DNA affects genomic damage of the cell and subsequent repair. Future developments of the model for the description of the time evolution of DNA damage response proteins, and more robust track structure models will be discussed.

  17. Novel Image Processing Interface to Relate DSB Spatial Distribution from Immunofluorescence Foci Experiments to the State-of-the-Art Models of DNA Breakage

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.

    2004-01-01

    A recently developed software (NASARadiationTrackImage) allows a quick and automatic segmentation of foci that indicate spatial localization of specific proteins that are visualized by immunofluorescence. Of interest are the spatial and temporal distribution of foci such as gammaH2AX, a signal of the phosphorylation of a variant of the histone H2A that has been shown to correspond to DSBs, or proteins involved in DSB processing, such as ATM, Rad51, and p53, following exposures of human cells to high charge and energy (HZE) ion irradiation. Experimental data are recorded as sets of two-dimensional images in color with cells and foci of gammaH2AX, ATM, Rad51 or others shown. Different cells, levels of radiation and timing after radiation were recorded. The software allows us to calculate the number of foci per cell, overall intensity of light in foci and their spatial organization. A simple statistical model allows for testing of foci overlap (eclipse). A more complex statistical model previously known as DNAbreak simulates track structure and random chromosome geometry. It has one adjustable parameter corresponding to an average intensity of DSB creation in cubic micrometers of DNA volume per particle track or unit dose. Its limitation is the low-resolution limit both in physical space and DSB's along DNA. It works adequately on the scale of a cell and provides further insights on how the geometry of tracks and DNA affects genomic damage of the cell and subsequent repair. Future developments of the model for the description of the time evolution of DNA damage response proteins, and more robust track structure models will be discussed.

  18. Identification of Naval Inhibitory Peptides of Protein-Protein Interactions Involved in DNA Repair as Potential Drugs in Breast Cancer Treatment

    DTIC Science & Technology

    2003-04-01

    importance of this pathway for the cell. The other major pathway for Double Strand Break (DSB) repair is the Ku70/80- mediated Non Homologous End Joining pathway...that should inhibit DNA binding activity. The Ku complex is the primary sensor for the Non Homologous End Joining (NHEJ) pathway of repairing double

  19. Functional diversity of three different DsbA proteins from Neisseria meningitidis.

    PubMed

    Sinha, Sunita; Langford, Paul R; Kroll, J Simon

    2004-09-01

    The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes - nmb0278, nmb0294 and nmb0407 - encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

  20. DNA repair by RNA: Templated, or not templated, that is the question.

    PubMed

    Meers, Chance; Keskin, Havva; Storici, Francesca

    2016-08-01

    Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    PubMed

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  2. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    PubMed

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  3. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair.

    PubMed

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness.

  4. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    DOE PAGES

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; ...

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

  5. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    SciTech Connect

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; van der Meulen, Annelot I.; Borden, Sean V.; van Steensel, Bas; Bindra, Ranjit S.; LaRocque, Jeannine R.; Karpen, Gary H.

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

  6. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    PubMed Central

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness. PMID:27446204

  7. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    PubMed Central

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; van der Meulen, Annelot I.; Borden, Sean V.; van Steensel, Bas; Bindra, Ranjit S.; LaRocque, Jeannine R.; Karpen, Gary H.

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio–temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

  8. Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

    PubMed

    Ohle, Corina; Tesorero, Rafael; Schermann, Géza; Dobrev, Nikolay; Sinning, Irmgard; Fischer, Tamás

    2016-11-03

    RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

  9. Dynamic monitoring of oxidative DNA double-strand break and repair in cardiomyocytes.

    PubMed

    Ye, Bo; Hou, Ning; Xiao, Lu; Xu, Yifan; Xu, Haodong; Li, Faqian

    2016-01-01

    DNA double-strand breaks (DSBs) are most dangerous lesions. To determine whether oxidative stress can induce DSBs and how they are repaired in cardiomyocytes (CMs), cultured neonatal rat CMs were treated with different doses of H2O2 and followed for up to 72 h for monitoring the spatiotemporal dynamics of DNA repair protein assembly/disassembly at DSB foci. The protein levels and foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) increased proportionally to 50, 100, and 200 μmol/L H2O2 after 30 min treatment. When H2O2 was at or above 400 μmol/L, γ-H2AX became predominantly pannuclear. After 30 min, 200 μmol/L of H2O2 treatment, γ-H2AX levels were highest within the first hour and then gradually declined during the recovery and returned to basal levels at 48 h. Among DNA damage transducer kinases, ataxia telangiectasia mutated (ATM) was significantly activated by H2O2 in contrast to mild activation of ATR (ATM and Rad3-related). A DSB binding protein, p53 binding protein 1, formed distinct nuclear foci that colocalized with γ-H2AX foci and phosphorylated ATM. Our findings indicate that DSBs can be induced by H2O2 and ATM is the main kinase to mediate DSB repair in CMs. Therefore, monitoring DSB repair can assess oxidative injury and response in CMs.

  10. Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair

    PubMed Central

    Yu, Helen; Pak, Helen; Hammond-Martel, Ian; Ghram, Mehdi; Rodrigue, Amélie; Daou, Salima; Barbour, Haithem; Corbeil, Luc; Hébert, Josée; Drobetsky, Elliot; Masson, Jean Yves; Di Noia, Javier M.; Affar, El Bachir

    2014-01-01

    The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI–induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function. PMID:24347639

  11. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates

    PubMed Central

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-01-01

    The RecA/Rad51 family of recombinases execute the critical step in homologous recombination (HR): the search for homologous DNA to serve as the template during DNA double-strand break (DSB) repair1–7. Although budding yeast Rad51 has been extensively characterized in vitro3,4,6–9, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. We analyzed Rad51-dependent break-induced replication (BIR) where the invading DSB end and its donor template share 108 bp homology and the donor carries different densities of single-bp mismatches (Fig. 1a). With every 8th bp mismatched, repair was ~14% compared to completely homologous sequences. With every 6th bp mismatched, repair was >5%. Thus completing BIR in vivo overcomes the apparent requirement for at least 6–8 consecutive paired bases inferred from in vitro studies6,8. When recombination occurs without a protruding nonhomologous 3′ tail, mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3′ protruding nonhomologous tail, Msh2 promotes rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favoring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase δ, although Msh2-Mlh1 and Exo1 influence the extent of correction. PMID:28405019

  12. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants.

    PubMed

    Edlinger, Bernd; Schlögelhofer, Peter

    2011-03-01

    Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

  13. Making Ends Meet: Repairing Breaks in Bacterial DNA by Non-Homologous End-Joining

    PubMed Central

    Bowater, Richard; Doherty, Aidan J

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages. PMID:16518468

  14. DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

    PubMed

    Rother, Magdalena B; van Attikum, Haico

    2017-10-05

    Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

  15. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  16. Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

    SciTech Connect

    Premkumar, Lakshmanane Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L.

    2013-10-01

    The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

  17. Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery

    PubMed Central

    Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W; Palle, Komaraiah

    2016-01-01

    Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB. PMID:26871286

  18. Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells

    PubMed Central

    Collis, S. J.; Sangar, V. K.; Tighe, A.; Roberts, S. A.; Clarke, N. W.; Hendry, J. H.; Margison, G. P.

    2002-01-01

    Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 ± 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 ± 3.5% for MGH-U1; 39.9 ± 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect. PMID:11788727

  19. Peroxisome proliferator-activated receptor γ enhances adiponectin secretion via up-regulating DsbA-L expression.

    PubMed

    Jin, Dan; Sun, Jun; Huang, Jing; Yu, Xiaoling; Yu, An; He, Yiduo; Li, Qiang; Yang, Zaiqing

    2015-08-15

    Disulfide-bond A oxidoreductase like-protein (DsbA-L) was identified as a molecular chaperone facilitating the assembly and secretion of adiponectin, an adipokine with multiple beneficial effects. In obesity the level of DsbA-L is reduced with a concomitant decrease of the circulating adiponectin level, especially of the high molecular weight form (HMW). Both rodent and human studies have shown that the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ agonists increase adiponectin levels in serum by activating PPARγ, which up-regulates critical endoplasmic reticulum (ER) chaperones thus facilitating protein folding. As shown in the present study, overexpression of PPARγ in human embryonic kidney (HEK) 293 cells elicited the cellular release of HMW adiponectin. PPARγ enhanced expression of DsbA-L by binding directly to peroxisome proliferator response element (PPRE) site within the DsbA-L promoter. Conversely, in differentiated 3T3-L1 cells, PPARγ knockdown resulted in decreased expression of Adiponectin, DsbA-L and ERp44. DsbA-L expression increased after PPARγ agonist treatment and decreased upon treatment with PPARγ antagonist in 3T3-L1 adipocytes. DsbA-L deficiency in differentiated 3T3-L1 cells impaired the secretion of adiponectin. We therefore propose that DsbA-L plays an important role in facilitating HMW adiponectin formation and release from cells under the regulation of PPARγ. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Structural and Biochemical Characterization of Xylella fastidiosa DsbA Family Members: New insightsinto the Enzyme-Substrate Interaction

    SciTech Connect

    Rinaldi, F.; Meza, A; Gulmarges, B

    2009-01-01

    Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

  1. INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

    PubMed

    Morrison, Ashby J; Highland, Jessica; Krogan, Nevan J; Arbel-Eden, Ayelet; Greenblatt, Jack F; Haber, James E; Shen, Xuetong

    2004-12-17

    While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

  2. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

    PubMed

    Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

    2014-04-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

  3. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair

    PubMed Central

    Morales, Julio C.; Richard, Patricia; Rommel, Amy; Fattah, Farjana J.; Motea, Edward A.; Patidar, Praveen L.; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N.; Chiang, Cheng-Ming; Manley, James L.; Boothman, David A.

    2014-01-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

  4. Effect of the cold pressor test on diffusing capacity. Comparison of normal subjects and those with Raynaud's disease and progressive systemic sclerosis.

    PubMed

    Miller, M J

    1983-09-01

    Single-breath carbon monoxide diffusing capacity (Dsb) was measured before and during immersion of one hand in ice water (cold pressor test) in the following three groups of subjects: (1) normal subjects; (2) patients with isolated Raynaud's disease; and (3) patients with Raynaud's phenomenon and progressive systemic sclerosis. No change in Dsb was found in normal subjects or patients with progressive systemic sclerosis. Patients with isolated Raynaud's disease showed a rise in Dsb during cold pressor testing, the mean increase being 8 percent. These results suggest that a rise in Dsb during exposure to cold is a response unique to patients with isolated Raynaud's disease or Raynaud's phenomenon without progressive systemic sclerosis, and not a normal physiologic response to cold. The lack of change in Dsb in response to cold in progressive systemic sclerosis, interpreted by other authors as an indicator of pulmonary vascular disease, resembles the normal response to a challenge with cold.

  5. G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status.

    PubMed

    Agarwal, Pallavi; Jackson, Stephen P

    2016-10-01

    Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide.

  6. Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

    PubMed

    Smith, Roxanne P; Paxman, Jason J; Scanlon, Martin J; Heras, Begoña

    2016-07-16

    Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.

  7. Analysis of chromatin structure at meiotic DSB sites in yeasts.

    PubMed

    Hirota, Kouji; Fukuda, Tomoyuki; Yamada, Takatomi; Ohta, Kunihiro

    2009-01-01

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion ofchromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

  8. Real-time analysis of double-strand DNA break repair by homologous recombination.

    PubMed

    Hicks, Wade M; Yamaguchi, Miyuki; Haber, James E

    2011-02-22

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.

  9. Real-time analysis of double-strand DNA break repair by homologous recombination

    PubMed Central

    Hicks, Wade M.; Yamaguchi, Miyuki; Haber, James E.

    2011-01-01

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion. PMID:21292986

  10. Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells.

    PubMed

    Golding, Sarah E; Rosenberg, Elizabeth; Khalil, Ashraf; McEwen, Alison; Holmes, Matthew; Neill, Steven; Povirk, Lawrence F; Valerie, Kristoffer

    2004-04-09

    To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.

  11. Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulfide bond formation to an Escherichia coli strain lacking DsbA

    PubMed Central

    Chng, Shu-Sin; Dutton, Rachel J.; Denoncin, Katleen; Vertommen, Didier; Collet, Jean-Francois; Kadokura, Hiroshi; Beckwith, Jonathan

    2012-01-01

    Summary Escherichia coli uses the DsbA/DsbB system for introducing disulfide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulfide bond formation but does not entirely eliminate it. Whether such background disulfide bond forming activity is enzyme-catalyzed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulfide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulfide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulfide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulfenic acid form and reacts with substrate proteins to form mixed disulfide adducts. DsbC either prevents the formation of these mixed disulfides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyze disulfide bond formation. Although this PspE/DsbC system is not responsible for the background disulfide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system. PMID:22809289

  12. The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies

    PubMed Central

    Mansour, Wael Y.; Rhein, Tim

    2010-01-01

    Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate. PMID:20483915

  13. Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair.

    PubMed

    Malewicz, Michal; Kadkhodaei, Banafsheh; Kee, Nigel; Volakakis, Nikolaos; Hellman, Ulf; Viktorsson, Kristina; Leung, Chuen Yan; Chen, Benjamin; Lewensohn, Rolf; van Gent, Dik C; Chen, David J; Perlmann, Thomas

    2011-10-01

    DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

  14. Nucleosome remodelers in double-strand break repair.

    PubMed

    Seeber, Andrew; Hauer, Michael; Gasser, Susan M

    2013-04-01

    ATP-dependent nucleosome remodelers use ATP hydrolysis to shift, evict and exchange histone dimers or octamers and have well-established roles in transcription. Earlier work has suggested a role for nucleosome remodelers such as INO80 in double-strand break (DSB) repair. This review will begin with an update on recent studies that explore how remodelers are recruited to DSBs. We then examine their impact on various steps of repair, focusing on resection and the formation of the Rad51-ssDNA nucleofilament. Finally, we will explore new studies that implicate remodelers in the physical movement of chromatin in response to damage.

  15. p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

    PubMed

    Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

    2015-03-01

    The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

  16. DNA damage and repair after high LET radiation

    NASA Astrophysics Data System (ADS)

    O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

    Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

  17. Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

    PubMed

    Han, Jianxun; Hendzel, Michael J; Allalunis-Turner, Joan

    2006-03-01

    Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

  18. ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.

    PubMed

    Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

    2013-10-11

    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

  19. Regulation of ATM in DNA double strand break repair accounts for the radiosensitivity in human cells exposed to high linear energy transfer ionizing radiation.

    PubMed

    Xue, Lian; Yu, Dong; Furusawa, Yoshiya; Okayasu, Ryuichi; Tong, Jian; Cao, Jianping; Fan, Saijun

    2009-11-02

    High linear energy transfer (LET) radiation shows different biological effects from low-LET radiation. The complex nature of high LET radiation-induced damage, especially the clustered DNA damage, brings about slow repair of DNA double strand breaks (DSBs), which finally lead to higher lethality and chromosome aberration. Ionizing radiation (IR) induced DNA DSBs are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways in mammalian cells. The novel function of ataxia telangiectasia-mutated (ATM) protein is its involvement in the DSB repair of slow kinetics for "dirty" breaks rejoining by NHEJ, this suggests that ATM may play a more important role in high LET radiation-induced DNA damage. We show here that KU55933, an ATM inhibitor could distinctly lower the clonogenic survival in normal human skin fibroblast cells exposed to carbon ion radiation and dramatically impair the normal process for DSB repair. We also implicated the involvement of ATM in the two pathways of DNA DSB repair, with DNA-PKcs and Rad51 as the representative proteins. The phosphorylation of DNA-PKcs at Thr-2609 with both immunoblotting and immunofluorescent staining indicated an ATM-dependent change, while for Rad51, KU55933 pretreatment could postpone the formation of nuclear Rad51 foci. Interestingly, we also found that pretreatment with chloroquine, an ATM stimulator could protect cells from carbon ion radiation only at lower doses. For doses over 1Gy, protection was no longer observed. There was a dose-dependent increase for ATM kinase activity, with saturation at about 1Gy. Chloroquine pretreatment prior to 1Gy of carbon ion radiation did not enhance the autophosphorylation of ATM at serine 1981. The function of ATM in G2/M checkpoint arrest facilitated DSB repair in high-LET irradiation. Our results provide a possible mechanism for the direct involvement of ATM in DSB repair by high-LET irradiation.

  20. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts.

    PubMed

    Aparicio, Tomas; Baer, Richard; Gottesman, Max; Gautier, Jean

    2016-02-15

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

  1. Nuclear position dictates DNA repair pathway choice

    PubMed Central

    Lemaître, Charlène; Grabarz, Anastazja; Tsouroula, Katerina; Andronov, Leonid; Furst, Audrey; Pankotai, Tibor; Heyer, Vincent; Rogier, Mélanie; Attwood, Kathleen M.; Kessler, Pascal; Dellaire, Graham; Klaholz, Bruno; Reina-San-Martin, Bernardo; Soutoglou, Evi

    2014-01-01

    Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus. PMID:25366693

  2. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates.

    PubMed

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-04-20

    The Rad51 (also known as RecA) family of recombinases executes the critical step in homologous recombination: the search for homologous DNA to serve as a template during the repair of DNA double-strand breaks (DSBs). Although budding yeast Rad51 has been extensively characterized in vitro, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. Here, in Saccharomyces cerevisiae, we analysed Rad51-dependent break-induced replication in which the invading DSB end and its donor template share a 108-base-pair homology region and the donor carries different densities of single-base-pair mismatches. With every eighth base pair mismatched, repair was about 14% of that of completely homologous sequences. With every sixth base pair mismatched, repair was still more than 5%. Thus, completing break-induced replication in vivo overcomes the apparent requirement for at least 6-8 consecutive paired bases that has been inferred from in vitro studies. When recombination occurs without a protruding nonhomologous 3' tail, the mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3' protruding nonhomologous tail, Msh2 promotes the rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favouring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase-δ, although the repair proteins Msh2, Mlh1 and Exo1 influence the extent of correction.

  4. 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

  5. Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria

    PubMed Central

    Um, Si-Hyeon; Kim, Jin-Sik; Song, Saemee; Kim, Nam Ah; Jeong, Seong Hoon; Ha, Nam-Chul

    2015-01-01

    In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae. PMID:26082031

  6. Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

    PubMed

    Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K; King, Gordon J; Halili, Maria A; Heras, Begoña; Martin, Jennifer L

    2014-07-18

    The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

  7. Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria.

    PubMed

    Um, Si-Hyeon; Kim, Jin-Sik; Song, Saemee; Kim, Nam Ah; Jeong, Seong Hoon; Ha, Nam-Chul

    2015-08-01

    In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

  8. Tendon repair

    MedlinePlus

    ... the area to see if there are any injuries to nerves and blood vessels. When the repair is complete, the wound is closed. If the tendon damage is too severe, the repair and reconstruction ... to repair part of the injury. Another surgery will be done at a later ...

  9. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

    PubMed

    di Masi, A; Cilli, D; Berardinelli, F; Talarico, A; Pallavicini, I; Pennisi, R; Leone, S; Antoccia, A; Noguera, N I; Lo-Coco, F; Ascenzi, P; Minucci, S; Nervi, C

    2016-07-28

    Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

  10. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

    PubMed Central

    di Masi, A; Cilli, D; Berardinelli, F; Talarico, A; Pallavicini, I; Pennisi, R; Leone, S; Antoccia, A; Noguera, N I; Lo-Coco, F; Ascenzi, P; Minucci, S; Nervi, C

    2016-01-01

    Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis. PMID:27468685

  11. Cytotoxic, mutagenic, and carcinogenic effects of energy-related agents in diploid human cells which differ in DNA repair capacity. Progress report, 1980-1983

    SciTech Connect

    McCormick, J.J.; Maher, V.M.

    1983-01-01

    The mechanisms of action of a series of energy-related chemical carcinogens to bring about cell killing, the induction of mutations, and the transformation of diploid human fibroblasts in culture were examined. Some of these studies have employed direct-acting compounds (reactive derivatives or metabolites of parent compounds) such as (+-)-7..beta..,8..cap alpha..-dihydroxy9..cap alpha.., 10..cap alpha..-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti BPDE), the 4,5-epoxide of benzo(a)pyrene(B(a)P 4,5-oxide), and aflatoxin B/sub 1/-dichloride (AFB/sub 1/-Cl/sub 2/). In other studies, human epithelial cell lines have served as source of the various metabolizing enzymes needed to convert parent compounds into reactive intermediates. We screened a series of 15 epithelial cell lines with unlimited lifespan (derived from various human carcinomas) for the ability to activate representative polycyclic aromatic hydrocarbons, aromatic amides, nitrogen heterocyclics, nitro samines, and/or aflatoxins. Candidate metabolizing cells were then combined with diploid human fibroblasts as target cells in a cell-mediated assay of the mutagenic and/or cytotoxic effect of particular energy-related chemical carcinogens, such as B(a)P, benzofluoranthenes, and dibenzo(c,g)carbazole. The number and kind of major DNA adducts formed in human cells by these chemicals were determined. Comparative studies of anti BPDE and B(a)P 4,5-oxide showed that in diploid human fibroblasts, both normal and DNA repair deficient, these agents do not differ significantly in mutagenic efficiency (per mean lethal event) or mutagenic effectiveness (per DNA adduct). In diploid Chinese hamster embryo fibroblasts anti BPDE had approx. 4-fold higher mutagenic efficiency than B(a)P 4,5-oxide in the latter cells. FA cells were significantly more sensitive than normal to killing by anti BPDE.

  12. DsbA directs efficient expression of outer membrane secretin EscC of the enteropathogenic Escherichia coli type III secretion apparatus.

    PubMed

    Miki, Tsuyoshi; Okada, Nobuhiko; Kim, Yeongsuk; Abe, Akio; Danbara, Hirofumi

    2008-02-01

    The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

  13. Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

    PubMed Central

    White, Ryan R.; Sung, Patricia; Vestal, C. Greer; Benedetto, Gregory; Cornelio, Noelle; Richardson, Christine

    2013-01-01

    Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation. PMID:24349572

  14. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  15. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  16. Repair of DNA double strand breaks: in vivo biochemistry.

    PubMed

    Sugawara, Neal; Haber, James E

    2006-01-01

    Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

  17. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair.

    PubMed

    Chai, Bob; Huang, Jian; Cairns, Bradley R; Laurent, Brehon C

    2005-07-15

    The failure of cells to repair damaged DNA can result in genomic instability and cancer. To efficiently repair chromosomal DNA lesions, the repair machinery must gain access to the damaged DNA in the context of chromatin. Here we report that both the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). RSC and Swi/Snf are each recruited to an in vivo DSB site but with distinct kinetics. We show that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.

  18. Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

    PubMed Central

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

    2014-01-01

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  19. A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli.

    PubMed Central

    Alksne, L E; Keeney, D; Rasmussen, B A

    1995-01-01

    The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation. PMID:7814337

  20. MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena.

    PubMed

    Lukaszewicz, Agnieszka; Howard-Till, Rachel A; Novatchkova, Maria; Mochizuki, Kazufumi; Loidl, Josef

    2010-10-01

    Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.

  1. Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

    PubMed

    Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay; Ahmed, Kazi Mokim; Sengupta, Shiladitya; Pandey, Arvind; Hegde, Pavana M; Tsai, Miaw-Sheue; Tainer, John A; Weinfeld, Michael; Hegde, Muralidhar L; Mitra, Sankar

    2017-03-17

    Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

    PubMed

    Li, Shuyi; Li, Zhentian; Shu, Feng-Jue; Xiong, Hairong; Phillips, Andrew C; Dynan, William S

    2014-09-01

    NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

  3. MRE11 is required for homologous synapsis and DSB processing in rice meiosis.

    PubMed

    Ji, Jianhui; Tang, Ding; Wang, Mo; Li, Yafei; Zhang, Lei; Wang, Kejian; Li, Ming; Cheng, Zhukuan

    2013-10-01

    Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1(RNAi) plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis.

  4. Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

    PubMed

    Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

    2017-04-01

    Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  5. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways.

    PubMed Central

    Boulton, S J; Jackson, S P

    1996-01-01

    Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes. Images PMID:8890183

  6. Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

    PubMed Central

    Zapotoczny, Grzegorz; Sekelsky, Jeff

    2017-01-01

    DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. PMID:28179392

  7. High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

    PubMed

    Missirian, Victor; Conklin, Phillip A; Culligan, Kevin M; Huefner, Neil D; Britt, Anne B

    2014-01-01

    Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants.

  8. Non-homologous end joining repair in Xenopus egg extract

    PubMed Central

    Zhu, Songli; Peng, Aimin

    2016-01-01

    Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair mechanism. We characterized here a series of plasmid-based DSB templates that were repaired in Xenopus egg extracts via the canonical, Ku-dependent NHEJ pathway. We showed that the template with compatible ends was efficiently repaired without end processing, in a manner that required the kinase activity of DNA-PKcs but not ATM. Moreover, non-compatible ends with blunt/3′-overhang, blunt/5′-overhang, and 3′-overhang/5′-overhang were predominantly repaired with fill-in and ligation without the removal of end nucleotides. In contrast, 3′-overhang/3′-overhang and 5′-overhang/5′-overhang templates were processed by resection of 3–5 bases and fill-in of 1–4 bases prior to end ligation. Therefore, the NHEJ machinery exhibited a strong preference for precise repair; the presence of neither non-compatible ends nor protruding single strand DNA sufficiently warranted the action of nucleases. ATM was required for the efficient repair of all non-compatible ends including those repaired without end processing by nucleases, suggesting its role beyond phosphorylation and regulation of Artemis. Finally, dephosphorylation of the 5′-overhang/3′-overhang template reduced the efficiency of DNA repair without increasing the risk of end resection, indicating that end protection via prompt end ligation is not the sole mechanism that suppresses the action of nucleases. PMID:27324260

  9. Double-strand break repair and colorectal cancer: gene variants within 3′ UTRs and microRNAs binding as modulators of cancer risk and clinical outcome

    PubMed Central

    Naccarati, Alessio; Rosa, Fabio; Vymetalkova, Veronika; Barone, Elisa; Jiraskova, Katerina; Di Gaetano, Cornelia; Novotny, Jan; Levy, Miroslav; Vodickova, Ludmila; Gemignani, Federica; Buchler, Tomas; Landi, Stefano

    2016-01-01

    Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome. PMID:26735576

  10. DNA DSB induced by iron ions in human fibroblasts: LET dependence and shielding efficiency

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Campa, A.; Dini, V.; Esposito, G.; Furusawa, Y.; Rydberg, B.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    Galactic Cosmic Rays represent one of the main sources of charged particle radiation outside the magnetic field of the Earth, containing particles of all charges, in a wide energy range. As HZE particles interact with a shield, they fragment and deposit energy at rates depending on the nature and energy of the incident particles, and on the nature and thickness of the shield. We have studied the DNA DSB induction in human fibroblasts by iron ions of different energies (5 GeV/u, 1 GeV/u, 500 MeV/u and 200 MeV) in the absence or presence of different shields (PMMA, Al and Pb). Measure of DNA DSB was performed by gel electrophoresis and .fragmentation analysis. The RBE for unshielded and shielded beams has been plotted as a function of the dose average LET, and the relationship shows a maximum of 1.7 at about 140 keV/μ m (5 GeV/u iron ions). The dose average LET seems to adequately describe the effectiveness of degraded beams since their RBEs fall roughly on the same relationship of the unshielded beams. The cross section for unshielded beams increases with LET. The shielding efficiency has been evaluated by the ratio between the cross sections for unshielded and shielded beams (SPF, shielding protection factor). When this ratio is plotted as a function of the shield thickness (g/cm2), different relationships were found for beams with different energy. For 500 MeV/u iron ions, the presence of the shields gives SPF lower than unity, independently of thickness and material. For 1 GeV/u iron beams, SPF values are always higher than unity. Moreover, PMMA shield gives a higher SPF than Al or Pb shields having the same residual range but different thickness. The higher SPF of PMMA has been confirmed in experiments performed with 5 GeV/u iron beams and different shields having the same thickness. The SPF found for 1 and 5 GeV/u iron ions remain constant also when the fragmentation analysis used for evaluating DNA DSB is extended to fragments of smaller size. The fragment

  11. Repair versus Checkpoint Functions of BRCA1 Are Differentially Regulated by Site of Chromatin Binding.

    PubMed

    Goldstein, Michael; Kastan, Michael B

    2015-07-01

    The product of the Brca1 tumor-suppressor gene is involved in multiple aspects of the cellular DNA damage response (DDR), including activation of cell-cycle arrests and DNA double-stranded break (DSB) repair by homologous recombination. Prior reports demonstrated that BRCA1 recruitment to areas of DNA breakage depended on RAP80 and the RNF8/RNF168 E3 ubiquitin ligases. Here, we extend these findings by showing that RAP80 is only required for the binding of BRCA1 to regions flanking the DSB, whereas BRCA1 binding directly to DNA breaks requires Nijmegen breakage syndrome 1 (NBS1). These differential recruitment mechanisms differentially affect BRCA1 functions: (i) RAP80-dependent recruitment of BRCA1 to chromatin flanking DNA breaks is required for BRCA1 phosphorylation at serine 1387 and 1423 by ATM and, consequently, for the activation of S and G(2) checkpoints; and (ii) BRCA1 interaction with NBS1 upon DSB induction results in an NBS1-dependent recruitment of BRCA1 directly to the DNA break and is required for nonhomologous end-joining repair. Together, these findings illustrate that spatially distinct fractions of BRCA1 exist at the DSB site, which are recruited by different mechanisms and execute different functions in the DDR.

  12. Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

    PubMed Central

    2011-01-01

    Background Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. Results In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. Conclusions The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of

  13. SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

    PubMed

    Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

    2014-03-14

    SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10.

  14. Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

    PubMed

    Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

    2012-10-01

    Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.

  15. Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.

    PubMed Central

    Rattray, A J; McGill, C B; Shafer, B K; Strathern, J N

    2001-01-01

    Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex. PMID:11333222

  16. An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations.

    PubMed

    Igarashi, Kento; Kobayashi, Junya; Katsumura, Takafumi; Urushihara, Yusuke; Hida, Kyohei; Watanabe-Asaka, Tomomi; Oota, Hiroki; Oda, Shoji; Mitani, Hiroshi

    2017-01-01

    Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

  17. An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations

    PubMed Central

    Igarashi, Kento; Kobayashi, Junya; Katsumura, Takafumi; Urushihara, Yusuke; Hida, Kyohei; Watanabe-Asaka, Tomomi; Oota, Hiroki; Oda, Shoji

    2017-01-01

    Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles. PMID:28107384

  18. Modulation of Biofilm-Formation in Salmonella enterica Serovar Typhimurium by the Periplasmic DsbA/DsbB Oxidoreductase System Requires the GGDEF-EAL Domain Protein STM3615

    PubMed Central

    Römling, Ute; Rhen, Mikael

    2014-01-01

    In Salmonella enterica serovar Typhimurium (S. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3′, 5′-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either dsbA or dsbB, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a red, dry and rough (rdar) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting csgD in either dsb mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the dsb mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene STM3615 (yhjK), previously not connected to rdar morphotype development, also abrogated the escalated rdar morphotype formation in dsb mutant backgrounds. Enhanced biofilm-formation in dsb mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both dsb mutants initially showed an escalated rdar morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in S. Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators. PMID:25153529

  19. BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite

    NASA Technical Reports Server (NTRS)

    Sanchez, Hugo; Lewis, Brian; Hanel, Robert

    2015-01-01

    We are designing and developing a 6U (10 x 22 x 34 cm; 14 kg) nanosatellite as a secondary payload to fly aboard NASAs Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinels 12- to 18-month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. In order to understand the relative contributions of the space environments two dominant biological perturbations, reduced gravity and ionizing radiation, results from deep space will be directly compared to data obtained in LEO (on ISS) and on Earth. These data points will be available for validation of existing biological radiation damage and repair models, and for extrapolation to humans, to assist in mitigating risks during future long-term exploration missions beyond LEO. The BioSentinel Payload occupies 4U of the spacecraft and will utilize the monocellular eukaryotic organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from ambient space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. Yeast was selected because of 1) its similarity to cells in higher organisms, 2) the well-established history of strains engineered to measure DSB repair, 3) its spaceflight heritage, and 4) the wealth of available ground and flight reference data. The S. cerevisiae flight strain will include engineered genetic defects to prevent growth and division until a radiation-induced DSB activates the yeasts DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its successful repair. The yeast will be carried in the dry state within the 1-atm PL container in 18 separate fluidics cards with each card

  20. Increased DNA double-strand break was associated with downregulation of repair and upregulation of apoptotic factors in rat hippocampus after alcohol exposure.

    PubMed

    Suman, Shubhankar; Kumar, Santosh; N'Gouemo, Prosper; Datta, Kamal

    2016-08-01

    Binge drinking is known to cause damage in critical areas of the brain, including the hippocampus, which is important for relational memory and is reported to be sensitive to alcohol toxicity. However, the roles of DNA double-strand break (DSB) and its repair pathways, homologous recombination (HR), and non-homologous end joining (NHEJ) in alcohol-induced hippocampal injury remain to be elucidated. The purpose of this first study was to assess alcohol-induced DNA DSB and the mechanism by which alcohol affects DSB repair pathways in rat hippocampus. Male Sprague-Dawley rats (8-10 weeks old) were put on a 4-day binge ethanol treatment regimen. Control animals were maintained under similar conditions but were given the vehicle without ethanol. All animals were humanely euthanized 24 h after the last dose of ethanol administration and the hippocampi were dissected for immunoblot and immunohistochemistry analysis. Ethanol exposure caused increased 4-hydroxynonenal (4-HNE) staining as well as elevated γH2AX and 53BP1 foci in hippocampal cells. Immunoblot analysis showed decreased Mre11, Rad51, Rad50, and Ku86 as well as increased Bax and p21 in samples from ethanol-treated rats. Additionally, we also observed increased activated caspase3 staining in hippocampal cells 24 h after ethanol withdrawal. Taken together, our data demonstrated that ethanol concurrently induced DNA DSB, downregulated DSB repair pathway proteins, and increased apoptotic factors in hippocampal cells. We believe these findings will provide the impetus for further research on DNA DSB and its repair pathways in relation to alcohol toxicity in brain.

  1. Physiologic determinants of exercise capacity in patients with different types of right-sided regurgitant lesions: Ebstein's malformation with tricuspid regurgitation and repaired tetralogy of Fallot with pulmonary regurgitation.

    PubMed

    Chen, S S M; Dimopoulos, K; Sheehan, F H; Gatzoulis, M A; Kilner, P J

    2016-02-15

    Exercise capacity relates to right ventricular (RV) volume overload in congenital heart disease and may improve after surgery. We herewith investigate the relation between exercise capacity, cardiac index, and RV volume overload due to tricuspid regurgitation (TR) in Ebstein's malformation and pulmonary regurgitation (PR) after repair of tetralogy of Fallot (rToF). We measured cardiac index and tricuspid/pulmonary regurgitant fraction by cardiovascular magnetic resonance in patients with Ebstein's malformation (n = 40) or rTOF (n = 53) with at least moderate TR/PR and 24 healthy controls. Exercise tolerance was determined by peak oxygen consumption (peak VO2) during cardiopulmonary exercise testing. TR and PR fraction were similar in Ebstein and rTOF patients (43 ± 17% versus 39 ± 12%, respectively). Cardiac index was reduced in Ebstein (2.7 ± 0.6L/min/m(2) compared to controls 3.5 ± 0.9L/min/m(2), p < 0.001) but not in rToF patients (3.2 ± 0.5L/min/m(2)). Multiple regression analysis revealed a significant correlation between peak VO2 and cardiac index in Ebstein. Furthermore, peak VO2 correlated with peak heart rate in both groups but not with regurgitation fraction. Despite comparable amounts of regurgitation from a right sided heart valve in patients with Ebstein and rToF, reduction of cardiac index was observed only in the former group. Greater physiologic complexity and adverse ventricular interaction with chronotropic incompetence in Ebstein's malformation may account for this. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

  2. Clubfoot repair

    MedlinePlus

    ... Clubfoot release; Talipes equinovarus - repair; Tibialis anterior tendon transfer ... complete blood count and check electrolytes or clotting factors) Always tell your child's provider: What drugs your ...

  3. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

    PubMed

    Deniz, Miriam; Kaufmann, Julia; Stahl, Andreea; Gundelach, Theresa; Janni, Wolfgang; Hoffmann, Isabell; Keimling, Marlen; Hampp, Stephanie; Ihle, Michaela; Wiesmüller, Lisa

    2016-11-01

    Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

  4. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    PubMed

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation