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1

Electrophoretic Mobility Shift Assays for Protein-DNA Complexes Involved in DNA Repair*  

PubMed Central

The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA–PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein–DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein–DNA complex. PMID:22941596

Tsai, Chun; Smider, Vaughn; Hwang, Byung Joon; Chu, Gilbert

2014-01-01

2

UC Davis researchers discover complexities of DNA repair:  

Cancer.gov

An international team of scientists led by UC Davis researchers has discovered that DNA repair in cancer cells is not a one-way street as previously believed. Their findings show instead that recombination, an important DNA repair process, has a self-correcting mechanism that allows DNA to make a virtual u-turn and start over.

3

Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes  

SciTech Connect

Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second objective is to locate the kinase domain of DNA-PKcs by determining the structure of a kinase deletion mutant both as an isolated protein and as part of a DNA-PKcs/Ku/DNA complex. A third objective is to pursue higher resolution studies of DNA-PKcs and the DNA-PKcs/Ku/DNA complex. If the crystal structure determination of DNA-PKcs is completed during the project period, the atomic coordinates of DNA-PKcs will be modeled within the cryo-EM structure of the complex. In order to achieve these goals, a collaborative effort is proposed between Dr. Phoebe Stewart at UCLA, whose laboratory has expertise in cryo-EM reconstruction methods, and Dr. David Chen at the Lawrence Berkeley National Laboratory, who has a long-standing interest in DNA repair. Advantages of the cryo-EM structural method include the fact that the sample is imaged in a frozen-hydrated and unstained state, avoiding artifacts associated with drying and staining in other EM approaches. Also crystals of the sample are not needed for the single particle reconstruction method and only microgram quantities of sample are required. Cryo-EM structural information of macromolecular assemblies is complementary to both atomic structures of individual component molecules, as well as low resolution information obtained from x-ray and neutron scattering. Knowledge of the geometrical arrangement of the complex, and the position of the essential DNA-PKcs kinase domain, should lead to a greater understanding of the molecular events in DNA double-strand break repair following exposure to low doses of radiation.

Phoebe L. Stewart

2005-06-27

4

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair  

SciTech Connect

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand (Montreal)

2010-09-07

5

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a dna substrate: Structural basis for damaged DNA recognition  

Microsoft Academic Search

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 Ĺ resolution. The atomic structure of the complex reveals the unique conformation of the

Dmitry G. Vassylyev; Tatsuki Kashiwagi; Yuriko Mikami; Mariko Ariyoshi; Shigenori Iwai; Eiko Ohtsuka; Kosuke Morikawa

1995-01-01

6

DNA excision repair pathways  

Microsoft Academic Search

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair

Tomas Lindahl; Peter Karran; Richard D Wood

1997-01-01

7

How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk  

SciTech Connect

Exposure to ionizing radiation increases the incidence of cancer. However, predicting which individuals are at most risk from radiation exposure is a distant goal. Predictive ability is needed to guide policies that regulate radiation exposure and ensure that medical treatments have maximum benefit and minimum risk. Differences between people in susceptibility to radiation are largely based on their genotype, the genes inherited from their parents. Among the important genes are those that produce proteins that repair DNA damaged by radiation. Base Excision Repair (BER) proteins repair single strand breaks and oxidized bases in DNA. Double Strand Break Repair proteins repair broken chromosomes. Using technologies and information from the Human Genome Project, we have previously determined that the DNA sequence of DNA repair genes varies within the human population. An average of 3-4 different variants were found that affect the protein for each of 37 genes studied. The average frequency of these variants is 5%. Given the many genes in each DNA repair pathway and their many variants, technical ability to determine an individual's repair genotype greatly exceeds ability to interpret the information. A long-term goal is to relate DNA repair genotypes to health risk from radiation. This study focused on the BER pathway. The BER genes are known, variants of the genes have been identified at LLNL, and LLNL had recently developed an assay for BER function using white blood cells. The goal of this initial effort was to begin developing data that could be used to test the hypothesis that many different genotypes have similar DNA repair capacity phenotypes (function). Relationships between genotype and phenotype could then be used to group genotypes with similar function and ultimately test the association of groups of genotypes with health risk from radiation. Genotypes with reduced repair function are expected to increase risk of radiation-induced health effects. The goal of this pilot project was to obtain preliminary data on genetic variation in DNA repair function in human cells that might encourage our efforts to establish a research program to relate DNA repair function to complex DNA repair genotype and ultimately to cancer risk of radiation exposure.

Jones, IM

2002-01-09

8

The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder.  

PubMed

Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia, a disorder characterized by sensitivity to DNA interstrand crosslinking agents. The clinical features of cytopenia, developmental defects, and tumor predisposition are similar in each group, suggesting that the gene products participate in a common pathway. The Fanconi anemia DNA repair pathway consists of an anchor complex that recognizes damage caused by interstrand crosslinks, a multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several downstream repair proteins including nucleases and homologous recombination enzymes. We review progress in the use of structural and biochemical approaches to understanding how each FANC protein functions in this pathway. PMID:24773018

Walden, Helen; Deans, Andrew J

2014-01-01

9

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway  

SciTech Connect

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P. (Harvard-Med); (Cornell); (MSKCC)

2011-08-29

10

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway  

SciTech Connect

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

2011-12-31

11

The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.  

PubMed

Cells respond to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin-remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs. PMID:20805320

Smeenk, Godelieve; Wiegant, Wouter W; Vrolijk, Hans; Solari, Aldo P; Pastink, Albert; van Attikum, Haico

2010-09-01

12

The Shu complex regulates Rad52 localization during rDNA repair Running Head: Shu1 promotes rDNA repair  

PubMed Central

The Shu complex, consisting of Rad51 paralogues, is an important regulator of homologous recombination, an error-free DNA repair pathway. Consequently, when members of this complex are disrupted, cells exhibit a mutator phenotype, sensitivity to DNA damage reagents and increased gross chromosomal rearrangements. Previously, we found that the Shu complex plays an important role in ribosomal DNA (rDNA) recombination when the Upstream Activating Factor (UAF) protein Uaf30 is disrupted. UAF30 encodes a protein needed for rDNA transcription and when deleted, rDNA recombination increases and the rDNA expands in a Shu1-dependent manner. Here we find using the uaf30-sensitized background that the central DNA repair protein Rad52, which is normally excluded from the nucleolus, frequently overlaps with the rDNA. This close association of Rad52 with the rDNA is dependent upon Shu1 in a uaf30 mutant. Previously, it was shown that in the absence of Rad52 sumoylation, Rad52 foci mislocalize to the nucleolus. Interestingly, here we find that using the uaf30 sensitized background the ability to regulate Rad52 sumoylation is important for Shu1 dependent rDNA recombination as well as Rad52 close association with rDNA. Our results suggest that in the absence of UAF30, the Shu complex plays a central role in Rad52 rDNA localization as long as Rad52 can be sumoylated. This discrimination is important for rDNA copy number homeostasis. PMID:23790361

Bernstein, Kara A.; Juanchich, Amelie; Sunjevaric, Ivana; Rothstein, Rodney

2013-01-01

13

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage  

PubMed Central

Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival. PMID:20966962

De Zio, D; Bordi, M; Tino, E; Lanzuolo, C; Ferraro, E; Mora, E; Ciccosanti, F; Fimia, G M; Orlando, V; Cecconi, F

2011-01-01

14

The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis.  

PubMed

All processes requiring interaction with DNA are attuned to occur within the context of the complex chromatin structure. As it does for programmed transcription and replication, this also holds true for unscheduled events, such as repair of DNA damage. Lesions such as double-strand breaks occur randomly; their repair requires that enzyme complexes access DNA at potentially any genomic site. This is achieved by chromatin remodeling factors that can locally slide, evict, or change nucleosomes. Here, we show that the Swi2/Snf2-related (SWR1 complex), known to deposit histone H2A.Z, is also important for DNA repair in Arabidopsis thaliana. Mutations in genes for Arabidopsis SWR1 complex subunits photoperiod-independent Early Flowering1, actin-related protein6, and SWR1 complex6 cause hypersensitivity to various DNA damaging agents. Even without additional genotoxic stress, these mutants show symptoms of DNA damage accumulation. The reduced DNA repair capacity is connected with impaired somatic homologous recombination, in contrast with the hyper-recombinogenic phenotype of yeast SWR1 mutants. This suggests functional diversification between lower and higher eukaryotes. Finally, reduced fertility and irregular gametogenesis in the Arabidopsis SWR1 mutants indicate an additional role for the chromatin-remodeling complex during meiosis. These results provide evidence for the importance of Arabidopsis SWR1 in somatic DNA repair and during meiosis. PMID:23780875

Rosa, Marisa; Von Harder, Mona; Cigliano, Riccardo Aiese; Schlögelhofer, Peter; Mittelsten Scheid, Ortrun

2013-06-01

15

The Arabidopsis SWR1 Chromatin-Remodeling Complex Is Important for DNA Repair, Somatic Recombination, and Meiosis[W][OPEN  

PubMed Central

All processes requiring interaction with DNA are attuned to occur within the context of the complex chromatin structure. As it does for programmed transcription and replication, this also holds true for unscheduled events, such as repair of DNA damage. Lesions such as double-strand breaks occur randomly; their repair requires that enzyme complexes access DNA at potentially any genomic site. This is achieved by chromatin remodeling factors that can locally slide, evict, or change nucleosomes. Here, we show that the Swi2/Snf2-related (SWR1 complex), known to deposit histone H2A.Z, is also important for DNA repair in Arabidopsis thaliana. Mutations in genes for Arabidopsis SWR1 complex subunits PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, ACTIN-RELATED PROTEIN6, and SWR1 COMPLEX6 cause hypersensitivity to various DNA damaging agents. Even without additional genotoxic stress, these mutants show symptoms of DNA damage accumulation. The reduced DNA repair capacity is connected with impaired somatic homologous recombination, in contrast with the hyper-recombinogenic phenotype of yeast SWR1 mutants. This suggests functional diversification between lower and higher eukaryotes. Finally, reduced fertility and irregular gametogenesis in the Arabidopsis SWR1 mutants indicate an additional role for the chromatin-remodeling complex during meiosis. These results provide evidence for the importance of Arabidopsis SWR1 in somatic DNA repair and during meiosis. PMID:23780875

Rosa, Marisa; Von Harder, Mona; Aiese Cigliano, Riccardo; Schlogelhofer, Peter; Mittelsten Scheid, Ortrun

2013-01-01

16

Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair  

PubMed Central

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair. PMID:24003026

Yang, Hui; Zhang, Tianlong; Tao, Ye; Wang, Fang; Tong, Liang; Ding, Jianping

2013-01-01

17

The BRCA1-RAP80 complex regulates DNA repair mechanism utilization by restricting end resection.  

PubMed

The tumor suppressor protein BRCA1 is a constituent of several different protein complexes and is required for homology-directed repair (HDR) of DNA double strand breaks (DSBs). The most recently discovered BRCA1-RAP80 complex is recruited to ubiquitin structures on chromatin surrounding the break. Deficiency of any member of this complex confers hypersensitivity to DNA-damaging agents by undefined mechanisms. In striking contrast to other BRCA1-containing complexes that are known to promote HDR, we demonstrate that the BRCA1-RAP80 complex restricts end resection in S/G(2) phase of the cell cycle, thereby limiting HDR. RAP80 or BRCC36 deficiency resulted in elevated Mre11-CtIP-dependent 5' end resection with a concomitant increase in HDR mechanisms that rely on 3' single-stranded overhangs. We propose a model in which the BRCA1-RAP80 complex limits nuclease accessibility to DSBs, thus preventing excessive end resection and potentially deleterious homology-directed DSB repair mechanisms that can impair genome integrity. PMID:21335604

Coleman, Kara A; Greenberg, Roger A

2011-04-15

18

The BRCA1-RAP80 Complex Regulates DNA Repair Mechanism Utilization by Restricting End Resection*  

PubMed Central

The tumor suppressor protein BRCA1 is a constituent of several different protein complexes and is required for homology-directed repair (HDR) of DNA double strand breaks (DSBs). The most recently discovered BRCA1-RAP80 complex is recruited to ubiquitin structures on chromatin surrounding the break. Deficiency of any member of this complex confers hypersensitivity to DNA-damaging agents by undefined mechanisms. In striking contrast to other BRCA1-containing complexes that are known to promote HDR, we demonstrate that the BRCA1-RAP80 complex restricts end resection in S/G2 phase of the cell cycle, thereby limiting HDR. RAP80 or BRCC36 deficiency resulted in elevated Mre11-CtIP-dependent 5? end resection with a concomitant increase in HDR mechanisms that rely on 3? single-stranded overhangs. We propose a model in which the BRCA1-RAP80 complex limits nuclease accessibility to DSBs, thus preventing excessive end resection and potentially deleterious homology-directed DSB repair mechanisms that can impair genome integrity. PMID:21335604

Coleman, Kara A.; Greenberg, Roger A.

2011-01-01

19

Regulation of DNA Repair through De-SUMOylation and SUMOylation of Replication Protein A Complex  

PubMed Central

The replication protein A complex (RPA) plays a crucial role in DNA replication and damage response. However, it is not known whether this complex is regulated by the SUMOylation pathway. Here we show that the 70kd subunit of RPA (RPA70) associates with a Sentrin/SUMO-specific protease, SENP6, in the nucleus to maintain RPA70 in a hypo-SUMOylated state during S phase. Campothecin (CPT), an inducer of replication stress, dissociates SENP6 from RPA70 allowing RPA70 to be modified by a small ubiquitin-like modifier 2/3 (SUMO-2/3). RPA70 SUMOylation facilitates recruitment of Rad51 to the DNA damage foci to initiate DNA repair through homologous recombination (HR). Cell lines that expressed a RPA70 mutant that cannot be SUMOylated are defective in HR and have a marked increase in sensitivity to CPT. These results demonstrate that SUMOylation status of RPA70 plays a critical role in the regulation of DNA repair through homologous recombination. PMID:20705237

Dou, Hong; Huang, Chao; Singh, Melissa; Carpenter, Phillip B; Yeh, Edward T. H.

2010-01-01

20

Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma  

PubMed Central

Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ) is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma (GBM). Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been described as the main modulator to determine the sensitivity of GBM to TMZ, a subset of GBM does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR), and the base excision repair (BER) pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break repair and double-strand break repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic. PMID:23227453

Yoshimoto, Koji; Mizoguchi, Masahiro; Hata, Nobuhiro; Murata, Hideki; Hatae, Ryusuke; Amano, Toshiyuki; Nakamizo, Akira; Sasaki, Tomio

2012-01-01

21

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability  

PubMed Central

Summary Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage. PMID:24746700

Savage, Kienan I.; Gorski, Julia J.; Barros, Eliana M.; Irwin, Gareth W.; Manti, Lorenzo; Powell, Alexander J.; Pellagatti, Andrea; Lukashchuk, Natalia; McCance, Dennis J.; McCluggage, W. Glenn; Schettino, Giuseppe; Salto-Tellez, Manuel; Boultwood, Jacqueline; Richard, Derek J.; McDade, Simon S.; Harkin, D. Paul

2014-01-01

22

DNA damage repair and transcription  

Microsoft Academic Search

During the course of DNA damage a complex repertoire of molecular signals, chromatin determinants and specific transcription factors are set in motion for repair. In many instances, the response pathway can be characterized by profound changes in molecular remodeling and is intimately linked with DNA replication and gene transcription. Our understanding of the molecular pathways has come from scientific developments

A. El-Osta

2004-01-01

23

Advances in understanding the complex mechanisms of DNA interstrand crosslink repair  

PubMed Central

DNA interstrand crosslinks (ICLs) are lesions caused by a variety of endogenous metabolites, environmental exposures, and cancer chemotherapeutic agents that have two reactive groups. The common feature of these diverse lesions is that two nucleotides on opposite strands are covalently joined. ICLs prevent the separation of two DNA strands and therefore essential cellular processes including DNA replication and transcription. ICLs are mainly detected in S phase when a replication fork stalls at an ICL. Damage signaling and repair of ICLs are promoted by the Fanconi anemia pathway and numerous posttranslational modifications of DNA repair and chromatin structural proteins. ICLs are also detected and repaired in non-replicating cells, although the mechanism is less clear. A unique feature of ICL repair is that both strands of DNA must be incised to completely remove the lesion. This is accomplished in sequential steps to prevent creating multiple double-strand breaks. Unhooking of an ICL from one strand is followed by translesion synthesis to fill the gap and create an intact duplex DNA, harboring a remnant of the ICL. Removal of the lesion from the second strand is likely accomplished by nucleotide excision repair. Inadequate repair of ICLs is particularly detrimental to rapidly dividing cells, explaining the bone marrow failure characteristic of Fanconi anemia and why cross-linking agents are efficacious in cancer therapy. Herein, recent advances in our understanding of ICLs and the biological responses they trigger are discussed. PMID:24224206

Clauson, Cheryl; Scharer, Orlando D.; Niedernhofer, Laura

2014-01-01

24

The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair  

PubMed Central

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass. PMID:25343618

Ball, Lindsay G.; Hanna, Michelle D.; Lambrecht, Amanda D.; Mitchell, Bryan A.; Ziola, Barry; Cobb, Jennifer A.; Xiao, Wei

2014-01-01

25

The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase  

PubMed Central

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2. PMID:21372173

Bernstein, Kara A.; Reid, Robert J.D.; Sunjevaric, Ivana; Demuth, Kimberly; Burgess, Rebecca C.; Rothstein, Rodney

2011-01-01

26

DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes ?  

PubMed Central

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways. PMID:21444718

Prudden, John; Perry, J. Jefferson P.; Nie, Minghua; Vashisht, Ajay A.; Arvai, Andrew S.; Hitomi, Chiharu; Guenther, Grant; Wohlschlegel, James A.; Tainer, John A.; Boddy, Michael N.

2011-01-01

27

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB.  

PubMed

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice. PMID:16482161

Yu, Bomina; Edstrom, William C; Benach, Jordi; Hamuro, Yoshitomo; Weber, Patricia C; Gibney, Brian R; Hunt, John F

2006-02-16

28

The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks  

Microsoft Academic Search

Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Non- homologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal

Eun Yong Shim; Jia-Lin Ma; Ji-Hyun Oum; Yvonne Yanez; Sang Eun Lee

2005-01-01

29

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts  

PubMed Central

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5?-base overhang. Regardless of the 5?-end structure, all bleomycin-induced DSBs possess 3?-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of ?0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires ?100 ng substrate DNA and ?50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA. PMID:11504886

Pastwa, Elzbieta; Neumann, Ronald D.; Winters, Thomas A.

2001-01-01

30

Recombinational DNA Repair in Bacteria  

E-print Network

Recombinational DNA Repair in Bacteria: Postreplication Kevin P Rice,University of Wisconsin Recombinational DNA repair represents the primary function for homologous DNA recombination in bacteria. Most of this repair occurs at replication forks that are stalled at sites of DNA damage. Introduction Deoxyribonucleic

Cox, Michael M.

31

ATM protein is indispensable to repair complex-type DNA double strand breaks induced by high LET heavy ion irradiation.  

NASA Astrophysics Data System (ADS)

ATM (ataxia telangiectasia-mutated) protein responsible for a rare genetic disease with hyperradiosensitivity, is the one of the earliest repair proteins sensing DNA double-strand breaks (DSB). ATM is known to phosphorylate DNA repair proteins such as MRN complex (Mre11, Rad50 and NBS1), 53BP1, Artemis, Brca1, gamma-H2AX, and MDC. We studied the interactions between ATM and DNA-PKcs, a crucial NHEJ repair protein, after cells exposure to high and low LET irradiation. Normal human (HFL III, MRC5VA) and AT homozygote (AT2KY, AT5BIVA, AT3BIVA) cells were irradiated with X-rays and high LET radiation (carbon ions: 290MeV/n initial energy at 70 keV/um, and iron ions: 500MeV/n initial energy at 200KeV/um), and several critical end points were examined. AT cells with high LET irradiation showed a significantly higher radiosensitivity when compared with normal cells. The behavior of DNA DSB repair was monitored by immuno-fluorescence techniques using DNA-PKcs (pThr2609, pSer2056) and ATM (pSer1981) antibodies. In normal cells, the phosphorylation of DNA-PKcs was clearly detected after high LET irradiation, though the peak of phosphorylation was delayed when compared to X-irradiation. In contrast, almost no DNA-PKcs phosphorylation foci were detected in AT cells irradiated with high LET radiation. A similar result was also observed in normal cells treated with 10 uM ATM kinase specific inhibitor (KU55933) one hour before irradiation. These data suggest that the phosphorylation of DNA-PKcs with low LET X-rays is mostly ATM-independent, and the phosphorylation of DNA-PKcs with high LET radiation seems to require ATM probably due to its complex nature of DSB induced. Our study indicates that high LET heavy ion irradiation which we can observe in the space environment would provide a useful tool to study the fundamental mechanism associated with DNA DSB repair.

Sekine, Emiko; Yu, Dong; Fujimori, Akira; Anzai, Kazunori; Okayasu, Ryuichi

32

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli*  

PubMed Central

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain. PMID:21454657

Winkler, Ines; Marx, Andreas D.; Lariviere, Damien; Heinze, Roger J.; Cristovao, Michele; Reumer, Annet; Curth, Ute; Sixma, Titia K.; Friedhoff, Peter

2011-01-01

33

A novel ruthenium(II)-polypyridyl complex inhibits cell proliferation and induces cell apoptosis by impairing DNA damage repair.  

PubMed

Ruthenium complexes are widely recognized as one of the most promising DNA damaging chemotherapeutic drugs. The main goal of this study was to explore the anticancer activity and underlying mechanisms of [Ru(phen)(2)(p-BrPIP)](ClO(4))(2), a novel chemically synthesized ruthenium (Ru) complex. To this end, we employed MTT assays to determine the anticancer activity of the complex, and performed single-cell gel electrophoresis (SCGE) and Western blotting to evaluate DNA damage. Our results showed that the Ru(II)-poly complex caused severe DNA damage, possibly by downregulating key factors involved in DNA repair pathways, such as proliferating cell nuclear antigen (PCNA) and ring finger protein 8 (RNF8). In addition, this complex induced cell apoptosis by upregulating both p21 and p53. Taken together, our findings demonstrate that the Ru(II)-poly complex exhibits antitumour activity by inducing cell apoptosis, which results from the accumulation of large amounts of unrepaired DNA damage. PMID:24070188

Yang, Qingyuan; Zhang, Zhao; Mei, Wenjie; Sun, Fenyong

2014-08-01

34

A novel ruthenium(II)–polypyridyl complex inhibits cell proliferation and induces cell apoptosis by impairing DNA damage repair  

PubMed Central

Ruthenium complexes are widely recognized as one of the most promising DNA damaging chemotherapeutic drugs. The main goal of this study was to explore the anticancer activity and underlying mechanisms of [Ru(phen)2(p-BrPIP)](ClO4)2, a novel chemically synthesized ruthenium (Ru) complex. To this end, we employed MTT assays to determine the anticancer activity of the complex, and performed single-cell gel electrophoresis (SCGE) and Western blotting to evaluate DNA damage. Our results showed that the Ru(II)–poly complex caused severe DNA damage, possibly by downregulating key factors involved in DNA repair pathways, such as proliferating cell nuclear antigen (PCNA) and ring finger protein 8 (RNF8). In addition, this complex induced cell apoptosis by upregulating both p21 and p53. Taken together, our findings demonstrate that the Ru(II)–poly complex exhibits antitumour activity by inducing cell apoptosis, which results from the accumulation of large amounts of unrepaired DNA damage. PMID:24070188

Yang, Qingyuan; Zhang, Zhao; Mei, Wenjie; Sun, Fenyong

2014-01-01

35

Mesoscale conformational changes in the DNA-repair complex Rad50\\/Mre11\\/Nbs1 upon binding DNA  

Microsoft Academic Search

The human Rad50\\/Mre11\\/Nbs1 complex (hR\\/M\\/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR\\/M\\/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their

Fernando Moreno-Herrero; Martijn de Jager; Nynke H. Dekker; Roland Kanaar; Claire Wyman; Cees Dekker

2005-01-01

36

Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage  

Microsoft Academic Search

The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple

Tadahide Izumi; Lee R. Wiederhold; Gargi Roy; Rabindra Roy; Arun Jaiswal; Kishor K. Bhakat; Sankar Mitra; Tapas K. Hazra

2003-01-01

37

Dynamics of DNA Mismatch Repair  

NASA Astrophysics Data System (ADS)

DNA mismatch repair protects the genome from spontaneous mutations by recognizing errors, excising damage, and re-synthesizing DNA in a pathway that is highly conserved. Mismatch recognition is accomplished by the MutS family of proteins which are weak ATPases that bind specifically to damaged DNA, but the specific molecular mechanisms by which these proteins recognize damage and initiate excision are not known. Previous structural investigations have implied that protein-induced conformational changes are central to mismatch recognition. Because damage detection is a highly dynamic process in which conformational changes of the protein-DNA complexes occur on a time scale of a few seconds, it is difficult to obtain meaningful kinetic information with traditional ensemble techniques. In this work, we use single molecule fluorescence resonance energy transfer (smFRET) to study the conformational dynamics of fluorescently labeled DNA substrates in the presence of the mismatch repair protein MutS from E. coli and its human homolog MSH2/MSH6. Our studies allow us to obtain quantitative kinetic information about the rates of binding and dissociation and to determine the conformational states for each protein-DNA complex.

Coats, Julie; Lin, Yuyen; Rasnik, Ivan

2009-11-01

38

Drug resistance and DNA repair  

Microsoft Academic Search

DNA repair confers resistance to anticancer drugs which kill cells by reacting with DNA. A review of our current information on the topic will be presented here. Our understanding of the molecular biology of repair of 06-alkylguanine adducts in DNA has advanced as a result of the molecular cloning of the E. coli ada gene but the precise role of

Margaret Fox; John J. Roberts

1987-01-01

39

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair  

PubMed Central

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway. PMID:23723247

Tomida, Junya; Itaya, Akiko; Shigechi, Tomoko; Unno, Junya; Uchida, Emi; Ikura, Masae; Masuda, Yuji; Matsuda, Shun; Adachi, Jun; Kobayashi, Masahiko; Meetei, Amom Ruhikanta; Maehara, Yoshihiko; Yamamoto, Ken-ichi; Kamiya, Kenji; Matsuura, Akira; Matsuda, Tomonari; Ikura, Tsuyoshi; Ishiai, Masamichi; Takata, Minoru

2013-01-01

40

A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair.  

PubMed

In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex. PMID:16168378

Matsushita, Nobuko; Kitao, Hiroyuki; Ishiai, Masamichi; Nagashima, Naoki; Hirano, Seiki; Okawa, Katsuya; Ohta, Tomohiko; Yu, David S; McHugh, Peter J; Hickson, Ian D; Venkitaraman, Ashok R; Kurumizaka, Hitoshi; Takata, Minoru

2005-09-16

41

Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems  

PubMed Central

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8. PMID:20981145

Morita, Rihito; Nakane, Shuhei; Shimada, Atsuhiro; Inoue, Masao; Iino, Hitoshi; Wakamatsu, Taisuke; Fukui, Kenji; Nakagawa, Noriko; Masui, Ryoji; Kuramitsu, Seiki

2010-01-01

42

Linking transcription with DNA repair, damage tolerance, and genome duplication  

E-print Network

complexes mask the DNA dam- age from recognition and removal by repair systems (3) (Fig. 1A). Transcription machinery, indicates that NusA promotes lesion removal by nucleotide ex- cision repair (7). The known/sensitivity of these RNAP mutants, lending strong support to the view that NusA promotes DNA dam- age repair in addition

Collins, James J.

43

Energy and Technology Review: Unlocking the mysteries of DNA repair  

SciTech Connect

DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

Quirk, W.A.

1993-04-01

44

Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro*  

E-print Network

(HRR)1 is an important pathway in repairing DNA double-strand breaks (DSBs) with high accuracy, which, a structural and functional homolog of Escherichia coli recombinase RecA (5, 6), plays a central role in HRR exchange (6­8). As a prerequisite step for the action of Rad51 in HRR, Rad51 binds DNA to form highly

Kowalczykowski, Stephen C.

45

DNA demethylation by DNA repair  

E-print Network

Active DNA demethylation underlies key facets of reproduction in flowering plants and mammals and serves a general genome housekeeping function in plants. A family of 5-methylcytosine DNA glycosylases catalyzes plant ...

Gehring, Mary

46

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage  

SciTech Connect

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

Noda, Taichi [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan) [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kondo, Natsuko [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan)] [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Mori, Eiichiro [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Ken [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan)] [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan); Zdzienicka, Malgorzata Z. [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland)] [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland); Thompson, Larry H. [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States)] [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States); Helleday, Thomas [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom) [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom); Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm (Sweden); Asada, Hideo [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); and others

2011-01-07

47

Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity  

PubMed Central

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET?=?370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET ?=?2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times ?1. Mdc1 accumulates faster (T0?=?17±2 s, ?1?=?98±11 s) than 53BP1 (T0?=?77±7 s, ?1?=?310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T0?=?73±16 s, ?1?=?1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time ?1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies. PMID:22860035

Hable, Volker; Drexler, Guido A.; Bruning, Tino; Burgdorf, Christian; Greubel, Christoph; Derer, Anja; Seel, Judith; Strickfaden, Hilmar; Cremer, Thomas; Friedl, Anna A.; Dollinger, Gunther

2012-01-01

48

DNA repair systems in rhabdomyosarcoma.  

PubMed

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in children and adolescent population. There are two major histological subtypes, embryonal (ERMS) and alveolar (ARMS), differing in cytogenetic and morphological features. RMS pathogenesis remains controversial and several cellular mechanisms and pathways have been implicated. Application of intense chemo- and radio-therapy improves survival rates for RMS patients, but significant efficacy has not been proved as DNA damage induced-resistance frequently occurs. The present review is aimed at summarizing the current evidence on DNA repair systems, implications in RMS development, focusing on gene expression alterations and point mutations of genes encoding for DNA repair enzymes. Understanding of DNA repair systems involvement in RMS pathogenesis could diversify RMS patients and provide novel individualized therapeutic targets. PMID:23516236

Tsioli, Panagiota G; Patsouris, Efstratios S; Giaginis, Constantinos; Theocharis, Stamatios E

2013-08-01

49

ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline.  

PubMed

Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to ?-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline. PMID:25329393

Kim, Hyun-Min; Colaiácovo, Monica P

2014-10-01

50

ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline  

PubMed Central

Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to ?-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline. PMID:25329393

Kim, Hyun-Min; Colaiacovo, Monica P.

2014-01-01

51

Inhibitors of DNA Repair and Response to Ionising Radiation  

Microsoft Academic Search

\\u000a Ionising radiation, and most chemotherapeutic agents currently used to treat cancer, target DNA to cause cytotoxicity. The\\u000a cellular response to DNA damage is a complex set of intra-cellular processes involving multiple DNA repair pathways, leading\\u000a either to cell death or to survival if the lesions are repaired or bypassed. Thus, the multiple and redundant pathways involved\\u000a in the repair of

Barbara Vischioni; Nils H. Nicolay; Ricky A. Sharma; Thomas Helleday

52

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.  

PubMed

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

2014-03-01

53

DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice  

PubMed Central

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

Schipler, Agnes; Iliakis, George

2013-01-01

54

Interactions between DNA damage, repair, and transcription  

Microsoft Academic Search

This review addresses a variety of mechanisms by which DNA repair interacts with transcription and vice versa. Blocking of transcriptional elongation is the best studied of these mechanisms. Transcription recovery after damage therefore has often been used as a surrogate marker of DNA repair in cells. However, it has become evident that relationships between DNA damage, repair, and transcription are

Andriy Khobta; Bernd Epe

55

Chromatin dynamics in DNA double-strand break repair  

PubMed Central

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. PMID:22285574

Shi, Lei; Oberdoerffer, Philipp

2012-01-01

56

DNA Repair Defects and Chromosomal Aberrations  

NASA Technical Reports Server (NTRS)

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 the DNA repair-defective cell lines were smaller than those of normal cells, with the DNA-PK-deficient cells having RBEs near unity. To further investigate the sensitivity differences that were observed in ATM and NBS deficient cells, chromosomal aberrations were analyzed in normal lung fibroblast cells treated with KU-55933 (a specific ATM kinase inhibitor) or Mirin (an Mre11- Rad50-Nbs1 complex inhibitor involved in activation of ATM). We also performed siRNA knockdown of these proteins. Preliminary data indicate that chromosome exchanges increase in cells treated with the specific ATM inhibitor. Possible cytogenetic signatures of acute and low dose-rate gamma irradiation in ATM or nibrin deficient and suppressed cells will be discussed.

Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

57

DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes  

Microsoft Academic Search

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination

David J Hosfield; Douglas S Daniels; Clifford D Mol; Christopher D Putnam; Sudip S Parikh; John A Tainer

2001-01-01

58

DNA Damage Induced by Alkylating Agents and Repair Pathways  

PubMed Central

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

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

2010-01-01

59

DNA repair pathways in human multiple myeloma  

PubMed Central

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment. PMID:23966156

Gourzones-Dmitriev, Claire; Kassambara, Alboukadel; Sahota, Surinder; Rčme, Thierry; Moreaux, Jérôme; Bourquard, Pascal; Hose, Dirk; Pasero, Philippe; Constantinou, Angelos; Klein, Bernard

2013-01-01

60

The hMre11/hRad50 protein complex and Nijmegen breakage syndrome:Linkage of double strand break repair to the cellular DNA damageresponse  

SciTech Connect

Nijmegen breakage syndrome (NBS) is an autosomal recessivedisorder characterized by increased cancer incidence, cell cyclecheckpoint defects, and ionizing radiation sensitivity. We have isolatedthe gene encoding p95, a member of the hMre11/hRad50 double-strand breakrepair complex. The p95 gene mapped to 8q21.3, the region that containsthe NBS locus, and p95 was absent from NBS cells established from NBSpatients, p95 deficiency in these cells completely abrogates theformation of hMre11/hRad50 ionizing radiation-induced foci. Comparison ofthe p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 areidentical. The implication of hMre11/hRad50/p95 protein complex in NBSreveals a direct molecular link between DSB repair and cell cyclecheckpoint functions.

Carney, James P.; Maser, Richard S.; Olivares, Heidi; Davis,Elizabeth M.; Le Beau, Michelle; Yates III, John R.; Hays, Lara; Morgan,William F.; Petrini, John H.J.

1998-04-01

61

DNA Repair 5 (2006) 145152 Mini review  

E-print Network

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 4. mtDNA damage leads to loss of membrane potential, ATP synthesis and cell deathDNA Repair 5 (2006) 145­152 Mini review Role of mitochondrial DNA in toxic responses to oxidative intermediates capable of damaging macromolecules. Mitochondrial DNA (mtDNA) represents a critical target

Santos, Janine H.

62

Mechanisms of human DNA repair: an update.  

PubMed

The human genome, comprising three billion base pairs coding for 30000-40000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which have evolved to remove or to tolerate pre-cytotoxic, pre-mutagenic and pre-clastogenic DNA lesions in an error-free, or in some cases, error-prone way. Defects in DNA repair give rise to hypersensitivity to DNA-damaging agents, accumulation of mutations in the genome and finally to the development of cancer and various metabolic disorders. The importance of DNA repair is illustrated by DNA repair deficiency and genomic instability syndromes, which are characterised by increased cancer incidence and multiple metabolic alterations. Up to 130 genes have been identified in humans that are associated with DNA repair. This review is aimed at updating our current knowledge of the various repair pathways by providing an overview of DNA-repair genes and the corresponding proteins, participating either directly in DNA repair, or in checkpoint control and signaling of DNA damage. PMID:14599765

Christmann, Markus; Tomicic, Maja T; Roos, Wynand P; Kaina, Bernd

2003-11-15

63

Cancer in xeroderma pigmentosum and related disorders of DNA repair  

Microsoft Academic Search

Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many

James E. Cleaver

2005-01-01

64

DNA repair pathways as targets for cancer therapy  

Microsoft Academic Search

DNA repair pathways can enable tumour cells to survive DNA damage that is induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA repair pathways might prove efficacious when used in combination with DNA-damaging chemotherapeutic drugs. In addition, alterations in DNA repair pathways that arise during tumour development can make some cancer cells reliant on a reduced set of DNA repair

Eva Petermann; Cecilia Lundin; Ben Hodgson; Ricky A. Sharma; Thomas Helleday

2008-01-01

65

Conditional DNA repair mutants enable highly precise genome engineering.  

PubMed

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. PMID:24500200

Nyerges, Ákos; Csorg?, Bálint; Nagy, István; Latinovics, Dóra; Szamecz, Béla; Pósfai, György; Pál, Csaba

2014-04-01

66

Conditional DNA repair mutants enable highly precise genome engineering  

PubMed Central

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. PMID:24500200

Nyerges, Akos; Csorgo, Balint; Nagy, Istvan; Latinovics, Dora; Szamecz, Bela; Posfai, Gyorgy; Pal, Csaba

2014-01-01

67

Functional characterization of dna repair proteins  

Microsoft Academic Search

Genetic material (DNA) is susceptible to spontaneous decomposition, to attack by reactive chemicals produced naturally in cells, and to assault by environmental and food mutagens. DNA modifications can lead to permanent genetic changes that promote human disease. To combat the deleterious effects of DNA damage, organisms are equipped with DNA repair systems. The focus of our investigations has been to

2000-01-01

68

Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex  

PubMed Central

The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections. PMID:23979587

Almond, Joshua R.; Stohr, Bradley A.; Panigrahi, Anil K.; Albrecht, Dustin W.; Nelson, Scott W.; Kreuzer, Kenneth N.

2013-01-01

69

Molecular mechanisms of DNA repair inhibition by caffeine  

SciTech Connect

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

Selby, C.P.; Sancar, A. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

1990-05-01

70

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4-signalosome complex to regulate DNA repair and cell death.  

PubMed

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1-3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN-CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4-CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

Rao, Feng; Xu, Jing; Khan, A Basit; Gadalla, Moataz M; Cha, Jiyoung Y; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H

2014-11-11

71

Enzymes of Direct, Excision, and Mismatch DNA Repair in Pro and Eukaryotes and Their Biological Role  

Microsoft Academic Search

DNA repair is of paramount importance to protect the integrity of the cell genome. Over the last decade, DNA repair enzymes have been intensely studied owing to their role in mutagenesis, carcinogenesis, and aging. This led to discoveries of novel enzymes, protein factors as well as their intermolecular complexes involved in the repair process. Alternative pathways of cell response to

N. L. Vasilenko; G. A. Nevinsky

2003-01-01

72

Nuclear position dictates DNA repair pathway choice  

PubMed Central

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

Lemaitre, Charlene; Grabarz, Anastazja; Tsouroula, Katerina; Andronov, Leonid; Furst, Audrey; Pankotai, Tibor; Heyer, Vincent; Rogier, Melanie; Attwood, Kathleen M.; Kessler, Pascal; Dellaire, Graham; Klaholz, Bruno; Reina-San-Martin, Bernardo; Soutoglou, Evi

2014-01-01

73

Repair of Topoisomerase I-Mediated DNA Damage  

PubMed Central

Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. PMID:16891172

Pommier, Yves; Barcelo, Juana; Rao, V. Ashutosh; Sordet, Olivier; Jobson, Andrew G.; Thibaut, Laurent; Miao, Zheyong; Seiler, Jennifer; Zhang, Hongliang; Marchand, Christophe; Agama, Keli; Redon, Christophe

2008-01-01

74

B-lymphoblastic leukemia/lymphoma: overexpression of nuclear DNA repair protein PARP-1 correlates with antiapoptotic protein Bcl-2 and complex chromosomal abnormalities.  

PubMed

Poly(ADP-ribose) polymerase-1 (PARP-1) and Bcl-2 are emerging as therapeutic targets in various cancers. The former is a DNA repair protein associated with genomic stability and apoptosis, whereas the latter is an antiapoptotic protein having a DNA repair function through inhibition of PARP-1. Because genomic stability is critical for prognosis in B-lymphoblastic leukemia/lymphoma (B-ALL), we studied the expression of PARP-1 and Bcl-2 proteins in patients with B-ALL of different ages and compared the results with cytogenetic data. The PARP-1 protein was overexpressed in about two-thirds (61%) of patients with B-ALL. It had a nuclear location, whereas Bcl-2 protein was cytosolic. Expression of the 2 proteins showed a highly positive correlation (? = 0.367; P < .001). Overexpression of PARP-1 correlated with a complex karyotype (P = .030), and this correlation remained significant for coexpression of PARP-1 and Bcl-2 proteins (?(2) = 7.498; P = .024) as well as after exclusion of pediatric patients (n = 9, P = .042). Overexpression of PARP-1 was not significantly more common in diploid versus aneuploid karyotypes (50% versus 59%, P = .610). The PARP-1 protein showed no correlation with specific chromosomal abnormalities associated with prognosis in B-ALL, as defined by the World Health Organization. In conclusion, high expression of the PARP-1 protein among patients with B-ALL is related to a complex karyotype and Bcl-2 positivity. Although these findings require validation in a larger population, the observations will be valuable in planning therapeutic trials (such as of PARP inhibitors and BH3 mimetics). PMID:24856976

Pournazari, Payam; Padmore, Ruth F; Kosari, Farid; Scalia, Peter; Shahbani-Rad, Meer-Taher; Shariff, Sami; Demetrick, Douglas J; Bosch, Mark; Mansoor, Adnan

2014-08-01

75

International congress on DNA damage and repair: Book of abstracts  

SciTech Connect

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

Not Available

1987-01-01

76

DNA repair mechanisms in Huntington's disease.  

PubMed

The human genome is under continuous attack by a plethora of harmful agents. Without the development of several dedicated DNA repair pathways, the genome would have been destroyed and cell death, inevitable. However, while DNA repair enzymes generally maintain the integrity of the whole genome by properly repairing mutagenic and cytotoxic intermediates, there are cases in which the DNA repair machinery is implicated in causing disease rather than protecting against it. One case is the instability of gene-specific trinucleotides, the causative mutations of numerous disorders including Huntington's disease. The DNA repair proteins induce mutations that are different from the genome-wide mutations that arise in the absence of repair enzymes; they occur at definite loci, they occur in specific tissues during development, and they are age-dependent. These latter characteristics make pluripotent stem cells a suitable model system for triplet repeat expansion disorders. Pluripotent stem cells can be kept in culture for a prolonged period of time and can easily be differentiated into any tissue, e.g., cells along the neural lineage. Here, we review the role of DNA repair proteins in the process of triplet repeat instability in Huntington's disease and also the potential use of pluripotent stem cells to investigate neurodegenerative disorders. PMID:23361256

Jonson, Ida; Ougland, Rune; Larsen, Elisabeth

2013-06-01

77

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways  

PubMed Central

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

78

Part 1: Mechanisms of DNA Repair  

NSDL National Science Digital Library

The most deleterious form of DNA damage is a double-strand break (DSB), which can arise from errors in DNA replication, from the failure of topoisomerases to complete their cycles of DNA cutting and rejoining, from mechanical stress and from the action of endonucleases that cleave DNA. Here we review how DSBs can be repaired either by nonhomologous end-joining mechanisms or by several homologous recombination pathways including single-strand annealing, gene conversion and break-induced replication.

Jim Haber (Brandeis University;Department of Biology and Rosenstiel Basic Medical Sciences Research Center)

2009-12-01

79

Chromatin Remodeling, DNA Damage Repair and Aging  

PubMed Central

Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

2012-01-01

80

DNA repair: the link between primary immunodeficiency and cancer.  

PubMed

The adaptive component of the immune system depends greatly on the generation of genetic diversity provided by lymphocyte-specific genomic rearrangements. V(D)J recombination, class switch recombination (CSR), and somatic hypermutation (SHM) constitute complex and vulnerable processes that are orchestrated by a multitude of DNA repair pathways. When inherited defects in certain DNA repair proteins are present, lymphocyte development can be compromised and, consequently, patients can develop primary immunodeficiencies (PIDs). PID patients often have a strong predisposition for cancer development as a result of genomic instability generated from defective DNA repair mechanisms. Tumors of lymphoid origin are one of the most common PID-associated cancers, likely due to DNA lesions resulting from defective V(D)J, CSR, and SHM. In this review, we describe PID syndromes that confer an increased risk for cancer development. Furthermore, we discuss the role of the affected proteins in tumorigenesis/lymphomagenesis. PMID:22236430

de Miranda, Noel Fcc; Björkman, Andrea; Pan-Hammarström, Qiang

2011-12-01

81

Breast cancer risk is associated with the genes encoding the DNA double-strand break repair Mre11/Rad50/Nbs1 complex.  

PubMed

The evolutionarily conserved Mre11-Rad50-Nbs1 (MRN) complex, consisting of proteins encoded by the genes Mre11, Rad50, and Nbs1, was recently shown to play a crucial role in DNA double-strand break (DSB) repair by recruiting the nuclear protein kinase ataxia telangiectasia mutated to DSB sites, leading to activation of this DNA repair network. Given the fact that carriers of defective mutation and polymorphic variants of ataxia telangiectasia mutated are at higher risk of developing breast cancer, we hypothesized a role of the MRN genes in determining breast cancer susceptibility. This hypothesis was examined both in a case control study of 559 breast cancer patients and 1,125 healthy women of single-nucleotide polymorphisms in Mre11, Rad50, and Nbs1 and by the in vivo detection of binding between Mre11 and BRCA1, encoded by the breast cancer susceptibility gene BRCA1. We were also interested in defining whether any association between MRN genes and breast cancer was modified by reproductive risk factors reflecting the level of estrogen exposure or susceptibility to estrogen exposure, as estrogen is known to initiate breast cancer development due to its metabolites causing DSB formation. Support for the hypothesis came from the observations that (a) one single-nucleotide polymorphism in Nbs1 was significantly associated with breast cancer risk, and a trend toward an increased risk of developing breast cancer was found in women harboring a greater number of putative high-risk genotypes of MRN genes (an adjusted odds ratio of 1.25 for each additional putative high-risk genotype; 95% confidence interval, 1.10-1.44); (b) this association between risk and the number of putative high-risk genotypes was stronger and more significant in women thought to be more susceptible to estrogen, i.e., those with no history of full-term pregnancy, those older (>or=26 years of age) at first full-term pregnancy, or those having had fewer (<2) full-term pregnancies; the risk effect conferred by harboring a higher number of high-risk genotypes of MRN genes was more significant in women without a history of breast feeding; and (c) Mre11 and BRCA1 were shown to form a complex in vivo, and this interaction was increased by irradiation. This study supports the role of the MRN pathway in breast cancer development, further strengthening the suggestion that mechanisms regulating DSB repair may play a mutator role driving breast cancer pathogenesis. PMID:17932350

Hsu, Huan-Ming; Wang, Hui-Chun; Chen, Sou-Tong; Hsu, Giu-Cheng; Shen, Chen-Yang; Yu, Jyh-Cherng

2007-10-01

82

Human DNA repair and recombination genes  

SciTech Connect

Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

Thompson, L.H.; Weber, C.A.; Jones, N.J.

1988-09-01

83

Association of CBP\\/p300 Acetylase and Thymine DNA Glycosylase Links DNA Repair and Transcription  

Microsoft Academic Search

DNA repair in chromatin is subject to topological constraints, suggesting a requirement for chromatin modification and remodeling activities. Thymine DNA glycosylase (TDG) initiates repair of G\\/T and G\\/U mismatches, commonly associated with CpG islands, by removing thymine and uracil moieties. We report that TDG associates with transcriptional coactivators CBP and p300 and that the resulting complexes are competent for both

Marc Tini; Arndt Benecke; Soo-Joong Um; Joseph Torchia; Ronald M Evans; Pierre Chambon

2002-01-01

84

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path  

E-print Network

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition information on, the complex process essential for DNA synthesis and repair. The five identified transition in organizing the active site for DNA synthesis (extension of primer strand by one base) and thereby helping

Schlick, Tamar

85

IDENTIFICATION AND CHARACTERIZATION OF MULTIPLE DNA LOOP REPAIR PATHWAYS IN HUMAN CELLS  

Microsoft Academic Search

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells

Scott D. McCulloch

2002-01-01

86

DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP  

PubMed Central

Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

2009-01-01

87

DNA interstrand crosslink repair and cancer  

Microsoft Academic Search

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that prevent transcription and replication by inhibiting DNA strand separation. Agents that induce ICLs were one of the earliest, and are still the most widely used, forms of chemotherapeutic drug. Only recently, however, have we begun to understand how cells repair these lesions. Important insights have come from studies of individuals with

Andrew J. Deans; Stephen C. West

2011-01-01

88

The Awakening of DNA Repair at Yale  

PubMed Central

As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease. PMID:24348216

Hanawalt, Philip C.

2013-01-01

89

Effect of nalidixic acid on DNA repair in rat hepatocytes.  

PubMed

Nalidixic acid, a DNA topoisomerase inhibitor, has been reported to inhibit DNA repair in some mammalian systems. To investigate the effect of nalidixic acid on DNA repair in cultured rat hepatocytes, DNA damage was induced by ultraviolet light or N-methyl-N-nitro-N'-nitrosoguanidine. The presence of aphidicolin, a DNA polymerase alpha inhibitor resulted in a decrease in DNA repair. Nalidixic acid had no inhibitory effect. Neither aphidicolin nor nalidixic acid induced DNA repair. These results indicate that nalidixic acid does not damage DNA or inhibit DNA repair processes in hepatocytes. PMID:2504447

McQueen, C A; Rosado, R R; Williams, G M

1989-06-01

90

DNA repair responses in human skin cells  

SciTech Connect

Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

1981-07-01

91

DNA Repair and Personalized Breast Cancer Therapy  

PubMed Central

Personalized cancer therapy is likely to be one of the next big advances in our search for a cure for cancer. To be able to treat people in an individualized manner, researchers need to know a great deal about their genetic constitution and the DNA repair status of their tumors. Specific knowledge is required regarding the polymorphisms individuals carry and how these polymorphisms influence responses to therapy. Researchers are actively engaged in biomarker discovery and validation for this purpose. In addition, the design of clinical trials must be reassessed to include new information on biomarkers and drug responses. In this review, we focus on personalized breast cancer therapy. The hypothesis we focus upon in this review is that there is connection between the DNA repair profile of individuals, their breast tumor subtypes, and their responses to cancer therapy. We first briefly review cellular DNA repair pathways that are likely to be impacted by breast cancer therapies. Next, we review the phenotypes of breast tumor subtypes with an emphasis on how a DNA repair deficiency might result in tumorigenesis itself and lead to the chemotherapeutic responses that are observed. Specific examples of breast tumor subtypes and their responses to cancer therapy are given, and we discuss possible DNA repair mechanisms that underlie the responses of tumors to various chemotherapeutic agents. Much is known about breast cancer subtypes and the way each of these subtypes responds to chemotherapy. In addition, we discuss novel design of clinical trials that incorporates rapidly emerging information on biomarkers. PMID:20872853

Li, Shu-Xia; Sjolund, Ashley; Harris, Lyndsay; Sweasy, Joann B.

2010-01-01

92

Single molecule techniques in DNA repair: a primer.  

PubMed

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit--searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. PMID:24819596

Hughes, Craig D; Simons, Michelle; Mackenzie, Cassidy E; Van Houten, Bennett; Kad, Neil M

2014-08-01

93

DNA damage and repair after high LET radiation  

NASA Astrophysics Data System (ADS)

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.

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

94

Isolating human DNA repair genes using rodent-cell mutants  

SciTech Connect

The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

1987-03-23

95

The ups and downs of DNA repair biomarkers for PARP inhibitor therapies  

PubMed Central

PARP inhibitors are emerging as a valuable new drug class in the treatment of cancer. Recent discoveries make a compelling case for the complexity of DNA repair biomarker evaluation and underscore the need to examine at multiple biomarkers in a relational manner. This review updates the current trends in DNA repair biomarker strategies in use for the PARP inhibitors and describes the impact of many DNA repair biomarkers on PARP inhibitor benefit in the cancer clinic. PMID:21968427

Wang, XiaoZhe; Weaver, David T

2011-01-01

96

DNA repair synthesis and ligation affect the processing of excised oligonucleotides generated by human nucleotide excision repair.  

PubMed

Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo. PMID:25107903

Kemp, Michael G; Gaddameedhi, Shobhan; Choi, Jun-Hyuk; Hu, Jinchuan; Sancar, Aziz

2014-09-19

97

Regulation of DNA repair by parkin  

SciTech Connect

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

Kao, Shyan-Yuan, E-mail: shyan-yuan_kao@meei.harvard.edu [Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114 (United States)] [Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114 (United States)

2009-05-01

98

Dealing with DNA damage: Relationships between checkpoint and repair pathways  

Microsoft Academic Search

Cell cycle checkpoint activation and DNA repair pathways govern genomic stability after genotoxic stress. Genotoxic insult results in activation of an interwoven network of DNA damage checkpoints and DNA repair pathways. Post-translational modifications on a number of proteins involved in both checkpoint activation and DNA repair play an important role in this cellular response. Genotoxic stress can induce a wide

Daniël O. Warmerdam; Roland Kanaar

2010-01-01

99

Sumoylation of the Rad1 nuclease promotes DNA repair and regulates its DNA association  

PubMed Central

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions. PMID:24753409

Sarangi, Prabha; Bartosova, Zdenka; Altmannova, Veronika; Holland, Cory; Chavdarova, Melita; Lee, Sang Eun; Krejci, Lumir; Zhao, Xiaolan

2014-01-01

100

Early Days of DNA Repair: Discovery of Nucleotide Excision Repair and Homology-Dependent Recombinational Repair  

PubMed Central

The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere. PMID:24348214

Rupp, W. Dean

2013-01-01

101

Electrically monitoring DNA repair by photolyase  

NASA Astrophysics Data System (ADS)

Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the ?-stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV/blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA. Author contributions: M.C.D. and J.K.B. designed research; M.C.D. performed research; A.S. contributed new reagents/analytic tools; M.C.D. analyzed data; and M.C.D. and J.K.B. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Abbreviations: T<>T, thymine dimer; CT, charge transport.

DeRosa, Maria C.; Sancar, Aziz; Barton, Jacqueline K.

2005-08-01

102

Five repair pathways in one context: chromatin modification during DNA repair  

Microsoft Academic Search

The eukaryotic cell is faced with more than 10 000 various kinds of DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Therefore, cells have developed 5 major repair path- ways in which different kinds of DNA damage can be detected and repaired: homologous recombination, nonhomologous end joining, nucleotide excision repair,

Yeganeh Ataian; Jocelyn E. Krebs

2006-01-01

103

DNA repair deficiency and neurological disease  

Microsoft Academic Search

The ability to respond to genotoxic stress is a prerequisite for the successful development of the nervous system. Mutations in various DNA repair factors can lead to human diseases that are characterized by pronounced neuropathology. In many of these syndromes the neurological component is among the most deleterious aspects of the disease. The nervous system poses a particular challenge in

Peter J. McKinnon

2009-01-01

104

Gallbladder Cancer Predisposition: A Multigenic Approach to DNA-Repair, Apoptotic and Inflammatory Pathway Genes  

Microsoft Academic Search

Gallbladder cancer (GBC) is a multifactorial disease with complex interplay between multiple genetic variants. We performed Classification and Regression Tree Analysis (CART) and Grade of Membership (GoM) analysis to identify combinations of alleles among the DNA repair, inflammatory and apoptotic pathway genetic variants in modifying the risk for GBC. We analyzed 16 polymorphisms in 8 genes involved in DNA repair,

Kshitij Srivastava; Anvesha Srivastava; Ashok Kumar; Balraj Mittal

2011-01-01

105

Introduction Telomere biology and DNA repair: Enemies with benefits  

E-print Network

Introduction Telomere biology and DNA repair: Enemies with benefits This special issue features in-depth reviews of telomere biology and DNA repair. Understanding how telomeres function requires insights into the nature and regulation of the cellular pathways that detect and repair DNA lesions. As telomeres block

de Lange, Titia

106

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage  

Microsoft Academic Search

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA?\\/?, FANCC?\\/?, FANCA?\\/?C?\\/?, FANCD2?\\/? and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell

Taichi Noda; Akihisa Takahashi; Natsuko Kondo; Eiichiro Mori; Noritomo Okamoto; Yosuke Nakagawa; Ken Ohnishi; Ma?gorzata Z. Zdzienicka; Larry H. Thompson; Thomas Helleday; Hideo Asada; Takeo Ohnishi

2011-01-01

107

DNA interstrand crosslink repair and cancer  

PubMed Central

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that prevent transcription and replication by inhibiting DNA strand separation. Agents that induce ICLs were one of the earliest, and are still the most widely used, forms of chemotherapeutic drug. Only recently, however, have we begun to understand how cells repair these lesions. Important insights have come from studies of individuals with Fanconi anaemia (FA), a rare genetic disorder that leads to ICL sensitivity. Understanding how the FA pathway links nucleases, helicases and other DNA-processing enzymes should lead to more targeted uses of ICL-inducing agents in cancer treatment and could provide novel insights into drug resistance. PMID:21701511

Deans, Andrew J.; West, Stephen C.

2013-01-01

108

DNA Repair-Protein Relocalization After Heavy Ion Exposure  

NASA Technical Reports Server (NTRS)

Ionizing radiation is good at making DNA double strand breaks, and high linear energy transfer (LET) radiations such as heavy ion particles are particularly efficient. For this reason, the proteins belonging to repair systems that deal with double strand breaks are of particular interest. One such protein is Ku, a component in the non-homologous recombination repair system. The Ku protein is an abundant, heterodimeric DNA end-binding complex, composed of one 70 and one 86 kDa subunit. Ku protein binds to DNA ends, nicks, gaps, and regions of transition between single and double-stranded structure. These binding properties suggest an important role in DNA repair. The Ku antigen is important in this study because it is present in relatively large copy numbers and it is part of a double-strand-break repair system. More importantly, we consistently measure an apparent upregulation in situ that is not verified by whole-cell-lysate immunoblot measurements. This apparent upregulation is triggered by very low doses of radiation, thus showing a potentially useful high sensitivity. However, elucidation of the mechanism underlying this phenomenon is still to be done.

Metting, N. F.

1999-01-01

109

A KTAP framework of DNA damage repair mechanism under Ion Radiation (IR) perturbation  

Microsoft Academic Search

In order to illustrate the cellular self-defense mechanisms in response to acute perturbations from outer environment, a mathematical model of DNA damage repair kinetics is proposed based on the method of Kinetic Theory of Active Particles (KTAP). Under acute Ion Radiation (IR), the complex interactions between the molecular pairs of DNA damage, repair mRNA, repair protein (RP), and double-strand breaks

Jinpeng Qi; Yongsheng Ding; Fang Han

2011-01-01

110

Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond  

PubMed Central

Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites. PMID:23386830

Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

2012-01-01

111

Alternative nucleotide incision repair pathway for oxidative DNA damage  

Microsoft Academic Search

The DNA glycosylase pathway, which requires the sequential action of two enzymes for the incision of DNA, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and\\/or blocking 3'-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated. Besides the logistical problems,

Alexander A. Ischenko; Murat K. Saparbaev

2002-01-01

112

Oxidative DNA damage repair in mammalian cells: A new perspective  

Microsoft Academic Search

Oxidatively induced DNA lesions have been implicated in the etiology of many diseases (including cancer) and in aging. Repair of oxidatively damaged bases in all organisms occurs primarily via the DNA base excision repair (BER) pathway, initiated with their excision by DNA glycosylases. Only two mammalian DNA glycosylases, OGG1 and NTH1 of E. coli Nth family, were previously characterized, which

Tapas K. Hazra; Aditi Das; Soumita Das; Sujata Choudhury; Yoke W. Kow; Rabindra Roy

2007-01-01

113

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics  

E-print Network

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics Soo­Yong Shin 1 , Eun Jeong the complexity of DNA computing. The complexity of any computational algorithm is typically measured in terms of time and space. In DNA computing, the time complexity can be measured by the total reaction time

114

The mismatch DNA repair heterodimer, hMSH2\\/6, regulates BLM helicase  

Microsoft Academic Search

The human MSH2\\/6 complex is essential for mismatch recognition during the repair of replication errors. Although mismatch repair components have been implicated in DNA homologous recombination repair, the exact function of hMSH2\\/6 in this pathway is unclear. Here, we show that the recombinant hMSH2\\/6 protein complex stimulated the ability of the Bloom's syndrome gene product, BLM, to process Holliday junctions

Qin Yang; Ran Zhang; Xin W Wang; Steven P Linke; Sagar Sengupta; Ian D Hickson; Graziella Pedrazzi; Claudia Perrera; Igor Stagljar; Susan J Littman; Paul Modrich; Curtis C Harris

2004-01-01

115

Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair  

PubMed Central

Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

2014-01-01

116

Tilting at windmills? The nucleotide excision repair of chromosomal DNA.  

PubMed

A typical view of how DNA repair functions in chromatin usually depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin on the repair process. It may be that in this current interpretation the repair mechanisms are 'tilting at windmills', fighting an imaginary foe. An emerging picture suggests that we should not consider chromatin as an inhibitory force to be overcome like some quixotic giant by the DNA repair processes. Instead we should now recognize that DNA repair and chromatin metabolism are inextricably and mechanistically linked. Here we discuss the latest findings which are beginning to reveal how changes in chromatin dynamics integrate with the DNA repair process in response to UV induced DNA damage, with an emphasis on events in the yeast Saccharomyces cerevisiae. PMID:19041427

Waters, Raymond; Teng, Yumin; Yu, Yachuan; Yu, Shirong; Reed, Simon H

2009-02-01

117

Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex  

PubMed Central

The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as ?-H2AX foci that colocalize with 53BP1, Mre11, Ser1981-pATM, and Thr68-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK–dependent ?-H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK-?-H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that ?-H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment. PMID:18632984

Guirouilh-Barbat, Josee; Redon, Christophe

2008-01-01

118

Repair of hydantoin lesions and their amine adducts in DNA by base and nucleotide excision repair  

PubMed Central

An important feature of the common DNA oxidation product 8;oxo;7,8;dihydroguanine (OG) is its susceptibility to further oxidation to produce guanidinohydantion (Gh) and spiroiminodihydantoin (Sp) lesions. In the presence of amines, G or OG oxidation produces hydantoin amine adducts. Such adducts may form in cells via interception of oxidized intermediates by protein;derived nucleophiles or naturally occurring amines that are tightly associated with DNA. Gh and Sp are known to be substrates for base excision repair (BER) glycosylases; however, large Sp;amine adducts would be expected to be more readily repaired by nucleotide excision repair (NER). A series of Sp adducts differing in size of the attached amine were synthesized to evaluate the relative processing by NER and BER. The UvrABC complex excised Gh, Sp and the Sp;amine adducts from duplex DNA, with the greatest efficiency for the largest Sp;amine adducts. The affinity of UvrA with all of the lesion duplexes was found to be similar, whereas the efficiency of UvrB loading tracked with the efficiency of UvrABC excision. In contrast, the human BER glycosylase NEIL1 exhibited robust activity for all Sp;amine adducts irrespective of size. These studies suggest that both NER and BER pathways mediate repair of a diverse set of hydantoin lesions in cells. PMID:23930966

McKibbin, Paige L.; Fleming, Aaron M.; Towheed, Mohammad Atif; Van Houten, Bennett; Burrows, Cynthia J.; David, Sheila S.

2013-01-01

119

Molecular mechanisms of DNA damage and repair: progress in plants.  

PubMed

Despite stable genomes of all living organisms, they are subject to damage by chemical and physical agents in the environment (e.g., UV and ionizing. radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. The DNA lesions produced by these damaging agents could be altered base, missing base, mismatch base, deletion or insertion, linked pyrimidines, strand breaks, intra- and inter-strand cross-links. These DNA lesions could be genotoxic or cytotoxic to the cell. Plants are most affected by the UV-B radiation of sunlight, which penetrates and damages their genome by inducing oxidative damage (pyrimidine hydrates) and cross-links (both DNA protein and DNA-DNA) that are responsible for retarding the growth and development. The DNA lesions can be removed by repair, replaced by recombination, or retained, leading to genome instability or mutations or carcinogenesis or cell death. Mostly organisms respond to genome damage by activating a DNA damage response pathway that regulates cell-cycle arrest, apoptosis, and DNA repair pathways. To prevent the harmful effect of DNA damage and maintain the genome integrity, all organisms have developed various strategies to either reverse, excise, or tolerate the persistence of DNA damage products by generating a network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway. The direct reversal and photoreactivation require single protein, all the rest of the repair mechanisms utilize multiple proteins to remove or repair the lesions. The base excision repair pathway eliminates single damaged base, while nucleotide excision repair excises a patch of 25- to 32-nucleotide-long oligomer, including the damage. The double-strand break repair utilizes either homologous recombination or nonhomologous endjoining. In plant the latter pathway is more error prone than in other eukaryotes, which could be an important driving force in plant genome evolution. The Arabidopsis genome data indicated that the DNA repair is highly conserved between plants and mammals than within the animal kingdom, perhaps reflecting common factors such as DNA methylation. This review describes all the possible mechanisms of DNA damage and repair in general and an up to date progress in plants. In addition, various types of DNA damage products, free radical production, lipid peroxidation, role of ozone, dessication damage of plant seed, DNA integrity in pollen, and the role of DNA helicases in damage and repair and the repair genes in Arabidopsis genome are also covered in this review. PMID:11563486

Tuteja, N; Singh, M B; Misra, M K; Bhalla, P L; Tuteja, R

2001-01-01

120

Ancient bacteria show evidence of DNA repair  

PubMed Central

Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability. PMID:17728401

Johnson, Sarah Stewart; Hebsgaard, Martin B.; Christensen, Torben R.; Mastepanov, Mikhail; Nielsen, Rasmus; Munch, Kasper; Brand, Tina; Gilbert, M. Thomas P.; Zuber, Maria T.; Bunce, Michael; R?nn, Regin; Gilichinsky, David; Froese, Duane; Willerslev, Eske

2007-01-01

121

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics  

E-print Network

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics Soo-Yong Shin1 , Eun Jeong of DNA computing. The complexity of any computational algorithm is typically measured in terms of time and space. In DNA computing, the time complexity can be measured by the total reaction time

122

Nucleotide excision repair of DNA: The very early history.  

PubMed

This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair. PMID:21600859

Friedberg, Errol C

2011-07-15

123

Low integrated DNA repair score and lung cancer risk.  

PubMed

DNA repair is a prime mechanism for preventing DNA damage, mutation, and cancers. Adopting a functional approach, we examined the association with lung cancer risk of an integrated DNA repair score, measured by a panel of three enzymatic DNA repair activities in peripheral blood mononuclear cells. The panel included assays for AP endonuclease 1 (APE1), 8-oxoguanine DNA glycosylase (OGG1), and methylpurine DNA glycosylase (MPG), all of which repair oxidative DNA damage as part of the base excision repair pathways. A blinded population-based case-control study was conducted with 96 patients with lung cancer and 96 control subjects matched by gender, age (±1 year), place of residence, and ethnic group (Jews/non-Jews). The three DNA repair activities were measured, and an integrated DNA repair OMA (OGG1, MPG, and APE1) score was calculated for each individual. Conditional logistic regression analysis revealed that individuals in the lowest tertile of the integrated DNA repair OMA score had an increased risk of lung cancer compared with the highest tertile, with OR = 9.7; 95% confidence interval (CI), 3.1-29.8; P < 0.001, or OR = 5.6; 95% CI, 2.1-15.1; P < 0.001 after cross-validation. These results suggest that pending validation, this DNA repair panel of risk factors may be useful for lung cancer risk assessment, assisting prevention and referral to early detection by technologies such as low-dose computed tomography scanning. PMID:24356339

Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

2014-04-01

124

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair  

SciTech Connect

Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA.UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for full-length UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.

Pakotiprapha, Danaya; Liu, Yi; Verdine, Gregory L.; Jeruzalmi, David; Harvard

2009-05-21

125

Brc1-Mediated DNA Repair and Damage Tolerance  

PubMed Central

The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1? cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1? epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination. PMID:15972456

Sheedy, Daniel M.; Dimitrova, Dora; Rankin, Jessica K.; Bass, Kirstin L.; Lee, Karen M.; Tapia-Alveal, Claudia; Harvey, Susan H.; Murray, Johanne M.; O'Connell, Matthew J.

2005-01-01

126

DNA damage repair – a new target for cancer therapy  

Microsoft Academic Search

Summary  DNA damage repair is essential for cellular homeostasis and cell survival. Cytostatic drugs and ionizing radiation cause an\\u000a enormous amount of DNA damage and efficient repair is crucial for sustained cell growth. Hence manipulating DNA damage repair\\u000a offers a new therapeutic principle, which together with conventional treatment strategies can increase treatment efficiency.\\u000a In this issue we would like to give

Oliver E. Bechter

2009-01-01

127

Chromatin Structure Following UV-Induced DNA Damage—Repair or Death?  

PubMed Central

In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury. PMID:22174650

Farrell, Andrew W.; Halliday, Gary M.; Lyons, James Guy

2011-01-01

128

DNA Repair and Resistance to UV-B Radiation in Western Spotted Frogs  

Microsoft Academic Search

We assessed DNA repair and resistance to solar radiation in eggs of members of the western spotted frog complex (Rana pretiosa and R. luteiventris), species whose populations are suffering severe range reductions and declines. Specifically, we measured the activity of photoreactivating enzyme (photolyase) in oocytes of spotted frogs. In some species, photoreactivation is the most important mechanism for repair of

Andrew R. Blaustein; John B. Hays; Peter D. Hoffman; Douglas P. Chivers; Joseph M. Kiesecker; William P. Leonard; Adolfo Marco; Deanna H. Olson; Jamie K. Reaser; Robert G. Anthony

1999-01-01

129

Role of nucleotide excision repair proteins in oxidative DNA damage repair: an updating  

Microsoft Academic Search

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage. Base excision repair (BER)\\u000a has an important role in preventing the deleterious effects of oxidative DNA damage, but recent evidence points to the involvement\\u000a of several repair pathways in this process. Oxidative damage may arise from endogenous and exogenous sources and may target\\u000a nuclear

B. Pascucci; M. D’Errico; E. Parlanti; S. Giovannini; E. Dogliotti

2011-01-01

130

Mutations in Two Ku Homologs Define a DNA End-Joining Repair Pathway inSaccharomyces cerevisiae  

Microsoft Academic Search

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiaegene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku80 associates with the product of theHDF1gene, forming the major DNA end-binding complex of yeast cells. DNA end

G. TODD MILNE; SHENGFANG JIN; KATIE B. SHANNON; ANDDAVID T. WEAVER

1996-01-01

131

Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair  

PubMed Central

Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in Xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER. PMID:23083890

Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Wani, Altaf

2012-01-01

132

Assessment of the role of DNA repair in damaged forensic samples.  

PubMed

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results. PMID:24792635

Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

2014-11-01

133

The Smc complexes in DNA damage response  

PubMed Central

The structural maintenance of chromosomes (Smc) proteins regulate nearly all aspects of chromosome biology and are critical for genomic stability. In eukaryotes, six Smc proteins form three heterodimers--Smc1/3, Smc2/4, and Smc5/6--which together with non-Smc proteins form cohesin, condensin, and the Smc5/6 complex, respectively. Cohesin is required for proper chromosome segregation. It establishes and maintains sister-chromatid cohesion until all sister chromatids achieve bipolar attachment to the mitotic spindle. Condensin mediates chromosome condensation during mitosis. The Smc5/6 complex has multiple roles in DNA repair. In addition to their major functions in chromosome cohesion and condensation, cohesin and condensin also participate in the cellular DNA damage response. Here we review recent progress on the functions of all three Smc complexes in DNA repair and their cell cycle regulation by posttranslational modifications, such as acetylation, phosphorylation, and sumoylation. An in-depth understanding of the mechanisms by which these complexes promote DNA repair and genomic stability may help us to uncover the molecular basis of genomic instability in human cancers and devise ways that exploit this instability to treat cancers. PMID:22369641

2012-01-01

134

DNA-PK: a dynamic enzyme in a versatile DSB repair pathway  

PubMed Central

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex. PMID:24680878

Davis, Anthony J.; Chen, Benjamin P.C.; Chen, David J.

2014-01-01

135

The effect of acrylamide on hepatocellular DNA repair.  

PubMed

Acrylamide has recently been reported to induce tumors in laboratory animals. The effect of acrylamide on unscheduled DNA synthesis using the hepatocyte primary culture (HPC)/DNA repair test was examined. Isolated hepatocytes were exposed to acrylamide and [3H]thymidine ( [3H]TdR) for 18 hr. Incorporation of [3H]TdR into DNA was determined by autoradiography. No DNA repair was observed at acrylamide concentrations up to 10(-2) M. These findings were confirmed using density gradients. Acrylamide concentrations exceeding 10(-2) M were cytotoxic to hepatocytes. Because both autoradiography and density gradients measure DNA repair as an endpoint, the ability of acrylamide to inhibit these repair processes was also determined. Acrylamide had no effect on the repair of UV-damaged DNA. These results show that acrylamide is not genotoxic in isolated hepatocytes. PMID:3943500

Miller, M J; McQueen, C A

1986-01-01

136

DNA Repair and Genome Maintenance in Bacillus subtilis  

PubMed Central

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

2012-01-01

137

The human Rad9\\/Rad1\\/Hus1 damage sensor clamp interacts with DNA polymerase b and increases its DNA substrate utilisation efficiency: implications for DNA repair  

Microsoft Academic Search

In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family pro- teins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 com- plex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins

Magali Toueille; Nazim El-Andaloussi; Isabelle Frouin; Raimundo Freire; Dorothee Funk; Igor Shevelev; Erica Friedrich-Heineken; Giuseppe Villani; Michael O. Hottiger

2004-01-01

138

DNA mismatch repair and oxidative DNA damage: implications for cancer biology and treatment.  

PubMed

Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting. PMID:25099886

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A

2014-01-01

139

DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment  

PubMed Central

Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting. PMID:25099886

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

2014-01-01

140

DNA repair systems as targets of cadmium toxicity  

SciTech Connect

Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity.

Giaginis, Constantinos [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece); Gatzidou, Elisavet [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece); Theocharis, Stamatios [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece)]. E-mail: theocharis@ath.forthnet.gr

2006-06-15

141

Arthroscopic Repairs of Triangular Fibrocartilage Complex Tears  

Microsoft Academic Search

Technical advancements in arthroscopic wrist procedures have improved our knowledge of normal and abnormal intraarticular wrist function. Triangular fibrocartilage complex (TFCC) tears from trauma injuries are a common source of ulnar-sided wrist pain. Fortunately, the TFCC is a structure that can be evaluated and treated arthroscopically with results that are comparable to open surgical procedures. Successful arthroscopic repairs of TFCC

Patricia Baehser-Griffith; John M. Bednar; A. Lee Osterman; Randall Culp

1997-01-01

142

Alkyltransferase-like proteins: Molecular switches between DNA repair pathways  

PubMed Central

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here, we review recent advances in unravelling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O6-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway. PMID:20502938

Tubbs, Julie L.; Tainer, John A.

2011-01-01

143

The BLM dissolvasome in DNA replication and repair  

PubMed Central

RecQ DNA helicases are critical for proper maintenance of genomic stability and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase III?, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems. PMID:23543275

Manthei, Kelly A.; Keck, James L.

2013-01-01

144

[Repair of ionizing radiation induced DNA double strand breaks].  

PubMed

DNA double-strand breaks (DSB) are created by ionizing radiation, an important environmental genotoxic agent. DSB are repaired by two mechanisms associated with recombination. In eukaryotic cells homologous recombination depends on genes belonging to the RAD52 epistatic group. Alternative pathway, DNA end-joining in non-homologous recombination involves DNA-dependent protein kinase (DNA-PK). PMID:10857377

Wid?ak, P

2000-01-01

145

DNA Damage Checkpoint, Damage Repair, and Genome Stability  

Microsoft Academic Search

Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance

Wei-Feng LIU; Shan-Shan YU; Guan-Jun CHEN; Yue-Zhong LI

2006-01-01

146

Targeting abnormal DNA double strand break repair in cancer  

PubMed Central

A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agents that selectively inhibit the DSB repair pathways that cancer cells are more dependent upon will facilitate the design of therapeutic strategies that exploit the differences in DSB repair between normal and cancer cells. Here, we discuss the pathways of DSB repair, alterations in DSB repair in cancer, inhibitors of DSB repair and future directions for cancer therapies that target DSB repair. PMID:20697770

Rassool, Feyruz V.

2010-01-01

147

Repair of endogenous DNA base lesions modulate lifespan in mice.  

PubMed

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O(6)-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm(-/-) mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity. PMID:24994062

Meira, Lisiane B; Calvo, Jennifer A; Shah, Dharini; Klapacz, Joanna; Moroski-Erkul, Catherine A; Bronson, Roderick T; Samson, Leona D

2014-09-01

148

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.  

PubMed

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes. PMID:9826691

Bertrand, P; Tishkoff, D X; Filosi, N; Dasgupta, R; Kolodner, R D

1998-11-24

149

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

PubMed

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

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

2013-08-30

150

DNA repair in cancer: emerging targets for personalized therapy  

PubMed Central

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer. PMID:24600246

Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

2014-01-01

151

DNA repair: Dynamic defenders against cancer and aging  

SciTech Connect

You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet (UV) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information.

Fuss, Jill O.; Cooper, Priscilla K.

2006-04-01

152

Homologous recombinational repair of DNA ensures mammalian chromosome stability  

Microsoft Academic Search

The process of homologous recombinational repair (HRR) is a major DNA repair pathway that acts on double-strand breaks and interstrand crosslinks, and probably to a lesser extent on other kinds of DNA damage. HRR provides a mechanism for the error-free removal of damage present in DNA that has replicated (S and G2 phases). Thus, HRR acts in a critical way,

Larry H Thompson; David Schild

2001-01-01

153

Homologous recombinational repair of DNA ensures mammalian chromosome stability.  

PubMed

The process of homologous recombinational repair (HRR) is a major DNA repair pathway that acts on double-strand breaks and interstrand crosslinks, and probably to a lesser extent on other kinds of DNA damage. HRR provides a mechanism for the error-free removal of damage present in DNA that has replicated (S and G2 phases). Thus, HRR acts in a critical way, in coordination with the S and G2 checkpoint machinery, to eliminate chromosomal breaks before the cell division occurs. Many of the human HRR genes, including five Rad51 paralogs, have been identified, and knockout mutants for most of these genes are available in chicken DT40 cells. In the mouse, most of the knockout mutations cause embryonic lethality. The Brca1 and Brca2 breast cancer susceptibility genes appear to be intimately involved in HRR, but the mechanistic basis is unknown. Biochemical studies with purified proteins and cell extracts, combined with cytological studies of nuclear foci, have begun to establish an outline of the steps in mammalian HRR. This pathway is subject to complex regulatory controls from the checkpoint machinery and other processes, and there is increasing evidence that loss of HRR gene function can contribute to tumor development. This review article is meant to be an update of our previous review [Biochimie 81 (1999) 87]. PMID:11376695

Thompson, L H; Schild, D

2001-06-01

154

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

PubMed Central

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

Boiteux, Serge; Jinks-Robertson, Sue

2013-01-01

155

Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair  

PubMed Central

Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. PMID:25389266

Li, Wentao; Selvam, Kathiresan; Ko, Tengyu; Li, Shisheng

2014-01-01

156

Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4  

SciTech Connect

Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

2009-01-01

157

Chromatin modifications and DNA repair: beyond double-strand breaks  

PubMed Central

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

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

2014-01-01

158

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation  

SciTech Connect

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.

Brandt, Patrick D. [Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Helt, Christopher E. [Department of Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Keng, Peter C. [Department of Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Bambara, Robert A. [Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States)]. E-mail: robert_bambara@urmc.rochester.edu

2006-08-18

159

A Phylogenomic Study of DNA Repair Genes, Proteins, and Processes  

PubMed Central

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence. PMID:10606811

Eisen, Jonathan A.; Hanawalt, Philip C.

2011-01-01

160

Fanconi Anemia: A Signal Transduction and DNA Repair Pathway  

PubMed Central

Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response. PMID:24348213

Kupfer, Gary M.

2013-01-01

161

DNA repair: a simple enzymatic assay for human cells  

Microsoft Academic Search

An assay is described based on the ability of certain endonucleases in ; Micrococcus luteus extracts to react specifically at sites of DNA damage, and ; produce single-strand breaks. The breaks were detected by sedimenting the DNA ; through alkali. DNA repair was assumed to occur if the number of endonuclease ; sensitive sites decreased during post-treatment incubation of cells.

R. J. Wilkins

1973-01-01

162

Ancient bacteria show evidence of DNA repair Sarah Stewart Johnson*  

E-print Network

Ancient bacteria show evidence of DNA repair Sarah Stewart Johnson* , Martin B. Hebsgaard , Torben authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long- term by an absence of convincing evidence for mecha- nisms by which a cell can withstand damage to DNA and other

Zuber, Maria

163

Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination  

PubMed Central

Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species. PMID:19593371

Delmas, Stephane; Shunburne, Lee; Ngo, Hien-Ping; Allers, Thorsten

2009-01-01

164

DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA.  

PubMed

The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair. PMID:25107472

Sung, Sihyun; Li, Fuyang; Park, Young Bong; Kim, Jin Seok; Kim, Ae-Kyoung; Song, Ok-Kyu; Kim, Jiae; Che, Jun; Lee, Sang Eun; Cho, Yunje

2014-10-16

165

Modeling Damage Complexity-Dependent Non-Homologous End-Joining Repair Pathway  

PubMed Central

Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF) model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET) radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed. PMID:24520318

Li, Yongfeng; Reynolds, Pamela; O'Neill, Peter; Cucinotta, Francis A.

2014-01-01

166

Enzymatic repair of UV-irradiated DNA in vitro.  

PubMed

Excision repair of UV-damaged Bacillus subtilis transforming DNA has been carried out by a sequential enzyme system in vitro. Incision adjacent to the pyrimidine dimer in the DNA strand by correndonuclease II-initiated excision of the damage by the 5' in equilibrium 3'-directed exonuclease of the Micrococcus luteus DNA polymerase. Reinsertion of nucleotides into the gap in the strand by the DNA polymerase at 10 degrees C terminated in a single-strand break which was sealed by a polynucleotide ligase, thereby repairing the DNA strand. This restored biological activity to damaged DNA up to doses resulting in 60% inactivation of transforming activity. At higher doses, less repair was achieved, due to the development of double-strand breaks during the in vitro incision and excision steps. PMID:811206

Hamilton, L D; Mahler, I; Grossman, L

1975-01-01

167

DNA damage and repair in human skin: Pathways and questions  

SciTech Connect

Skin is assaulted daily with physical and chemical carcinogens, promoters, and modifiers of biological responses to such agents. DNA is the principal target for most carcinogens, and DNA in skin is particularly at risk. It is subject to damage not only from ingested compounds and their metabolic products, but also from externally applied or encountered chemicals, as well as from physical carcinogens such as sunlight and cosmetic or medical source of ultraviolet radiation. Three major factors determine the balance between damage to DNA of skin and the biological consequences of that damage: the frequencies and types of lesions, the ability of the individual to repair a lesion, and the strategy that skin employs to deal with the different spectra of lesions inflicted under varying environmental conditions. Thus, cellular responses to DNA damage, including repair of DNA lesions, are critical factors in determining the final level of damage and its consequences. This paper discusses DNA damage and repair in human skin. 35 refs.

Sutherland, B.M.; Hacham, H.; Sutherland, J.C. (Brookhaven National Lab., Upton, NY (USA)); Gange, R.W.; Maytum, D. (Harvard Medical School, Boston, MA (USA). Dept. of Dermatology)

1989-01-01

168

Induced DNA repair pathway in mammalian cells  

SciTech Connect

The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-..beta..-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells.

Overberg, R.

1985-01-01

169

Toward Compact Inherently Self-Repairing DNA Lattices Using Reversibility  

E-print Network

Toward Compact Inherently Self-Repairing DNA Lattices Using Reversibility Urmi Majumder and John H. Reif Abstract Self-repair provides the ability to remain functional in spite of gradual damage. Self importance to the self-assembly of large-scale synthetic biomolecular systems. Prior methods for transforming

Reif, John H.

170

DNA excision repair and transcription: implications for genome evolution  

Microsoft Academic Search

The past two years have seen a substantial increase in knowledge regarding the enzymology of DNA excision repair. These data support a growing body of information which suggests that transcribed nucleotide sequences are preferentially subject to excision repair. It is possible that these mechanisms, or related ones, are relevant to the molecular evolution of sequences that appear not to evolve

David T. Sullivan

1995-01-01

171

Transcript-RNA-templated DNA recombination and repair.  

PubMed

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. PMID:25186730

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

2014-11-20

172

DNA repair\\/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis  

Microsoft Academic Search

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute

Carol Bernstein; Harris Bernstein; Claire M. Payne; Harinder Garewal

2002-01-01

173

Intelligent Design | Overwhelming Evidence Human DNA repair process video -by chance?  

E-print Network

Intelligent Design | Overwhelming Evidence Human DNA repair process video - by chance? Uncommon Descent - Sat, 2009-01-31 00:42 More details of DNA repair have been revealed. See: Human DNA repair process recorded in action (Video) (PhysOrg.com) -- A key phase in the repair process of damaged human DNA

Kowalczykowski, Stephen C.

174

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay  

PubMed Central

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (?20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase ?, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

175

MicroRNAs in the DNA Damage/Repair Network and Cancer  

PubMed Central

Cancer is a multistep process characterized by various and different genetic lesions which cause the transformation of normal cells into tumor cells. To preserve the genomic integrity, eukaryotic cells need a complex DNA damage/repair response network of signaling pathways, involving many proteins, able to induce cell cycle arrest, apoptosis, or DNA repair. Chemotherapy and/or radiation therapy are the most commonly used therapeutic approaches to manage cancer and act mainly through the induction of DNA damage. Impairment in the DNA repair proteins, which physiologically protect cells from persistent DNA injury, can affect the efficacy of cancer therapies. Recently, increasing evidence has suggested that microRNAs take actively part in the regulation of the DNA damage/repair network. MicroRNAs are endogenous short noncoding molecules able to regulate gene expression at the post-transcriptional level. Due to their activity, microRNAs play a role in many fundamental physiological and pathological processes. In this review we report and discuss the role of microRNAs in the DNA damage/repair and cancer. PMID:24616890

Tessitore, Alessandra; Cicciarelli, Germana; Del Vecchio, Filippo; Gaggiano, Agata; Verzella, Daniela; Fischietti, Mariafausta; Vecchiotti, Davide; Capece, Daria; Zazzeroni, Francesca; Alesse, Edoardo

2014-01-01

176

[Ionizing radiation-induced DNA damage and its repair in human cells]. Progress report, [April 1, 1993--February 28, 1994  

SciTech Connect

The excision of radiation-induced lesions in DNA by a DNA repair enzyme complex, namely the UvrABC nuclease complex, has been investigated. Irradiated DNA was treated with the enzyme complex. DNA fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results showed that a number pyrimidine- and purine-derived lesions in DNA were excised by the UvrABC nuclease complex and that the enzyme complex does not act on radiation-induced DNA lesions as a glycosylase. This means that it does not excise individual base products, but it excises oligomers containing these lesions. A number of pyrimidine-derived lesions that were no substrates for other DNA repair enzymes investigated in our laboratory were substrates for the UvrABC nuclease complex.

Not Available

1994-07-01

177

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.  

PubMed

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency. PMID:24754999

Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

2014-07-01

178

Inhibition of DNA damage repair by artificial activation of PARP with siDNA  

PubMed Central

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. PMID:23761435

Croset, Amelie; Cordelieres, Fabrice P.; Berthault, Nathalie; Buhler, Cyril; Sun, Jian-Sheng; Quanz, Maria; Dutreix, Marie

2013-01-01

179

Inhibition of DNA damage repair by artificial activation of PARP with siDNA.  

PubMed

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. PMID:23761435

Croset, Amelie; Cordeličres, Fabrice P; Berthault, Nathalie; Buhler, Cyril; Sun, Jian-Sheng; Quanz, Maria; Dutreix, Marie

2013-08-01

180

Heat Shock Protein 90? (Hsp90?) Is Phosphorylated in Response to DNA Damage and Accumulates in Repair Foci*  

PubMed Central

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90? (Hsp90?) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90? then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90? was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90?. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90? were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90? may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone. PMID:22270370

Quanz, Maria; Herbette, Aurelie; Sayarath, Mano; de Koning, Leanne; Dubois, Thierry; Sun, Jian-Sheng; Dutreix, Marie

2012-01-01

181

DNA repair mechanisms in dividing and non-dividing cells.  

PubMed

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease. PMID:23684800

Iyama, Teruaki; Wilson, David M

2013-08-01

182

DNA repair mechanisms in dividing and non-dividing cells  

PubMed Central

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye towards how these pathways may regulate the development of neurological disease. PMID:23684800

Iyama, Teruaki; Wilson, David M.

2013-01-01

183

DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair  

PubMed Central

Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not ?-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair. PMID:25299602

Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

2014-01-01

184

Mitochondrial DNA damage and impaired base excision repair during epileptogenesis  

PubMed Central

Oxidative stress and mitochondrial dysfunction are acute consequences of status epilepticus (SE). However, the role of mitochondrial oxidative stress and genomic instability during epileptogenesis remains unknown. Using the kainate animal model of temporal lobe epilepsy, we investigated oxidative mitochondrial DNA (mtDNA) damage and changes in the mitochondrial base excision repair pathway (mtBER) in the rat hippocampus for a period of 3 months after SE. Acute seizure activity caused a time-dependent increase in mitochondrial, but not nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG/2dG) levels and a greater frequency of mtDNA lesions. This was accompanied by increased mitochondrial H2O2 production and a transient decrease in mtDNA repair capacity. The mtBER proteins 8-oxoguanine glycosylase (Ogg1) and DNA polymerase gamma (Pol ?) demonstrated elevated expression at mRNA and protein levels shortly after SE and this was followed by a gradual improvement in mtDNA repair capacity. Recurrent seizures associated with the chronic phase of epilepsy coincided with the accumulation of mtDNA damage, increased mitochondrial H2O2 levels, decreased expression of Ogg1 and Pol ? and impaired mtDNA repair capacity. Together, increased oxidative mtDNA damage, mitochondrial H2O2 production and alterations in the mtBER pathway provide evidence for mitochondrial oxidative stress in epilepsy and suggest that mitochondrial injury may contribute to epileptogenesis. PMID:18295498

Jarrett, Stuart G.; Liang, Li-Ping; Hellier, Jennifer L.; Staley, Kevin J.; Patel, Manisha

2009-01-01

185

Genotoxicity and DNA repair processes of zinc oxide nanoparticles.  

PubMed

Two different sizes of zinc oxide nanoparticles (ZnO NP, ? 35 nm and 50-80 nm) were tested in the human lymphoblastoid cell line TK6 to increase our knowledge on their genotoxic potential. The comet assay was the system used, and the results obtained showed that the highest concentration tested (100 ?g/ml) for the two selected compounds was genotoxic. The percent DNA in tail obtained after treatment with ZnO NP (? 35 nm) was significantly higher than that of ZnO NP (50-80 nm) at all concentrations tested. To investigate the nature of the induced genotoxic damage, specific enzymes recognizing oxidized DNA bases were used. Treatments with endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (FPG) demonstrated that only ZnO NP (50-80 nm) were able to induce significant levels of net oxidative DNA damage. Further DNA repair kinetics studies revealed that DNA damage initially induced was removed in approximately 5 h. DNA damage induced by ZnO NP was repaired more slowly than damage following microparticulated ZnO exposure. No marked differences in repair kinetics of both forms of ZnO NP were observed. Evidence indicates that a high proportion of DNA damage induced by ZnO NP (50-80 nm) correlated with induction of oxidative damage, and that both forms of ZnO NP interfere with mechanisms involved in DNA damage repair. PMID:25268556

Demir, E?ref; Creus, Amadeu; Marcos, Ricard

2014-01-01

186

BCR-ABL down-regulates the DNA repair protein DNA-PKcs  

Microsoft Academic Search

This study demonstrates in both stable and inducible BCR-ABL-expressing he- matopoietic cells a down-regulation of the major mammalian DNA repair protein DNA-PKcs by BCR-ABL. Similar results were found in BCR-ABL CD341 cells from patients with chronic myelogenous leuke- mia (CML). DNA-PKcs down-regulation is a proteasome-dependent degradation that requires tyrosine kinase activity and is associated with a marked DNA repair deficiency

Eric Deutsch; Aymeric Dugray; Bassam AbdulKarim; Elisabetta Marangoni; Laurence Maggiorella; Sabine Vaganay; Radia M'Kacher; Setha Douc Rasy; Francois Eschwege; William Vainchenker; Ali G. Turhan; Jean Bourhis

187

DNA repair: a changing geography? (1964-2008).  

PubMed

This article aims to explain the current state of DNA Repair studies' global geography by focusing on the genesis of the community. Bibliometric data is used to localize scientific activities related to DNA Repair at the city level. The keyword "DNA Repair" was introduced first by American scientists. It started to spread after 1964 that is to say, after P. Howard-Flanders (Yale University), P. Hanawalt (Stanford University) and R. Setlow (Oak Ridge Laboratories) found evidence for Excision Repair mechanisms. It was the first stage in the emergence of an autonomous scientific community. In this article, we will try to assess to what extent the geo-history of this scientific field is determinant in understanding its current geography. In order to do so, we will localize the places where the first "DNA Repair" publications were signed fifty years ago and the following spatial diffusion process, which led to the current geography of the field. Then, we will focus on the evolution of the research activity of "early entrants" in relation to the activity of "latecomers". This article is an opportunity to share with DNA Repair scientists some research results of a dynamic field in Science studies: spatial scientometrics. PMID:23669398

Maisonobe, Marion; Giglia-Mari, Giuseppina; Eckert, Denis

2013-07-01

188

Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration  

PubMed Central

The pathogenesis of age-related macular degeneration (AMD) is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133) in the hOGG1 gene and the c.972G>C polymorphism (rs3219489) in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395) and c.–32A>G (rs3087404) polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2), XRCC1 and ERCC6 (CSB) have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis. PMID:23202958

Blasiak, Janusz; Synowiec, Ewelina; Salminen, Antero; Kaarniranta, Kai

2012-01-01

189

Loss of urokinase receptor sensitizes cells to DNA damage and delays DNA repair.  

PubMed

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders. PMID:24987841

Narayanaswamy, Pavan B; Hodjat, Mahshid; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

2014-01-01

190

The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.  

PubMed

The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the remaining four genes (XPC, ERCC2, XRCC1, and MLH1) in either the experimental or control animals. PMID:21080036

Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

2011-03-01

191

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair  

PubMed Central

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER. PMID:23890570

German, Peter; Szaniszlo, Peter; Hajas, Gyorgy; Radak, Zsolt; Bacsi, Attila; Hazra, Tapas K.; Hegde, Muralidhar L.; Ba, Xueqing; Boldogh, Istvan

2013-01-01

192

Alterations of DNA damage repair pathways resulting from JCV infection  

Microsoft Academic Search

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disorder of the CNS caused by infection of glial cells with the polyomavirus, JCV. Here we report that genomic stability and DNA repair are significantly dysregulated by JCV infection of human astrocytes. Metaphase spreads exhibited increased ploidy correlating with duration of infection. Increased micronuclei formation and phospho-Histone2AX expression also indicated DNA damage.

Armine Darbinyan; Martyn K. White; Selma Akan; Sujatha Radhakrishnan; Luis Del Valle; Shohreh Amini; Kamel Khalili

2007-01-01

193

Premature Aging in Mice Deficient in DNA Repair and Transcription  

Microsoft Academic Search

One of the factors postulated to drive the aging process is the accumulation of DNA damage. Here, we provide strong support for this hypothesis by de- scribing studies of mice with a mutation in XPD, a gene encoding a DNA helicase that functions in both repair and transcription and that is mutated in the human disorder trichothiodystrophy (TTD). T TD

Jan de Boer; Jaan Olle Andressoo; Jan de Wit; Jan Huijmans; Rudolph B. Beems; Harry van Steeg; Geert Weeda; Wibeke van Leeuwen; Axel P. N. Themmen; Morteza Meradji; Jan H. J. Hoeijmakers

2008-01-01

194

DNA repair defects associated with chromosomal translocation breaksite regions  

SciTech Connect

Using an assay that measures the removal of UV-induced pyrimidine dimers in specific DNA sequences, we have found that the Pvt-1, immunoglobulin H-C{alpha} (IgH-C{alpha}), and IgL-{kappa} loci are poorly repaired in normal B lymphoblasts from plasmacytoma-susceptible BALB/cAnPt mice. Breaksites in these genes are associated with the chromosomal translocations that are found in >95% of BALB/cAnPt plasmacytomas. In contrast to those from BALB/cAnPt mice, B lymphoblasts from plasmacytoma-resistant DBA/2N mice rapidly repair Pvt-1, IgH-C{alpha} and IgL-{kappa}. Further, (BALB/cAnPt x DBA/2N)F{sub 1} hybrids, which are resistant to plasmacytoma development, carry an efficient (DBA/2N-like) repair phenotype. Analysis of allele-specific repair in the IgH-C{alpha} locus indicates that efficient repair is controlled by dominant, transacting factors. In the F{sub 1} heterozygotes, these factors promote efficient repair of BALB/cAnPt IgH-C{alpha} gene sequences. The same sequences are poorly repaired in the BALB/cAnPt parental strain. Analysis of the strand specificity of repair indicates that both strand-selective and nonselective forms of repair determine repair efficiency at the gene level in nonimmortalized murine B lymphoblasts. 36 refs., 3 figs., 6 tabs.

Beecham, E.J.; Link, C.; Bohr, V.A. [National Cancer Inst., Bethesda, MD (United States)] [and others

1994-02-01

195

Cancer-specific defects in DNA repair pathways as targets for personalized therapeutic approaches.  

PubMed

Defects in DNA repair pathways enable cancer cells to accumulate genomic alterations that contribute to their aggressive phenotype. However, tumors rely on residual DNA repair capacities to survive the damage induced by genotoxic stress. This dichotomy might explain why only isolated DNA repair pathways are inactivated in cancer cells. Accordingly, synergism has been observed between DNA-damaging drugs and targeted inhibitors of DNA repair. DNA repair pathways are generally thought of as mutually exclusive mechanistic units handling different types of lesions in distinct cell cycle phases. Recent preclinical studies, however, provide strong evidence that multifunctional DNA repair hubs, which are involved in multiple conventional DNA repair pathways, are frequently altered in cancer. We therefore propose that targeted anticancer therapies should not only exploit synthetic lethal interactions between two single genes but also consider alterations in DNA repair hubs. Such a network-based approach considerably increases the opportunities for targeting DNA repair-defective tumors. PMID:25017190

Dietlein, Felix; Thelen, Lisa; Reinhardt, H Christian

2014-08-01

196

Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.  

PubMed

Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3' flap, 5' flap, and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular toolkit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure. PMID:19596235

Svendsen, Jennifer M; Smogorzewska, Agata; Sowa, Mathew E; O'Connell, Brenda C; Gygi, Steven P; Elledge, Stephen J; Harper, J Wade

2009-07-10

197

DNA repair in murine embryonic stem cells and differentiated cells  

SciTech Connect

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

Tichy, Elisia D. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)], E-mail: tichyed@email.uc.edu; Stambrook, Peter J. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)

2008-06-10

198

DNA repair pathway gene expression score correlates with repair proficiency and tumor sensitivity to chemotherapy.  

PubMed

Mutagenesis is a hallmark of malignancy, and many oncologic treatments function by generating additional DNA damage. Therefore, DNA damage repair is centrally important in both carcinogenesis and cancer treatment. Homologous recombination (HR) and nonhomologous end joining are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The recombination proficiency score (RPS) is based on the expression levels for four genes involved in DNA repair pathway preference (Rif1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLCs). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression contributes to the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteract the adverse prognosis associated with low RPS. Therefore, RPS may help oncologists select which therapies will be effective for individual patients, thereby enabling more personalized care. PMID:24670686

Pitroda, Sean P; Pashtan, Itai M; Logan, Hillary L; Budke, Brian; Darga, Thomas E; Weichselbaum, Ralph R; Connell, Philip P

2014-03-26

199

Mechanism of oxidative DNA damage repair and relevance to human pathology  

Microsoft Academic Search

Since DNA is prone to oxidative attack cells have evolved multiple protective strategies to prevent the deleterious effects of DNA oxidation. Base excision repair is the major mechanism for repair of DNA base damage by reactive oxygen species but recent evidence indicate that nucleotide excision repair proteins, that are mutated in human syndromes, are involved too. The mechanisms of repair

Mariarosaria D’Errico; Eleonora Parlanti; Eugenia Dogliotti

2008-01-01

200

Parathyroid hormone suppresses osteoblast apoptosis by augmenting DNA repair  

PubMed Central

Daily injection of parathyroid hormone (PTH) is a clinically approved treatment for osteoporosis. It suppresses apoptosis of bone forming osteoblasts although its exact anti-apoptotic mechanism(s) is incompletely understood. In this study, PTH treatment of cultured osteoblasts blocked the pro-apoptotic effects of serum withdrawal and nutrient deprivation; hydrogen peroxide induced oxidative stress, and UV irradiation. We hypothesized that PTH might suppress osteoblast apoptosis by enhancing DNA repair. Evidence is provided showing that post-confluent, non-proliferating osteoblasts treated with PTH exhibited a protein kinase A-mediated activation of two proteins that regulate DNA repair processes (proliferating cell nuclear antigen and forkhead box transcription factor 3a) as well as a suppression of the pro-apoptotic growth arrest and DNA damage protein 153. Additional proof of a connection between DNA damage and osteoblast apoptosis came from an unexpected finding whereby a majority of fixed PTH-treated osteoblasts scored weakly positive for Terminal Deoxynucleotidyl dUTP Nick-End Labeling (TUNEL), even though similar cultures were determined to be viable via a trypsin replating strategy. TUNEL identifies DNA excision repair, not just apoptotic DNA fragmentation, and the most likely explanation of these TUNEL results is that PTH's activation of DNA repair processes would permit nucleotide incorporation as a result of enhanced excision repair. This explanation was confirmed by an enhanced incorporation of bromodeoxyuridine in PTH-treated cells even though a majority of the cell population was determined to be non-replicating. An augmentation of DNA repair by PTH is an unreported finding, and provides an additional explanation for its anti-apoptotic mechanism(s). PMID:19450716

Schnoke, Matthew; Midura, Sharon B.; Midura, Ronald J.

2009-01-01

201

3 Xeroderma pigmentosum and related disorders: Defects in DNA repair and transcription  

Microsoft Academic Search

The genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) are all associated with defects in nucleotide excision repair (NER) of DNA damage. Their clinical features are very different, however, XP being a highly cancer-prone skin disorder, whereas CS and TTD are cancer-free multisystem disorders. All three are genetically complex, with at least eight complementation groups for XP

Mark Berneburgl; Alan R Lehmann

2001-01-01

202

Effect of donor age on DNA repair by articular chondrocytes  

SciTech Connect

The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by two different measures: unscheduled DNA synthesis (UDS) and O/sup 6/-methylguanine acceptor protein (MGAP) activity. UDS following damage by 254 nm ultraviolet irradiation (20J/m/sup 2/) was examined in intact articular cartilage from rabbits of different ages. Semiconservative DNA synthesis was suppressed with hydroxurea and repair followed by the incorporation of (/sup 3/H)-thymidine ((/sup 3/H)-dThd). After repair the cartilage was digested in proteinase K (0.5mg/ml) with dodecyl sodium sulfate (0.2%) and DNA determined with Hoechst 33258 dye. UDS (dpm (/sup 3/H)-dThd/..mu..g DNA) was greater in articular cartilage from 3- than 39-month-old rabbits. MGAP was studied in cell extracts of cultured human and rabbit chondrocytes by transfer of (/sup 3/H) O/sup 6/-methyl groups from exogenous DNA to protein. It was significantly less in rabbit than in human cells on a per protein or DNA basis. There was no decline in this activity in human chondrocytes from newborn to 60 years of age; and rabbits from 3- to 36-months-old. The data indicate that in the two different repair mechanisms, age differences are found with resting but not dividing chondrocytes.

Lipman, J.M.

1986-05-01

203

DNA repair is limiting for haematopoietic stem cells during ageing  

Microsoft Academic Search

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4Y288C

Anastasia Nijnik; Lisa Woodbine; Caterina Marchetti; Sara Dawson; Teresa Lambe; Cong Liu; Neil P. Rodrigues; Tanya L. Crockford; Erik Cabuy; Alessandro Vindigni; Tariq Enver; John I. Bell; Predrag Slijepcevic; Christopher C. Goodnow; Penelope A. Jeggo; Richard J. Cornall

2007-01-01

204

Alkyltransferase-like proteins: molecular switches between DNA repair pathways  

Microsoft Academic Search

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation\\u000a damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O\\u000a 6-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its\\u000a mechanism for cell protection was previously unknown. Here we review recent advances

Julie L. TubbsJohn; John A. Tainer

2010-01-01

205

Metal Complexes for DNA-Mediated Charge Transport  

PubMed Central

In all organisms, oxidation threatens the integrity of the genome. DNA-mediated charge transport (CT) may play an important role in the generation and repair of this oxidative damage. In studies involving long-range CT from intercalating Ru and Rh complexes to 5?-GG-3? sites, we have examined the efficiency of CT as a function of distance, temperature, and the electronic coupling of metal oxidants bound to the base stack. Most striking is the shallow distance dependence and the sensitivity of DNA CT to how the metal complexes are stacked in the helix. Experiments with cyclopropylamine-modified bases have revealed that charge occupation occurs at all sites along the bridge. Using Ir complexes, we have seen that the process of DNA-mediated reduction is very similar to that of DNA-mediated oxidation. Studies involving metalloproteins have, furthermore, shown that their redox activity is DNA-dependent and can be DNA-mediated. Long range DNA-mediated CT can facilitate the oxidation of DNA-bound base excision repair proteins to initiate a redox-active search for DNA lesions. DNA CT can also activate the transcription factor SoxR, triggering a cellular response to oxidative stress. Indeed, these studies show that within the cell, redox-active proteins may utilize the same chemistry as that of synthetic metal complexes in vitro, and these proteins may harness DNA-mediated CT to reduce damage to the genome and regulate cellular processes. PMID:21643528

Barton, Jacqueline K.; Olmon, Eric D.; Sontz, Pamela A.

2010-01-01

206

Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair.  

PubMed

Recruitment of DNA repair factors and modulation of chromatin structure at sites of DNA double-strand breaks (DSBs) is a complex and highly orchestrated process. We developed a system that can induce DSBs rapidly at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Distinct advantages of this system over focus formation/disappearance assays for assessing DSB repair are demonstrated. Using ChIP, we found that nucleosomes are partially disassembled around DSBs during nonhomologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its arginine-glycine rich domain and is recruited to DSBs rapidly in an MRE11-NBS1-RAD50 complex-dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and the repair of the DSB, demonstrating the functional importance of nucleosome disruption in DSB repair and identifying a chromatin-remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for recruitment of replication protein A to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair. PMID:24082117

Goldstein, Michael; Derheimer, Frederick A; Tait-Mulder, Jacqueline; Kastan, Michael B

2013-10-15

207

Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase  

PubMed Central

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

2014-01-01

208

DNA double-strand break repair at--15{degrees}C.  

PubMed

The survival of microorganisms in ancient glacial ice and permafrost has been ascribed to their ability to persist in a dormant, metabolically inert state. An alternative possibility, supported by experimental data, is that microorganisms in frozen matrices are able to sustain a level of metabolic function that is sufficient for cellular repair and maintenance. To examine this experimentally, frozen populations of Psychrobacter arcticus 273-4 were exposed to ionizing radiation (IR) to simulate the damage incurred from natural background IR sources in the permafrost environment from over ?225 kiloyears (ky). High-molecular-weight DNA was fragmented by exposure to 450 Gy of IR, which introduced an average of 16 double-strand breaks (DSBs) per chromosome. During incubation at -15°C for 505 days, P. arcticus repaired DNA DSBs in the absence of net growth. Based on the time frame for the assembly of genomic fragments by P. arcticus, the rate of DNA DSB repair was estimated at 7 to 10 DSBs year(-1) under the conditions tested. Our results provide direct evidence for the repair of DNA lesions, extending the range of complex biochemical reactions known to occur in bacteria at frozen temperatures. Provided that sufficient energy and nutrient sources are available, a functional DNA repair mechanism would allow cells to maintain genome integrity and augment microbial survival in icy terrestrial or extraterrestrial environments. PMID:24077718

Dieser, Markus; Battista, John R; Christner, Brent C

2013-12-01

209

Electronic Pathways in Photoactivated Repair of UV Mutated DNA  

NASA Astrophysics Data System (ADS)

An investigation of the physics, underlying the damage caused to DNA by UV radiation and its subsequent repair via a photoreactivation mechanism, is presented in this study. Electronic pathways, starting from the initial damage to the final repair process, are presented. UV radiation is absorbed to create a hole-excited thymine or other pyrimidine that subsequently is responsible for the formation of a dimer. The negative-ion of the cofactor riboflavin, FADH-, formed by the exposure of the photolyase protein to visible light, interacts with the hole-excited electronic orbital of the thymine dimer inducing a photon-less Auger transition, which restores the two thymines to the ground state, thereby detaching the lesion and repairing the DNA. Density functional theoretical calculations supporting the theory are presented. The mechanism involves the least amount of energy dissipation and is charge neutral. It also avoids radiation damage in the repair process. Recent experimental data are compatible with this theory.

Bohr, Henrik; Jalkanen, K. J.; Bary Malik, F.

210

Monitoring populations for DNA repair deficiency and for cancer susceptibility.  

PubMed Central

The induction of a mutator phenotype has been hypothesized to cause the accumulation of multiple mutations in the development of cancer. Recent evidence suggests that the mutator phenotype is associated with DNA repair deficiencies. We have been using a challenge assay to study exposed populations to test our hypothesis that exposure to environmental toxicants induce DNA repair deficiency in somatic cells. In this assay, lymphocytes were irradiated in vitro to challenge cells to repair the radiation-induction DNA strand breaks. An increase of chromosome aberrations in the challenged cells from toxicant-exposed populations compared to nonexposed populations is used to indicate abnormal DNA repair response. From studies of cigarette smokers, butadiene-exposed workers, and uranium-exposed residents, the assay showed that these exposed populations had mutagen-induced abnormal DNA repair response. The phenomenon was also demonstrated using experimental animals. Mice were exposed in vivo to two different doses of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) and their lymphocytes were challenged with one dose of a radiomimetic chemical, bleomycin, in vitro. These challenged lymphocytes showed an MNNG dose-dependent increase of abnormal DNA repair response. In a population that was potentially exposed to teratogens--mothers having children with neural tube defects--lymphocytes from these mothers did not have the abnormal response in our assay. In studies with patients, we reported that lymphocytes from Down's syndrome patients have the abnormal DNA repair response. Lymphocytes from skin cancer-prone patients (epidermodysplasia verruciformis) have normal response to gamma-ray challenge but abnormal response to UV-light challenge. These patient studies also indicate that the challenge assay is useful in documenting the radiosensitivity of Down's syndrome and the UV sensitivity in EV patients. In most cases, the challenge assay is more sensitive in detecting biological effects than the standard chromosome aberration assay. Our series of studies indicates that the challenge assay can be used to document biological effects from exposure to mutagens and that the effect is an abnormal DNA repair response. This abnormality can increase the risk for development of cancer. The repair deficiency is currently being validated using a plasmid transfection (host-reactivation) assay. The need to integrate chromosome aberration and the challenge assays with other relevant assays for better documentation of biological effects and for more precise prediction of health risk will be presented. Our experience in using genetic polymorphism and host-reactivation assays will be discussed. PMID:8781386

Au, W W; Wilkinson, G S; Tyring, S K; Legator, M S; el Zein, R; Hallberg, L; Heo, M Y

1996-01-01

211

Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival  

PubMed Central

A clustered DNA lesion, also known as a multiply damaged site, is defined as ? 2 damages in the DNA within 1–2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990’s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed. PMID:21185841

Sage, Evelyne; Harrison, Lynn

2011-01-01

212

Repair of mismatched basepairs in mammalian DNA  

SciTech Connect

We have concentrated on three specific areas of our research plan. Our greatest emphasis is on the role of single strand nicks in influencing template strand selection in mismatch repair. We have found, that the ability of a nick in one strand to influence which strand is repaired is not a simple function of distance from the mismatched site but rather that an hot spot where a nick is more likely to have an influence can exist. The second line was production of single-genotype heteroduplexes in order to examine independently the repair of T/G and A/C mispairs within the same sequence context as in our mixed mispair preparations. We have shown preparations of supercoiled heteroduplex can be prepared that were exclusively T/G or exclusively A/C at the mispair site. The third effort has been to understand the difference in repair bias of different cell lines or different transfection conditions as it may relate to different repair systems in the cell. We have identified some of the sources of variation, including cell cycle position. We hope to continue this work to more precisely identify the phase of the cell cycle.

Taylor, J.H.; Hare, J.T.

1991-08-01

213

Bacterial DNA repair by non-homologous end joining  

Microsoft Academic Search

The capacity to rectify DNA double-strand breaks (DSBs) is crucial for the survival of all species. DSBs can be repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). The long-standing notion that bacteria rely solely on HR for DSB repair has been overturned by evidence that mycobacteria and other genera have an NHEJ system that depends on a

Michael S. Glickman; Stewart Shuman

2007-01-01

214

Charge transport-mediated recruitment of DNA repair enzymes  

E-print Network

Damaged or mismatched bases in DNA can be repaired by Base Excision Repair (BER) enzymes that replace the defective base. Although the detailed molecular structures of many BER enzymes are known, how they colocalize to lesions remains unclear. One hypothesis involves charge transport (CT) along DNA [Yavin, {\\it et al.}, PNAS, {\\bf 102}, 3546, (2005)]. In this CT mechanism, electrons are released by recently adsorbed BER enzymes and travel along the DNA. The electrons can scatter (by heterogeneities along the DNA) back to the enzyme, destabilizing and knocking it off the DNA, or, they can be absorbed by nearby lesions and guanine radicals. We develop a stochastic model to describe the electron dynamics, and compute probabilities of electron capture by guanine radicals and repair enzymes. We also calculate first passage times of electron return, and ensemble-average these results over guanine radical distributions. Our statistical results provide the rules that enable us to perform implicit-electron Monte-Carlo simulations of repair enzyme binding and redistribution near lesions. When lesions are electron absorbing, we show that the CT mechanism suppresses wasteful buildup of enzymes along intact portions of the DNA, maximizing enzyme concentration near lesions.

Pak-Wing Fok; Chin-Lin Guo; Tom Chou

2008-11-18

215

Mitochondrial DNA replication and repair: all a flap.  

PubMed

The mitochondrial genome is dwarfed by its neighbour in the nucleus, and, thus, it has been sensible for far more resources to be invested in the study of nuclear, rather than mitochondrial, DNA metabolism. Furthermore, few researchers have considered using mitochondrial DNA (mtDNA) as a model system for nuclear DNA metabolism. A quick look into the history of mtDNA provides ready answers as to why this was the case; however, recently mitochondria have been found to contain several nuclear replication and repair factors, so is there any potential to adopt the mitochondrion as a tool to unravel some of the intricacies of replication and repair in higher-order eukaryotes? Perhaps it is now time to invite the Cinderella genome to the ball. PMID:19559620

Holt, Ian J

2009-07-01

216

Functional Aspects of PARP1 in DNA Repair and Transcription  

PubMed Central

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP-ribosylating enzyme essential for initiating various forms of DNA repair. Inhibiting its enzyme activity with small molecules thus achieves synthetic lethality by preventing unwanted DNA repair in the treatment of cancers. Through enzyme-dependent chromatin remodeling and enzyme-independent motif recognition, PARP1 also plays important roles in regulating gene expression. Besides presenting current findings on how each process is individually controlled by PARP1, we shall discuss how transcription and DNA repair are so intricately linked that disturbance by PARP1 enzymatic inhibition, enzyme hyperactivation in diseases, and viral replication can favor one function while suppressing the other. PMID:24970148

Ko, Hui Ling; Ren, Ee Chee

2012-01-01

217

The DNA repair component Metnase regulates Chk1 stability  

PubMed Central

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest. PMID:25024738

2014-01-01

218

Microhomology Directs Diverse DNA Break Repair Pathways and Chromosomal Translocations  

PubMed Central

Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called “microhomology,” yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements. PMID:23144625

Villarreal, Diana D.; Lee, Kihoon; Deem, Angela; Shim, Eun Yong; Malkova, Anna; Lee, Sang Eun

2012-01-01

219

Base Excision Repair of Oxidative DNA Damage  

PubMed Central

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

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

2010-01-01

220

Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus  

SciTech Connect

Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5? E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

2005-01-01

221

Compound K suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair in human keratinocytes.  

PubMed

Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage of mammalian cells. The purpose of this study is to investigate the functional role of ginsenoside compound K on HaCaT cells (a keratinocyte-derived permanent cell line) irradiated by UV. Hoechst 33258 staining were performed in analyzing UV-induced apoptosis on keratinocytes which were treated with compound K. ImmunoDotBlot assay was used in detecting cyclobutane pyrimidine dimers, the main DNA damage. Western blot analysis was applied for analyzing XPC and ERCC1, two of the NER proteins. Compound K inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In agreement with this, compound K induced the expression of particular components of the NER complex, such as XPC and ERCC1. Our results demonstrate that compound K can protect cells from apoptosis induced by UV radiation by inducing DNA repair. PMID:19023546

Cai, Bao-Xiang; Luo, Dan; Lin, Xiang-Fei; Gao, Jie

2008-11-01

222

Recombinational DNA repair and human disease  

Microsoft Academic Search

We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in

Larry H Thompson; David Schild

2002-01-01

223

DNA Repair 2 (2003) 947954 Mini review  

E-print Network

element in their promoter or intronic segments. The p53-regulated cyclin-dependent kinase inhibitor p21, the p53 tumor suppressor gene product is activated and regulates a number of downstream cellular role for p53 in NER, mediating expression of the global genomic repair (GGR)-specific damage

Ford, James

224

The repair of DNA methylation damage in Saccharomyces cerevisiae  

Microsoft Academic Search

The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion.\\u000a An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the\\u000a damaged base, followed by an Apn1 apurinic\\/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent\\u000a repair. MMS is also

Wei Xiao; Barbara L. Chow

1996-01-01

225

ATM-mediated phosphorylation of polynucleotide kinase\\/phosphatase is required for effective DNA double-strand break repair  

Microsoft Academic Search

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase\\/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5?-OH

Hava Segal-Raz; Gilad Mass; Keren Baranes-Bachar; Yaniv Lerenthal; Shih-Ya Wang; Young Min Chung; Shelly Ziv-Lehrman; Cecilia E Ström; Thomas Helleday; Mickey C.-T. Hu; David J Chen; Yosef Shiloh

2011-01-01

226

Exploiting the homologous recombination DNA repair network for targeted cancer therapy  

PubMed Central

Genomic instability is a characteristic of cancer cells. In order to maintain genomic integrity, cells have evolved a complex DNA repair system to detect, signal and repair a diversity of DNA lesions. Homologous recombination (HR)-mediated DNA repair represents an error-free repair mechanism to maintain genomic integrity and ensure high-fidelity transmission of genetic information. Deficiencies in HR repair are of tremendous importance in the etiology of human cancers and at the same time offer great opportunities for designing targeted therapeutic strategies. The increase in the number of proteins identified as being involved in HR repair has dramatically shifted our concept of the proteins involved in this process: traditionally viewed as existing in a linear and simple pathway, today they are viewed as existing in a dynamic and interconnected network. Moreover, exploration of the targets within this network that can be modulated by small molecule drugs has led to the discovery of many effective kinase inhibitors, such as ATM, ATR, DNA-PK, CHK1, and CHK2 inhibitors. In preclinical studies, these inhibitors have been shown to sensitize cancer cells to chemotherapy and radiation therapy. The most exciting discovery in the field of HR repair is the identification of the synthetic lethality relationship between poly (ADP-ribose) polymerase (PARP) inhibitors and HR deficiency. The promises of clinical applications of PARP inhibitors and the concept of synthetic lethality also bring challenges into focus. Future research directions in the area of HR repair include determining how to identify the patients most likely to benefit from PARP inhibitors and developing strategies to overcome resistance to PARP inhibitors. PMID:21603316

Peng, Guang; Lin, Shiaw-Yih

2011-01-01

227

WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN  

SciTech Connect

The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of XPG as well as the C-terminal region of WRN. The physical interaction between XPG and WRN links NER, (made evident by the disease XP) with DSBR, which imparts additional knowledge of the overlapping nature of these two proteins and the previously distinct DNA repair pathways they are associated with. Since genomic integrity is constantly threatened by both endogenous and exogenous (internal and external) damage, understanding the roles of these proteins in coordinating DNA repair processes with replication will signifi cantly further understanding how defects instigate physiological consequences in response to various DNA damaging sources. This ultimately contributes to our understanding of cancer and premature aging.

Rangaraj, K.; Cooper, P.K.; Trego, K.S.

2009-01-01

228

DNA Repair at Telomeres: Keeping the Ends Intact  

PubMed Central

The molecular era of telomere biology began with the discovery that telomeres usually consist of G-rich simple repeats and end with 3? single-stranded tails. Enormous progress has been made in identifying the mechanisms that maintain and replenish telomeric DNA and the proteins that protect them from degradation, fusions, and checkpoint activation. Although telomeres in different organisms (or even in the same organism under different conditions) are maintained by different mechanisms, the disparate processes have the common goals of repairing defects caused by semiconservative replication through G-rich DNA, countering the shortening caused by incomplete replication, and postreplication regeneration of G tails. In addition, standard DNA repair mechanisms must be suppressed or modified at telomeres to prevent their being recognized and processed as DNA double-strand breaks. Here, we discuss the players and processes that maintain and regenerate telomere structure. PMID:23732473

Webb, Christopher J.; Wu, Yun; Zakian, Virginia A.

2013-01-01

229

DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)  

SciTech Connect

Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of (6-/sup 3/H)thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.

Ammermann, D.

1988-05-01

230

DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts.  

PubMed

Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts. PMID:24056763

Simonatto, M; Marullo, F; Chiacchiera, F; Musaró, A; Wang, J Y J; Latella, L; Puri, P L

2013-12-01

231

Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis.  

PubMed

Double-strand breaks in genomic DNA (DSB) are potentially lethal lesions which separate parts of chromosome arms from their centromeres. Repair of DSB by recombination can generate mutations and further chromosomal rearrangements, making the regulation of recombination and the choice of recombination pathways of the highest importance. Although knowledge of recombination mechanisms has considerably advanced, the complex interrelationships and regulation of pathways are far from being fully understood. We analyse the different pathways of DSB repair acting in G2/M phase nuclei of irradiated plants, through quantitation of the kinetics of appearance and loss of ?-H2AX foci in Arabidopsis mutants. These analyses show the roles for the four major recombination pathways in post-S-phase DSB repair and that non-homologous recombination pathways constitute the major response. The data suggest a hierarchical organisation of DSB repair in these cells: C-NHEJ acts prior to B-NHEJ which can also inhibit MMEJ. Surprisingly the quadruple ku80 xrcc1 xrcc2 xpf mutant can repair DSB, although with severely altered kinetics. This repair leads to massive genetic instability with more than 50% of mitoses showing anaphase bridges following irradiation. This study thus clarifies the relationships between the different pathways of DSB repair in the living plant and points to the existence of novel DSB repair processes. PMID:21530420

Charbonnel, Cyril; Allain, Elisabeth; Gallego, Maria Eugenia; White, Charles I

2011-06-10

232

Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.  

PubMed Central

Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG. PMID:9034344

Evans, E; Fellows, J; Coffer, A; Wood, R D

1997-01-01

233

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis  

E-print Network

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair ...

Nagel, Zachary D.

234

Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF.  

PubMed

Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5' to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes. PMID:14729965

Tian, Ming; Shinkura, Reiko; Shinkura, Nobuhiko; Alt, Frederick W

2004-02-01

235

At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence  

PubMed Central

It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure, and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence. PMID:23967007

Ohsawa, Ryosuke; Seol, Ja-Hwan; Tyler, Jessica K.

2013-01-01

236

A role for nuclear envelope-bridging complexes in homology-directed repair  

PubMed Central

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency. PMID:24943839

Swartz, Rebecca K.; Rodriguez, Elisa C.; King, Megan C.

2014-01-01

237

Effects of Extracellular Matix on DNA Repair in Vivo.  

National Technical Information Service (NTIS)

DNA damage in the form of double-strand breaks is caused by exposure to endogenous factors as well as in response to radiation therapy in breast cancer patients. Double-strand breaks can be repaired by homologous recombination or nonhomologous end joining...

A. Rizki

2006-01-01

238

Human Premature Aging Disorders and Dysfunction of DNA Repair  

Microsoft Academic Search

Werner?s syndrome (WS) and Cockayne syndrome (CS) are rare human autosomal recessive disorders classified as segmental progeroid disorders. WS is marked by premature onset of age-related phenotypic changes (such as cataract and greying of hair etc) and genome instability. Cells derived from CS patients are defective in DNA repair, and CS patients display severe neurological abnormalities and certain features of

Byungchan Ahn; Vilhelm A. Bohr

239

Recognition of DNA alterations by the mismatch repair system.  

PubMed Central

Misincorporation of non-complementary bases by DNA polymerases is a major source of the occurrence of promutagenic base-pairing errors during DNA replication or repair. Base-base mismatches or loops of extra bases can arise which, if left unrepaired, will generate point or frameshift mutations respectively. To counteract this mutagenic potential, organisms have developed a number of elaborate surveillance and repair strategies which co-operate to maintain the integrity of their genomes. An important replication-associated correction function is provided by the post-replicative mismatch repair system. This system is highly conserved among species and appears to be the major pathway for strand-specific elimination of base-base mispairs and short insertion/deletion loops (IDLs), not only during DNA replication, but also in intermediates of homologous recombination. The efficiency of repair of different base-pairing errors in the DNA varies, and appears to depend on multiple factors, such as the physical structure of the mismatch and sequence context effects. These structural aspects of mismatch repair are poorly understood. In contrast, remarkable progress in understanding the biochemical role of error-recognition proteins has been made in the recent past. In eukaryotes, two heterodimers consisting of MutS-homologous proteins have been shown to share the function of mismatch recognition in vivo and in vitro. A first MutS homologue, MSH2, is present in both heterodimers, and the specificity for mismatch recognition is dictated by its association with either of two other MutS homologues: MSH6 for recognition of base-base mismatches and small IDLs, or MSH3 for recognition of IDLs only. Mismatch repair deficiency in cells can arise through mutation, transcriptional silencing or as a result of imbalanced expression of these genes. PMID:9931291

Marra, G; Schar, P

1999-01-01

240

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

NASA Technical Reports Server (NTRS)

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 gamma-radiation the induction of gamma-H2AX foci is similar in M059J and M059K cells. However, the repair rate of DSBs is slower in M059J cells than in M059K as shown previously but faster than seen with DSB induced by 56Fe ions. Vanillin, an inhibitor of DNA-PK(sub cs), reduces significantly the rate of DSB repair in HF19 cells following 1 Gy gamma-radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

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

2007-01-01

241

Dynamics and pathway of electron tunneling in repair of damaged DNA by photolyase  

NASA Astrophysics Data System (ADS)

Through electron tunneling, photolyase, a photoenzyme, restores damaged DNA into normal bases. Here, we report our systematic characterization and analyses of three electron transfer processes in thymine dimer restoration by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repairing and for the final electron return to restore the active cofactor and complete the repair photocycle. Significantly, we found that the adenine moiety of the unusual bent cofactor is essential to mediating all electron-transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure these elementary dynamics in synergy in this complex photorepair machinery to achieve the maximum repair efficiency close to unity.

Liu, Zheyun; Guo, Xunmin; Tan, Chuang; Li, Jiang; Kao, Ya-Ting; Wang, Lijuan; Sancar, Aziz; Zhong, Dongping

2013-03-01

242

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

PubMed

Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account. PMID:25012975

Grasso, Stefano; Tell, Gianluca

2014-09-01

243

GADD45? inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair  

PubMed Central

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45?. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45?, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45? during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45? increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45? specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45?. Since GADD45? binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

2012-01-01

244

Polymorphisms in DNA repair genes, recreational physical activity and breast cancer risk.  

PubMed

The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p < 0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR = 0.54; 95% CI, 0.36-0.81) and XPF (OR = 0.62; 95% CI, 0.44-0.87), as well as among active women who carried at least one variant allele in XPG (OR = 0.46; 95% CI, 0.29-0.77) and MLH1 (OR = 0.46; 95% CI, 0.30-0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation. PMID:23852586

McCullough, Lauren E; Santella, Regina M; Cleveland, Rebecca J; Millikan, Robert C; Olshan, Andrew F; North, Kari E; Bradshaw, Patrick T; Eng, Sybil M; Terry, Mary Beth; Shen, Jing; Crew, Katherine D; Rossner, Pavel; Teitelbaum, Susan L; Neugut, Alfred I; Gammon, Marilie D

2014-02-01

245

INO80 and ?-H2AX Interaction Links ATP-Dependent Chromatin Remodeling to DNA Damage Repair  

Microsoft Academic Search

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

Ashby J. Morrison; Jessica Highland; Nevan J. Krogan; Ayelet Arbel-Eden; Jack F. Greenblatt; James E. Haber; Xuetong Shen

2004-01-01

246

E2F-7 couples DNA damage-dependent transcription with the DNA repair process  

PubMed Central

The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy. PMID:23974101

Zalmas, Lykourgos-Panagiotis; Coutts, Amanda S; Helleday, Thomas; La Thangue, Nicholas B

2013-01-01

247

DNA repair in spermatocytes and spermatids of the mouse  

SciTech Connect

When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced.

Sega, G.A.

1982-01-01

248

Spatiotemporal analysis of DNA repair using charged particle radiation.  

PubMed

Approaches to visualise the dynamics of the DNA lesion processing substantially contributes to the understanding of the hierarchical organisation of the DNA damage response pathways. Charged particle irradiation has recently emerged as a tool to generate discrete sites of subnuclear damage by its means of extremely localised dose deposition at low energies, thus facilitating the spatiotemporal analysis of repair events. In addition, they are of high interest for risk estimations of human space exploration (e.g. mars mission) in the high energy regime (HZE). In this short review we will give examples for the application of charged particle irradiation to study spatiotemporal aspects of DNA damage recognition and repair in the context of recent achievements in this field. Beamline microscopy allows determining the exact kinetics of repair-related proteins after irradiation with different charged particles that induce different lesion densities. The classification into fast recruited proteins like DNA-PK or XRCC1 or slower recruited ones like 53BP1 or MDC1 helps to establish the hierarchical organisation of damage recognition and subsequent repair events. Additionally, motional analysis of DNA lesions induced by traversing particles proved information about the mobility of DSBs. Increased mobility or the absence of large scale motion has direct consequences on the formation of chromosomal translocations and, thus, on mechanisms of cancer formation. Charged particle microbeams offer the interesting perspective of precise nuclear or subnuclear targeting with a defined number of ions, avoiding the Poisson distribution of traversals inherent to broad beam experiments. With the help of the microbeam, geometrical patterns of traversing ions can be applied facilitating the analysis of spatial organisation of repair. PMID:19944777

Tobias, F; Durante, M; Taucher-Scholz, G; Jakob, B

2010-01-01

249

Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress.  

PubMed

To maintain genome stability, cells have evolved various DNA repair pathways to deal with oxidative DNA damage. DNA damage response (DDR) pathways, including ATM-Chk2 and ATR-Chk1 checkpoints, are also activated in oxidative stress to coordinate DNA repair, cell cycle progression, transcription, apoptosis, and senescence. Several studies demonstrate that DDR pathways can regulate DNA repair pathways. On the other hand, accumulating evidence suggests that DNA repair pathways may modulate DDR pathway activation as well. In this review, we summarize our current understanding of how various DNA repair and DDR pathways are activated in response to oxidative DNA damage primarily from studies in eukaryotes. In particular, we analyze the functional interplay between DNA repair and DDR pathways in oxidative stress. A better understanding of cellular response to oxidative stress may provide novel avenues of treating human diseases, such as cancer and neurodegenerative disorders. PMID:24947324

Yan, Shan; Sorrell, Melanie; Berman, Zachary

2014-10-01

250

Word-based characterization of promoters involved in human DNA repair pathways  

Microsoft Academic Search

BACKGROUND: DNA repair genes provide an important contribution towards the surveillance and repair of DNA damage. These genes produce a large network of interacting proteins whose mRNA expression is likely to be regulated by similar regulatory factors. Full characterization of promoters of DNA repair genes and the similarities among them will more fully elucidate the regulatory networks that activate or

Jens Lichtenberg; Edwin Jacox; Joshua D Welch; Kyle Kurz; Xiaoyu Liang; Mary Qu Yang; Frank Drews; Klaus Ecker; Stephen S Lee; Laura Elnitski; Lonnie R Welch

2009-01-01

251

Regulatory network nodes of check point factors in DNA repair pathways  

Microsoft Academic Search

DNA repair pathways and associated cell-cycle checkpoint factors are critical to normal cellular function. Although the roles of many of the member genes of the DNA repair pathways are well known, little is known about the transcriptional regulation of these genes. We hypothesize that the genes involved in DNA repair processes have a common regulatory control system. To address the

Laura Elnitski; Jens Lichtenberg; Lonnie R. Welch

2010-01-01

252

Induction and processing of complex DNA damage in human breast cancer cells MCF7 and nonmalignant MCF10A cells  

Microsoft Academic Search

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair

Dave C. Francisco; Prakash Peddi; Jessica M. Hair; Brittany A. Flood; Angela M. Cecil; Peter T. Kalogerinis; George Sigounas; Alexandros G. Georgakilas

2008-01-01

253

Theoretical study of fast repair of DNA damage by cistanoside C and analogs: mechanism and docking.  

PubMed

Experiments show that the natural substances phenylpropanoid glycosides (PPGs) extracted from pelicularis spicata are capable of repairing DNA damaged by oxygen radicals. Based on kinetic measurements and experiments on tumor cells, a theoretical study of the interaction between PPG molecules and isolated DNA bases, as well as a DNA fragment has been performed. An interaction mechanism reported early has been refined. The docking calculations performed using junction minimization of nucleic acids (JUMNA) software showed that the PPG molecules can be docked into the minor groove of DNA and form complexes with the geometry suitable for an electron transfer between guanine radical and the ligand. Such complexes can be formed without major distortions of DNA structure and are further stabilized by the interaction with the rhamnosyl side-groups. PMID:12071653

Sperandio, O; Fan, B T; Zakrzewska, K; Jia, Z J; Zheng, R L; Panaye, A; Doucet, J P; El Fassi, N

2002-03-01

254

Single-molecule fluorescence microscopy on nucleotide excision repair complexes using GFP fusion proteins  

NASA Astrophysics Data System (ADS)

Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these DNA-protein complexes and to study dynamic changes that occur in these structures. For this purpose NER- complexes are partly reconstituted onto DNA-substrates using NER-proteins fused to different Green Fluorescent Protein mutants. Here we describe the recombinant production of NER- GFP fusion proteins. Characterization of GFP fluorescence is shown together with results of GFP single molecule imaging. First results with NER-GFP fusion proteins are presented as well.

Segers-Nolten, Ine; Rademakers, Suzanne; Vermeulen, Wim; Lenferink, Aufried; Otto, Cees; Hoeijmakers, Jan; Greve, Jan

2000-12-01

255

FIGNL1-containing protein complex is required for efficient homologous recombination repair  

PubMed Central

The RAD51 recombinase plays a central role in homologous recombination (HR), which is critical for repair of DNA double-strand breaks, maintenance of genomic stability, and prevention of developmental disorders and cancer. Here, we report the identification of an RAD51-binding protein fidgetin-like 1 (FIGNL1). FIGNL1 specifically interacts with RAD51 through its conserved RAD51 binding domain. Cells depleted of FIGNL1 show defective HR repair. Interestingly, FIGNL1 is recruited to sites of DNA damage in a manner that is independent of breast cancer 2, early onset, RAD51, and probably, RAD51 paralogs. Conversely, FIGNL1 depletion does not affect the loading of RAD51 onto ssDNA. Our additional analysis uncovered KIAA0146, also known as scaffolding protein involved in DNA repair (SPIDR), as a binding partner of FIGNL1 and established that KIAA0146/SPIDR acts with FIGNL1 in HR repair. Collectively, our study uncovers a protein complex, which consists of FIGNL1 and KIAA0146/SPIDR, in DNA repair and provides potential directions for cancer diagnosis and therapy. PMID:23754376

Yuan, Jingsong; Chen, Junjie

2013-01-01

256

Hypomorphic PCNA mutation underlies a human DNA repair disorder.  

PubMed

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

Baple, Emma L; Chambers, Helen; Cross, Harold E; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A; Harlalka, Gaurav V; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K; Last, James I; Taylor, A Malcolm R; Jackson, Andrew P; Ogi, Tomoo; Lehmann, Alan R; Green, Catherine M; Crosby, Andrew H

2014-07-01

257

Hypomorphic PCNA mutation underlies a human DNA repair disorder  

PubMed Central

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

2014-01-01

258

Reduction in DNA repair capacity following differentiation of murine proadipocytes  

SciTech Connect

It has been suggested that terminally differentiated mammalian cells have a decreased DNA repair capacity, compared with proliferating stem cells. To investigate this hypothesis, we have examined {gamma}-ray-induced DNA strand breaks and their repair in the murine proadipocyte stem cell line 3T3-T. By exposure to human plasma, 3T3-T cells can be induced to undergo nonterminal and then terminal differentiation. DNA strand breaks were evaluated using the technique of alkaline elution. No difference was detected among stem, nonterminally differentiated, and terminally differentiated cells in the initial levels of radiation-induced DNA strand breaks. Each of the strand break dose responses increased as a linear function of {gamma}-ray dose. The strand breaks induced by 4 Gy rejoined following biphasic kinetics for each cell type. At each time point examined after irradiation, however, the percentage of strand breaks that had not rejoined in terminally differentiated cells was three to six times greater than in stem cells. The rate of strand break rejoining in nonterminally differentiated cells was of an intermediate value between that of the stem and of the terminally differentiated cells. These results indicate that, at least for 3T3-T cells, differentiated cells have a reduced capacity for DNA repair.

Tofilon, P.J.; Meyn, R.E. (Univ. of Texas M.D. Anderson Hospital and Tumor Institute, Houston (USA))

1988-02-01

259

Histone Modifications and DNA Double-Strand Break Repair after Exposure to Ionizing Radiations  

PubMed Central

Ionizing radiation exposure induces highly lethal DNA double-strand breaks (DSBs) in all phases of the cell cycle. After DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining (NHEJ), which re-ligates the broken ends of the DNA and (2) homologous recombination (HR), that makes use of an undamaged identical DNA sequence as a template to maintain the fidelity of DNA repair. DNA DSB repair must occur within the context of the natural cellular DNA structure. Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. The overall chromatin structure regulates DNA damage responses since chromatin status can impede DNA damage site access by repair proteins. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery. The DNA DSB response is also facilitated by hierarchical signaling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple enzymatic activities to repair broken DNA ends. During DNA damage sensing and repair, histones undergo posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation and ubiquitylation. Such histone modifications represent a histone code that directs the recruitment of proteins involved in DNA damage sensing and repair processes. In this review, we summarize histone modifications that occur during DNA DSB repair processes. PMID:23373901

Hunt, Clayton R.; Ramnarain, Deepti; Horikoshi, Nobuo; Iyengar, Puneeth; Pandita, Raj K.; Shay, Jerry W.; Pandita, Tej K.

2014-01-01

260

ATM Protein-dependent Phosphorylation of Rad50 Protein Regulates DNA Repair and Cell Cycle Control*  

PubMed Central

The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity. PMID:21757780

Gatei, Magtouf; Jakob, Burkhard; Chen, Philip; Kijas, Amanda W.; Becherel, Olivier J.; Gueven, Nuri; Birrell, Geoff; Lee, Ji-Hoon; Paull, Tanya T.; Lerenthal, Yaniv; Fazry, Shazrul; Taucher-Scholz, Gisela; Kalb, Reinhard; Schindler, Detlev; Waltes, Regina; Dork, Thilo; Lavin, Martin F.

2011-01-01

261

Molecular Understanding of Efficient DNA Repair Machinery of Photolyase  

NASA Astrophysics Data System (ADS)

Photolyases repair the UV-induced pyrimidine dimers in damage DNA with high efficiency, through a cylic light-driven electron transfer radical mechanism. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of five active-site residues. The significant loss of repair efficiency by the mutation indicates that those active-site residues play an important role in the DNA repair by photolyase. To understand how the active-site residues modulate the efficiency, we mapped out the entire evolution of each elementary step during the repair in those photolyase mutants with femtosecond resolution. We completely analyzed the electron transfer dynamics using the Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.

Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

2012-06-01

262

The MRN complex in Double-Strand Break Repair and Telomere Maintenance  

PubMed Central

Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction. PMID:20655309

Lamarche, Brandon J; Orazio, Nicole I; Weitzman, Matthew D

2010-01-01

263

Operational readiness of a complex system under priority repair disciplines  

Microsoft Academic Search

Reliability of a complex system under priority repair disciplines viz. head of line, preemptive resume and preemptive repeat\\u000a repair disciplines have been considered. Mathematical models have been developed to find the probability of system being in\\u000a up-state and down-state. Supplementary variable technique and discrete transforms have been used in solving the problem.

A. K. Govil

1970-01-01

264

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

PubMed Central

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

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

2011-01-01

265

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia  

PubMed Central

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair. PMID:20613862

Slabicki, Mikolaj; Theis, Mirko; Krastev, Dragomir B.; Samsonov, Sergey; Mundwiller, Emeline; Junqueira, Magno; Paszkowski-Rogacz, Maciej; Teyra, Joan; Heninger, Anne-Kristin; Poser, Ina; Prieur, Fabienne; Truchetto, Jeremy; Confavreux, Christian; Marelli, Cecilia; Durr, Alexandra; Camdessanche, Jean Philippe; Brice, Alexis; Shevchenko, Andrej; Pisabarro, M. Teresa; Stevanin, Giovanni; Buchholz, Frank

2010-01-01

266

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity  

SciTech Connect

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

Iwanaga, Kentaro [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sueoka, Naoko [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sato, Akemi [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Hayashi, Shinichiro [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sueoka, Eisaburo [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan)]. E-mail: sueokae@post.saga-med.ac.jp

2005-08-05

267

Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells  

PubMed Central

Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 ?M) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

2014-01-01

268

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection.  

PubMed

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends. PMID:21947284

Gospodinov, Anastas; Vaissiere, Thomas; Krastev, Dragomir B; Legube, Gaëlle; Anachkova, Boyka; Herceg, Zdenko

2011-12-01

269

Crystal structure of the human Hsmar1-derived transposase domain in the DNA repair enzyme Metnase.  

PubMed

Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity. PMID:20521842

Goodwin, Kristie D; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M

2010-07-13

270

Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase  

SciTech Connect

Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M. (Indiana-Med)

2010-08-12

271

DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK  

Microsoft Academic Search

Two processes involving DNA double-strand breaks (DSBs) are the repair of DNA damage induced by ionizing radiation, and V(D)J recombination, the genomic rearrangement that creates antigen-receptor diversity in vertebrates. Recent evidence indicates that DNA-dependent protein kinase (DNA-PK), which is activated by DNA ends, is a central component of both the DNA DSB repair and V(D)J recombination machineries.

Stephen P. Jackson; Penny A. Jeggo

1995-01-01

272

A DNA-dependent protease involved in DNA-protein crosslink repair.  

PubMed

Toxic DNA-protein crosslinks (DPCs) arise by ionizing irradiation and UV light, are particularly caused by endogenously produced reactive compounds such as formaldehyde, and also occur during compromised topoisomerase action. Although nucleotide excision repair and homologous recombination contribute to cell survival upon DPCs, hardly anything is known about mechanisms that target the protein component of DPCs directly. Here, we identify the metalloprotease Wss1 as being crucial for cell survival upon exposure to formaldehyde and topoisomerase 1-dependent DNA damage. Yeast mutants lacking Wss1 accumulate DPCs and exhibit gross chromosomal rearrangements. Notably, in vitro assays indicate that substrates such as topoisomerase 1 are processed by the metalloprotease directly and in a DNA-dependent manner. Thus, our data suggest that Wss1 contributes to survival of DPC-harboring cells by acting on DPCs proteolytically. We propose that DPC proteolysis enables repair of these unique lesions via downstream canonical DNA repair pathways. PMID:24998930

Stingele, Julian; Schwarz, Michael S; Bloemeke, Nicolas; Wolf, Peter G; Jentsch, Stefan

2014-07-17

273

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals  

PubMed Central

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. PMID:24101514

Hura, Greg L.; Tsai, Chi-Lin; Claridge, Shelley A.; Mendillo, Marc L.; Smith, Jessica M.; Williams, Gareth J.; Mastroianni, Alexander J.; Alivisatos, A. Paul; Putnam, Christopher D.; Kolodner, Richard D.; Tainer, John A.

2013-01-01

274

Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer  

PubMed Central

DNA instability is an important contributor to cancer development. Previously, defects in the chromosome segregation and excessive DNA double strand breaks due to the replication or oxidative stresses were implicated in DNA instability in cancer. Here, we demonstrate that DNA instability can directly result from the oncogene-induced senescence signaling. Expression of the activated form of Her2 oncogene, NeuT, in immortalized breast epithelial cells led to downregulation of the major DNA repair factor histone H2AX and a number of other components of the HR and NHEJ double strand DNA breaks repair pathways. H2AX expression was regulated at the transcriptional level via a senescence pathway involving p21-mediated regulation of CDK and Rb1. The p21-dependent downregulation of H2AX was seen both in cell culture and the MMTV-neu mouse model of Her2-positive breast cancer. Importantly, downregulation of H2AX upon Her2/NeuT expression impaired repair of double strand DNA breaks. This impairment resulted in both increased DNA instability in the form of somatic copy number alterations, and in increased sensitivity to the chemotherapeutic drug doxorubicin. Overall, these findings indicate that the Her2/NeuT oncogene signaling directly potentiates DNA instability and increases sensitivity to DNA damaging treatments. PMID:25252808

Yaglom, Julia A.; McFarland, Christopher; Mirny, Leonid; Sherman, Michael Y.

2014-01-01

275

Human DNA repair process recorded in action | Science Codex http://sciencecodex.com/human_dna_repair_process_recorded_in_action (1 of 3) [1/29/2009 4:07:04 PM  

E-print Network

with one of 'nature's antibiotics' Search #12;Human DNA repair process recorded in action | Science CodexHuman DNA repair process recorded in action | Science Codex http://sciencecodex.com/human_dna_repair_process_recorded_in_action (1 of 3) [1/29/2009 4:07:04 PM] Science Codex Human DNA repair process recorded in action Posted On

Kowalczykowski, Stephen C.

276

Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair  

Microsoft Academic Search

Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disease that shares some common defects with ataxia-telangiectasia. The gene product mutated in NBS, named NBS1, is a component of the Mre11 complex that is involved in DNA strand-break repair. To elucidate the physiological roles of NBS1, we disrupted the N-terminal exons of the NBS1 gene in mice. NBS1(m\\/m) mice are

J. Kang; R T Bronson; Y Xu

2002-01-01

277

Targeting abnormal DNA repair in therapy-resistant breast cancers  

PubMed Central

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently we showed that, relative to non-tumorigenic breast epithelial MCF10A cells, estrogen receptor- and progesterone receptor-positive (ER/PR+) MCF7 breast cancer cells have reduced steady state levels of DNA ligase IV, a component of the major DNA-PK dependent non-homologous end-joining (NHEJ) pathway, whereas the steady state level of DNA ligase III?, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER/PR- cells have even higher steady state levels of DNA ligase III? and increased levels of poly (ADP-ribose) polymerase (PARP1), another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double strand breaks (DSB)s and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER/PR- cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER/PR- tumors had elevated levels of ALT NHEJ and reduced levels of DNA-PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. PMID:22112941

Tobin, Lisa A.; Robert, Carine; Nagaria, Pratik; Chumsri, Saranya; Twaddell, William; Ioffe, Olga B.; Greco, George E.; Brodie, Angela H.; Tomkinson, Alan E.; Rassool, Feyruz V.

2012-01-01

278

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide  

SciTech Connect

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.

Hagensee, M.E.; Bryan, S.K.; Moses, R.E.

1987-10-01

279

Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes.  

PubMed Central

Cockayne's syndrome (CS) is a disease characterized by developmental and growth defects, sunlight sensitivity, and a defect in transcription-coupled nucleotide excision repair. The two principle proteins involved in CS, CSA and CSB/ERCC6, have been hypothesized to bind RNA polymerase II (Pol II) and link transcription to DNA repair. We have tested CSA and CSB in assays designed to determine their role in transcription-coupled repair. Using a unique oligo(dC)-tailed DNA template, we provide biochemical evidence that CSB/ERCC6 interacts with Pol II molecules engaged in ternary complexes containing DNA and nascent RNA. CSB is a DNA-activated ATPase, and hydrolysis of the ATP beta-gamma phosphoanhydride bond is required for the formation of a stable Pol II-CSB-DNA-RNA complex. Unlike CSB, CSA does not directly bind Pol II. PMID:9372911

Tantin, D; Kansal, A; Carey, M

1997-01-01

280

Detection of the early stage of recombinational DNA repair by silicon nanowire transistors.  

PubMed

A silicon nanowire-based biosensor has been designed and applied for label-free and ultrasensitive detection of the early stage of recombinational DNA repair by RecA protein. Silicon nanowires transistors were fabricated by atomic force microscopy nanolithography and integrated into a microfluidic environment. The sensor operates by measuring the changes in the resistance of the nanowire as the biomolecular reactions proceed. We show that the nanoelectronic sensor can detect and differentiate several steps in the binding of RecA to a single-stranded DNA filament taking place on the nanowire-aqueous interface. We report relative changes in the resistance of 3.5% which are related to the interaction of 250 RecA·single-stranded DNA complexes. Spectroscopy data confirm the presence of the protein-DNA complexes on the functionalized silicon surfaces. PMID:22364265

Chiesa, Marco; Cardenas, Paula P; Otón, Francisco; Martinez, Javier; Mas-Torrent, Marta; Garcia, Fernando; Alonso, Juan C; Rovira, Concepció; Garcia, Ricardo

2012-03-14

281

Positive Regulation of DNA Double Strand Break Repair Activity during Differentiation of Long Life Span Cells: The Example of Adipogenesis  

PubMed Central

Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue. Our results show that DNA DSBs repair activity is up regulated during the early commitment into adipogenesis due to an up-regulation of DNA-PK expression and activity. In opposition to the general view that DNA DSBs repair is decreased during differentiation, our results demonstrate that an up-regulation of this process might be observed in post-mitotic long-lived cells. PMID:18846213

Meulle, Aline; Salles, Bernard; Daviaud, Daniele; Valet, Philippe; Muller, Catherine

2008-01-01

282

The Cyclin F-Ribonucleotide Reductase M2 axis controls genome integrity and DNA repair  

PubMed Central

Summary F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as a new interactor of the F-box protein Cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCFCyclin F to maintain balanced dNTP pools and genome stability. After DNA damage, Cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of Cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a non-degradable RRM2 mutant. In summary, we have identified a novel biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress. PMID:22632967

D'Angiolella, Vincenzo; Donato, Valerio; Forrester, Frances M.; Jeong, Yeon-Tae; Pellacani, Claudia; Kudo, Yasusei; Saraf, Anita; Florens, Laurence; Washburn, Michael P.; Pagano, Michele

2012-01-01

283

DNA Damage, Homology-Directed Repair, and DNA Methylation  

Microsoft Academic Search

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP

Concetta Cuozzo; Antonio Porcellini; Tiziana Angrisano; Annalisa Morano; Bongyong Lee; Alba Di Pardo; Samantha Messina; Rodolfo Iuliano; Alfredo Fusco; Maria R Santillo; Mark T Muller; Lorenzo Chiariotti; Max E Gottesman; Enrico V Avvedimento

2007-01-01

284

DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage  

SciTech Connect

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

2008-02-21

285

DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage  

SciTech Connect

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

2007-12-01

286

Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair  

PubMed Central

Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer. PMID:24553445

Peng, Guang; Lin, Curtis Chun-Jen; Mo, Wei; Dai, Hui; Park, Yun-Yong; Kim, Soo-Mi; Peng, Yang; Mo, Qianxing; Siwko, Stefan; Hu, Ruozhen; Lee, Ju-Seog; Hennessy, Bryan; Hanash, Samir; Mills, Gordon B.; Lin, Shiaw-Yih

2014-01-01

287

Regulation of DNA double-strand break repair pathway choice  

Microsoft Academic Search

DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including

Meena Shrivastav; Leyma P De Haro; Jac A Nickoloff

2008-01-01

288

Radiation Induced DNA-Damage\\/Repair and Associated Signaling Pathways  

Microsoft Academic Search

Radiation-induced DNA damage and related repair mechanisms are described in this chapter. The emerging connection with growth\\u000a factor induced signal transduction is described, with important implications for radiotherapy. The prospect of developing\\u000a targeting agents, which selectively deliver radioactivity to the tumor and at the same time radiosensitize the tumor cells\\u000a is discussed in some detail.

Bo Stenerlöw; Lina Ekerljung; Jörgen Carlsson; Johan Lennartsson

289

Decreased repair of gamma damaged DNA in progeria  

Microsoft Academic Search

A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced

A. J. Rainbow; M. Howes

1977-01-01

290

DNA-repair capacity and lipid peroxidation in chronic alcoholics.  

PubMed

The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p less than 0.01) and LPO in plasma was significantly higher (p less than 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = -0.078 x vit. C + 1.9; p less than 0.05) and between UDS and LPO values (UDS = -0.384 x LPO + 4.1; p less than 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol. PMID:2067553

Topinka, J; Binková, B; Srám, R J; Fojtíková, I

1991-07-01

291

Beyond DNA Repair: Additional Functions of PARP-1 in Cancer  

PubMed Central

Poly(ADP-ribose) polymerases (PARPs) are DNA-dependent nuclear enzymes that transfer negatively charged ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) to a variety of protein substrates, altering protein–protein and protein-DNA interactions. The most studied of these enzymes is poly(ADP-ribose) polymerase-1 (PARP-1), which is an excellent therapeutic target in cancer due to its pivotal role in the DNA damage response. Clinical studies have shown susceptibility to PARP inhibitors in DNA repair defective cancers with only mild adverse side effects. Interestingly, additional studies are emerging which demonstrate a role for this therapy in DNA repair proficient tumors through a variety of mechanisms. In this review, we will discuss additional functions of PARP-1 – including regulation of inflammatory mediators, cellular energetics and death pathways, gene transcription, sex hormone- and ERK-mediated signaling, and mitosis – and the role these PARP-1-mediated processes play in oncogenesis, cancer progression, and the development of therapeutic resistance. As PARP-1 can act in both a pro- and anti-tumor manner depending on the context, it is important to consider the global effects of this protein in determining when, and how, to best use PARP inhibitors in anticancer therapy. PMID:24350055

Weaver, Alice N.; Yang, Eddy S.

2013-01-01

292

[DNA polymerases beta and lambda, and their roles in the DNA replication and repair].  

PubMed

One of the key stages of life of a cell is genome duplication. The main enzymes which lead this process are DNA-dependent DNA polymerases. At the moment, 19 DNA polymerases with striking properties are listed in the eukaryotic cells. Mitochondrial DNA polymerase gamma from A family and most of the nuclear enzymes from B family are high fidelity DNA polymerases which are participate in genome DNA replication process as well as in DNA repair. Among the other 1 5 proteins, the D N A polymerases belonging to the X and Y families have a special place. They participate in a different repair processes such as base excision repair and non-homologous end joining. Moreover, some of them play a specific role in the replication of the damaged DNA templates. This process is referred as translesion synthesis or TLS. The DNA polymerases beta and lambda members of X family are enclosed in polyfunctional enzymes, and their properties and functions will be discussed in this review. PMID:21290819

Belousova, E A; Lavrik, O I

2010-01-01

293

Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes  

PubMed Central

DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on >95% inhibition of 8-oxodeoxyguosine (8-oxodG) and other unidentified oxidative DNA adducts induced by 4-hydroxy-17ß-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 ?M) than that for 8-oxodG (100 ?M). In the in vivo study, female CD-1 mice (n=6) were fed either a control diet or diet supplemented with ellagic acid (400 ppm) and dehydrated berries (5% w/w) with varying ellagic acid contents – blueberry (low), strawberry (medium) and red raspberry (high), for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%). However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001) and 48% (p < 0.01), respectively. Both diets also resulted in a 3–8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA), DNA excision repair protein (ERCC5) and DNA ligase III (DNL3). These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair. PMID:19325752

Aiyer, Harini S.; Vadhanam, Manicka V.; Stoyanova, Radka; Caprio, Gerard D.; Clapper, Margie L.; Gupta, Ramesh C.

2008-01-01

294

A hormone-DNA repair circuit governs the response to genotoxic insult  

PubMed Central

Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings demonstrate that androgen receptor (AR) activity is induced by DNA damage, and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNAPKcs was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance, and establish a new node for therapeutic intervention in advanced disease. PMID:24027197

Goodwin, Jonathan F.; Schiewer, Matthew J.; Dean, Jeffry L.; Schrecengost, Randy S.; de Leeuw, Renee; Han, Sumin; Ma, Teng; Den, Robert B.; Dicker, Adam P.; Feng, Felix Y.; Knudsen, Karen E.

2013-01-01

295

Ovarian cancer and DNA repair: DNA ligase IV as a potential key  

PubMed Central

Ovarian cancer (OC) is the sixth most common cancer and the seventh cause of death from cancer in women. The etiology and the ovarian carcinogenesis still need clarification although ovulation may be determinant due to its carcinogenic role in ovarian surface epithelium. The link between ovarian carcinogenesis and DNA repair is well established and it became clear that alterations in DNA damage response may affect the risk to develop OC. Polymorphisms are variations in the DNA sequence that exist in normal individuals of a population and are capable to change, among other mechanisms, the balance between DNA damage and cellular response. Consequently, genetic variability of the host has a great role in the development, progression and consequent prognosis of the oncologic patient as well as in treatment response. Standard treatment for OC patients is based on cytoreductive surgery, followed by chemotherapy with a platinum agent and a taxane. Although 80% of the patients respond to the first-line therapy, the development of resistance is common although the mechanisms underlying therapy failure remain mostly unknown. Because of their role in oncology, enzymes involved in the DNA repair pathways, like DNA Ligase IV (LIG4), became attractive study targets. It has been reported that variations in LIG4 activity can lead to a hyper-sensitivity to DNA damage, deregulation of repair and apoptosis mechanisms, affecting the susceptibility to cancer development and therapy response. To overcome resistance mechanisms, several investigations have been made and the strategy to target crucial molecular pathways, such as DNA repair, became one of the important areas in clinical oncology. This review aims to elucidate the link between DNA repair and OC, namely which concerns the role of LIG4 enzyme, and how genetic polymorphisms in LIG4 gene can modulate the activity of the enzyme and affect the ovarian carcinogenesis and treatment response. Moreover, we try to understand how LIG4 inhibition can be a potential contributor for the development of new cancer treatment strategies. PMID:23538968

Assis, Joana; Pereira, Deolinda; Medeiros, Rui

2013-01-01

296

AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis.  

PubMed

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair. PMID:24795278

Yuan, Dongke; Lai, Jianbin; Xu, Panglian; Zhang, Shengchun; Zhang, Juanjuan; Li, Chunxin; Wang, Yaqin; Du, Jinju; Liu, Yiyang; Yang, Chengwei

2014-09-01

297

Genetic Interactions of DNA Repair Pathways in the Pathogen Neisseria meningitidis  

Microsoft Academic Search

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational

Tonje Davidsen; Hanne K. Tuven; M. Bjoras; E. A. Rodland; T. Tonjum

2007-01-01

298

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM  

EPA Science Inventory

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanism Anuradha Mudipalli. Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

299

Defective DNA Strand Break Repair after DNA Damage in Prostate Cancer Cells: Implications for Genetic Instability and Prostate Cancer Progression  

Microsoft Academic Search

Together with cell cycle checkpoint control, DNA repair plays a pivotal role in protecting the genome from endogenous and exogenous DNA damage. Although increased genetic instability has been associated with prostate cancer progression, the relative role of DNA double-strand break repair in malignant versus normal prostate epithelial cells is not known. In this study, we determined the RNA and protein

Rong Fan; Tirukalikundram S. Kumaravel; Farid Jalali; Paula Marrano; Jeremy A. Squire; Robert G. Bristow

2004-01-01

300

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination  

PubMed Central

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR. PMID:24662483

Gao, Min; Wei, Wei; Li, Ming-Ming; Wu, Yong-Sheng; Ba, Zhaoqing; Jin, Kang-Xuan; Li, Miao-Miao; Liao, You-Qi; Adhikari, Samir; Chong, Zechen; Zhang, Ting; Guo, Cai-Xia; Tang, Tie-shan; Zhu, Bing-Tao; Xu, Xing-Zhi; Mailand, Niels; Yang, Yun-Gui; Qi, Yijun; Danielsen, Jannie M Rendtlew

2014-01-01

301

Resection is a major repair pathway of heavy ion-induced DNA lesions  

NASA Astrophysics Data System (ADS)

Space radiation include densely ionizing heavy ions, which can produce clustered DNA damage with high frequency in human cells. Repair of these complex lesions is generally assumed to be more difficult than for simple double-strand breaks. We show here that human cells use break resection with increasing frequency after exposure to heavy ions. Resection can lead to misrepair of the DNA lesion, via microhomology mediated end-joining. Resection can therefore be responsible for the increased effectiveness of heavy ions in the induction of mutations and genetic late effects.

Durante, Marco; Averbeck, Nicole; Taucher-Scholz, Gisela

302

Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones  

Microsoft Academic Search

Much effort has long been devoted to unraveling the coordinated cellular response to genotoxic insults. In view of the difficulty of obtaining human biological samples of homogeneous origin, I have established a set of stable human clones where one DNA repair gene has been stably silenced by means of RNA interference. I used pEBVsiRNA plasmids that greatly enhance long-term gene

D. S. F. Biard

2007-01-01

303

Replication protein A: Single-stranded DNA's first responder: Dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.  

PubMed

Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. PMID:25171654

Chen, Ran; Wold, Marc S

2014-12-01

304

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.  

PubMed

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity. PMID:25066234

Kakarougkas, Andreas; Ismail, Amani; Chambers, Anna L; Riballo, Enriqueta; Herbert, Alex D; Künzel, Julia; Löbrich, Markus; Jeggo, Penny A; Downs, Jessica A

2014-09-01

305

Reading, writing, and repair: the role of ubiquitin and the ubiquitin-like proteins in DNA damage signaling and repair  

PubMed Central

Genomic instability is both a hallmark of cancer and a major contributing factor to tumor development. Central to the maintenance of genome stability is the repair of DNA damage, and the most toxic form of DNA damage is the DNA double-strand break. As a consequence the eukaryotic cell harbors an impressive array of protein machinery to detect and repair DNA breaks through the initiation of a multi-branched, highly coordinated signaling cascade. This signaling cascade, known as the DNA damage response (DDR), functions to integrate DNA repair with a host of cellular processes including cell cycle checkpoint activation, transcriptional regulation, and programmed cell death. In eukaryotes, DNA is packaged in chromatin, which provides a mechanism to regulate DNA transactions including DNA repair through an equally impressive array of post-translational modifications to proteins within chromatin, and the DDR machinery itself. Histones, as the major protein component of chromatin, are subject to a host of post-translational modifications including phosphorylation, methylation, and acetylation. More recently, modification of both the histones and DDR machinery by ubiquitin and other ubiquitin-like proteins, such as the small ubiquitin-like modifiers, has been shown to play a central role in coordinating the DDR. In this review, we explore how ubiquitination and sumoylation contribute to the “writing” of key post-translational modifications within chromatin that are in turn “read” by the DDR machinery and chromatin-remodeling factors, which act together to facilitate the efficient detection and repair of DNA damage. PMID:23554604

Pinder, Jordan B.; Attwood, Kathleen M.; Dellaire, Graham

2013-01-01

306

Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations  

SciTech Connect

The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

2007-02-07

307

Opportunities for translation: targeting DNA repair pathways in pancreatic cancer.  

PubMed

Pancreatic ductal adenocarcinoma (PDAC) remains one of the poorest prognosis neoplasms. It is typified by high levels of genomic aberrations and copy-number variation, intra-tumoural heterogeneity and resistance to conventional chemotherapy. Improved therapeutic options, ideally targeted against cancer-specific biological mechanisms, are urgently needed. Although induction of DNA damage and/or modulation of DNA damage response pathways are associated with the activity of a number of conventional PDAC chemotherapies, the effectiveness of this approach in the treatment of PDAC has not been comprehensively reviewed. Here, we review chemotherapeutic agents that have shown anti-cancer activity in PDAC and whose mechanisms of action involve modulation of DNA repair pathways. In addition, we highlight novel potential targets within these pathways based on the emerging understanding of PDAC biology and their exploitation as targets in other cancers. PMID:24727386

Maginn, Elaina N; de Sousa, Camila H; Wasan, Harpreet S; Stronach, Euan A

2014-08-01

308

DNA Bending Propensity in the Presence of Base Mismatches: Implications for DNA Repair  

PubMed Central

DNA bending is believed to facilitate the initial recognition of the mismatched base for repair. The repair efficiencies are dependent on both the mismatch type and neighboring nucleotide sequence. We have studied bending of several DNA duplexes containing canonical matches: A:T, G:C, various mismatches: A:A, A:C, G:A, G:G, G:T, C:C, C:T, T:T, and a bis-abasic site: X:X. Free energy profiles were generated for DNA bending using umbrella sampling. The highest energetic cost associated with DNA bending is observed for canonical matches while bending free energies are lower in the presence of mismatches, with the lowest value for the abasic site. In all of the sequences, DNA duplexes bend towards the major groove with widening of the minor groove. For homoduplexes, DNA bending is observed to occur via smooth deformations, whereas for heteroduplexes, kinks are observed at the mismatch site during strong bending. In general, pyrimidine:pyrimidine mismatches are the most destabilizing, while purine:purine mismatches lead to intermediate destabilization and purine:pyrimidine mismatches are the least destabilizing. The ease of bending is partially correlated with the binding affinity of MutS to the mismatch pairs and subsequent repair efficiencies, indicating that intrinsic DNA bending propensities are a key factor of mismatch recognition. PMID:23621762

Sharma, Monika; Predeus, Alexander V.; Mukherjee, Shayantani; Feig, Michael

2013-01-01

309

MERIT40 facilitates BRCA1 localization and DNA damage repair  

PubMed Central

The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization. PMID:19261748

Feng, Lin; Huang, Jun; Chen, Junjie

2009-01-01

310

MERIT40 facilitates BRCA1 localization and DNA damage repair.  

PubMed

The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization. PMID:19261748

Feng, Lin; Huang, Jun; Chen, Junjie

2009-03-15

311

Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair  

SciTech Connect

The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens.

Oliveri, D.R.

1989-01-01

312

In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair.  

PubMed

In vivo single-molecule experiments offer new perspectives on the behaviour of DNA binding proteins, from the molecular level to the length scale of whole bacterial cells. With technological advances in instrumentation and data analysis, fluorescence microscopy can detect single molecules in live cells, opening the doors to directly follow individual proteins binding to DNA in real time. In this review, we describe key technical considerations for implementing in vivo single-molecule fluorescence microscopy. We discuss how single-molecule tracking and quantitative super-resolution microscopy can be adapted to extract DNA binding kinetics, spatial distributions, and copy numbers of proteins, as well as stoichiometries of protein complexes. We highlight experiments which have exploited these techniques to answer important questions in the field of bacterial gene regulation and transcription, as well as chromosome replication, organisation and repair. Together, these studies demonstrate how single-molecule imaging is transforming our understanding of DNA-binding proteins in cells. PMID:24859634

Stracy, Mathew; Uphoff, Stephan; Garza de Leon, Federico; Kapanidis, Achillefs N

2014-10-01

313

RPA Antagonizes Microhomology-Mediated Repair of DNA Double-Strand Breaks  

PubMed Central

Microhomology-mediated end joining (MMEJ) is a Ku and Ligase IV independent mechanism for repair of DNA double-strand breaks, which contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the ssDNA overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity. PMID:24608368

Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino; Greene, Eric C; Symington, Lorraine S

2014-01-01

314

Interactions between branched DNAs and peptide inhibitors of DNA repair  

PubMed Central

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (Kd = 14 nM) and ?4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility. PMID:18689438

Kepple, Kevin V.; Patel, Namita; Salamon, Peter; Segall, Anca M.

2008-01-01

315

DNA repair and its relation to recombination-deficient and other mutations in Bacillus subtilis.  

PubMed

DNA repair processes operating in Bacillus subtilis are similar to other transformable bacterial systems. Radiation-sensitive, recombination-deficient mutants are blocked in distinct steps leading to recombination. DNA polymerase I is essential for the repair of X-ray-induced damage to DNA but not for recombination. PMID:811208

Ganesan, A T

1975-01-01

316

Repair of 06-Alkylguanine during DNA Synthesis in Murine Bone Marrow Hematopoietic Precursors1  

Microsoft Academic Search

O'-AIkylguanine, a DNA adduci formed by nitrosoureas, becomes the site of a point mutation during DNA synthesis by preferentially base mispairing with thymine rather than correctly base pairing with cytosine. To repair this adduct, cells contain a limited amount of 06-alkylguanine- DNA alkyltransferase (alkyltransferase), a protein which removes the alkyl group in a stoichiometric reaction. To prevent mutations, repair must

Stanton L. Gerson; Joan E. Trey; Kathleen Miller; Evan Benjamin

317

Dynamic flexibility of DNA repair pathways in growth arrested Escherichia coli  

Microsoft Academic Search

The DNA of all organisms is constantly damaged by exogenous and endogenous agents. Base excision repair (BER) is important for the removal of several non-bulky lesions from the DNA, however not much is known about the contributions of other DNA repair pathways to the processing of non-bulky lesions. Here we utilized a luciferase reporter system to assess the contributions of

Cheryl L. Clauson; Tina T. Saxowsky; Paul W. Doetsch

2010-01-01

318

DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair  

Microsoft Academic Search

DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endo- nucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep

Nuray Akyuz; Gisa S. Boehden; Silke Susse; Andreas Rimek; Ute Preuss; Karl-Heinz Scheidtmann; Lisa Wiesmuller

2002-01-01

319

Genotoxic stress in plants: Shedding light on DNA damage, repair and DNA repair helicases  

Microsoft Academic Search

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone

Narendra Tuteja; Parvaiz Ahmad; Brahma B. Panda; Renu Tuteja

2009-01-01

320

Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG), in breast cancer  

Microsoft Academic Search

We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes N-alkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2–24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased

Sonia R Cerda; Patrick W Turk; Ann D Thor; Sigmund A Weitzman

1998-01-01

321

Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton  

SciTech Connect

The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlated with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. 54 refs., 4 figs., 2 tabs.

Malloy, K.D.; Holman, M.A. [Northeastern Univ., Boston, MA (United States)] [Northeastern Univ., Boston, MA (United States); Mitchell, D. [Univ. of Texas, Smithville, TX (United States)] [and others] [Univ. of Texas, Smithville, TX (United States); and others

1997-02-18

322

Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton.  

PubMed

The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlate with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. PMID:9037040

Malloy, K D; Holman, M A; Mitchell, D; Detrich, H W

1997-02-18

323

Mutations in the MutS? interaction interface of MLH1 can abolish DNA mismatch repair  

PubMed Central

MutL?, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutS? and assembles and controls further repair enzymes. We tested if the interaction of MutL? with DNA-bound MutS? is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutL? for MutS? is located on the edge of an extensive ?-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutL?–MutS? interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development. PMID:17135187

Plotz, Guido; Welsch, Christoph; Giron-Monzon, Luis; Friedhoff, Peter; Albrecht, Mario; Piiper, Albrecht; Biondi, Ricardo M.; Lengauer, Thomas; Zeuzem, Stefan; Raedle, Jochen

2006-01-01

324

DNA double-strand break repair: From mechanistic understanding to cancer treatment  

Microsoft Academic Search

Accurate repair of DNA double-strand breaks is essential to life. Indeed, defective DNA double-strand break repair can lead to toxicity and large scale sequence rearrangements that cause cancer and promote premature aging. Here, we highlight the two major repair systems for handling DNA double-strand breaks: homologous recombination and non-homologous end joining. To clarify recombination mechanisms, we present animations that illustrate

Thomas Helleday; Justin Lo; Dik C. van Gent; Bevin P. Engelward

2007-01-01

325

The potential of exploiting DNA-repair defects for optimizing lung cancer treatment  

Microsoft Academic Search

The tumor genome is commonly aberrant as a consequence of mutagenic insult and incomplete DNA repair. DNA repair as a therapeutic target has recently received considerable attention owing to the promise of drugs that target tumor-specific DNA-repair enzymes and potentiate conventional cytotoxic therapy through mechanism-based approaches, such as synthetic lethality. Treatment for non-small-cell lung cancer (NSCLC) consists mainly of platinum-based

Sophie Postel-Vinay; Elsa Vanhecke; Ken A. Olaussen; Christopher J. Lord; Alan Ashworth; Jean-Charles Soria

2012-01-01

326

DNA damage action spectroscopy and DNA repair in intact organisms: Alfalfa seedlings  

Microsoft Academic Search

Understanding the effects of UV, and increased levels of UV, on DNA in living organisms requires knowledge of both the frequency of damages induced by the quantities and quality (wavelength composition) of the damaging radiation, and of the capacity of the organisms to carry out efficient and accurate repair. The major levels of uncertainty in understanding the responses of intact

B. M. Sutherland; F. E. Quaite; J. C. Sutherland

1993-01-01

327

Calculation of complex DNA damage induced by ions  

NASA Astrophysics Data System (ADS)

This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

2011-11-01

328

Calculation of complex DNA damage induced by ions  

SciTech Connect

This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

Surdutovich, Eugene [Department of Physics, Oakland University, Rochester, Michigan 48309 (United States); Frankfurt Institute for Advanced Studies, Ruth-Moufang-Strasse 1, D-60438 Frankfurt am Main (Germany); Gallagher, David C. [Department of Physics, Oakland University, Rochester, Michigan 48309 (United States); Solov'yov, Andrey V. [Frankfurt Institute for Advanced Studies, Ruth-Moufang-Strasse 1, D-60438 Frankfurt am Main (Germany)

2011-11-15

329

Global genome repair is required to activate KIN17, a UVC-responsive gene involved in DNA replication  

Microsoft Academic Search

UV light provokes DNA lesions that interfere with replication and transcription. These lesions may compromise cell viability and usually are removed by nucleotide excision repair (NER). In humans, inactivation of NER is associated with three rare autosomal recessive inherited disorders: xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. The NER earliest step is lesion recognition by a complex formed by XPC

Christel Masson; Farid Menaa; Ghislaine Pinon-Lataillade; Yveline Frobert; Sylvie Chevillard; J. Pablo Radicella; Alain Sarasin; Jaime F. Angulo

2003-01-01

330

DNA Repair Scaffolds Dampen Checkpoint Signaling by Counteracting the Rad9 Adaptor  

PubMed Central

In response to genotoxic stress, a transient arrest in cell cycle progression enforced by the DNA damage checkpoint (DDC) signaling pathway positively contributes to genome maintenance1. Because hyperactivated DDC can lead to a persistent and detrimental cell cycle arrest2,3, cells must tightly regulate the activity of DDC kinases. Despite their importance, the mechanisms for monitoring and modulating DDC signaling are not fully understood. Here we show that DNA repair scaffolding proteins Slx4 and Rtt107 prevent lesions generated during DNA replication from aberrantly hyperactivating DDC signaling in Saccharomyces cerevisiae. Upon replication stress, cells lacking Slx4 or Rtt107 exhibit hyperactivation of the downstream DDC kinase Rad53 while activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phospho-H2A, two positive regulators of Rad9-dependent Rad53 activation. Reduction of DDC signaling by hypomorphic mutations in RAD53 and H2A rescue the hyper-sensitivity of slx4? or rtt107? cells to replication stress. We propose that the Slx4-Rtt107 complex modulates Rad53 activation via a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings reveal that DDC signaling is monitored and modulated through the direct action of DNA repair factors. PMID:23160493

Ohouo, Patrice Y.; Bastos de Oliveira, Francisco M.; Liu, Yi; Ma, Chu Jian; Smolka, Marcus B.

2012-01-01

331

Interaction of novel bis(platinum) complexes with DNA.  

PubMed Central

Bis(platinum) complexes [[cis-PtCl2(NH3)]2H2N(CH2)nNH2] are a novel series of potential anticancer agents in which two cis-diamine(platinum) groups are linked by an alkyldiamine of variable length. These complexes are potentially tetrafunctional, a unique feature in comparison with known anticancer agents. Studies of DNA interactions of bis(platinum) complexes in comparison with cisplatin demonstrate significant differences. Investigations of interstrand crosslink formation in which crosslinking of a short DNA fragment is detected by gel electrophoresis under denaturing conditions demonstrate that interstrand crosslinks are 250 fold more frequent among bis(platinum) adducts than among cisplatin-derived adducts under the conditions examined. These investigations indicate that bis(platinum) adducts contain a high frequency of structurally novel interstrand crosslinks formed through binding of the two platinum centers to opposite DNA strands. Unlike cisplatin, bis(platinum) complex binding does not unwind supercoiled DNA. Studies with the E. coli UvrABC nuclease complex demonstrate that both linear and supercoiled DNA containing bis(platinum) adducts are subject to incision by the repair enzyme complex. Initial studies using UvrABC nuclease as a probe to define the base and sequence specificity for bis(platinum) complex binding suggest that the specificity of the bis(platinum)s is similar, but not identical, to that of cisplatin. Images PMID:2690006

Roberts, J D; Van Houten, B; Qu, Y; Farrell, N P

1989-01-01

332

Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions  

PubMed Central

The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps. PMID:23469115

Tobias, Frank; Löb, Daniel; Lengert, Nicor; Durante, Marco; Drossel, Barbara; Taucher-Scholz, Gisela; Jakob, Burkhard

2013-01-01

333

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair.  

PubMed

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of ?H2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas ?H2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair. PMID:24351651

Min, Sunwoo; Jo, Sujin; Lee, Ho-Soo; Chae, Sunyoung; Lee, Jong-Soo; Ji, Jae-Hoon; Cho, Hyeseong

2014-02-15

334

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency  

PubMed Central

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage–fusion–bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling “chromoanasynthesis,” a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. PMID:25030888

Meier, Bettina; Cooke, Susanna L.; Weiss, Joerg; Bailly, Aymeric P.; Alexandrov, Ludmil B.; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R.; Campbell, Peter J.

2014-01-01

335

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency.  

PubMed

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling "chromoanasynthesis," a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. PMID:25030888

Meier, Bettina; Cooke, Susanna L; Weiss, Joerg; Bailly, Aymeric P; Alexandrov, Ludmil B; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R; Gartner, Anton; Campbell, Peter J

2014-10-01

336

Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance.  

PubMed

The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases. PMID:12944481

Ueno, Masaru; Nakazaki, Tomofumi; Akamatsu, Yufuko; Watanabe, Kikuo; Tomita, Kazunori; Lindsay, Howard D; Shinagawa, Hideo; Iwasaki, Hiroshi

2003-09-01

337

RAD50, an SMC family member with multiple roles in DNA break repair: how does ATP affect function?  

Microsoft Academic Search

The protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process\\u000a DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member\\u000a of the structural maintenance of chromosome (SMC) protein family. Like SMC proteins, Rad50 function depends on ATP binding\\u000a and hydrolysis.

Eri Kinoshita; Eddy van der Linden; Humberto Sanchez; Claire Wyman

2009-01-01

338

DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood  

PubMed Central

DNA damage and repair are hallmarks of cellular responses to ionizing radiation. We hypothesized that monitoring the expression of DNA repair-associated genes would enhance the detection of individuals exposed to radiation versus other forms of physiological stress. We employed the human blood ex vivo radiation model to investigate the expression responses of DNA repair genes in repeated blood samples from healthy, non-smoking men and women exposed to 2 Gy of X-rays in the context of inflammation stress mimicked by the bacterial endotoxin lipopolysaccharide (LPS). Radiation exposure significantly modulated the transcript expression of 12 genes of 40 tested (2.2E-06DNA repair gene expression may be helpful to identify biodosimeters of exposure to radiation, especially within high-complexity exposure scenarios. PMID:23144912

Marchetti, Francesco; Mannion, Brandon; Bhatnagar, Sandhya; Kwoh, Ely; Tan, Yuande; Wang, Shan X.; Blakely, William F.; Coleman, Matthew; Peterson, Leif; Wyrobek, Andrew J.

2012-01-01

339

A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M.  

PubMed

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA. PMID:16116422

Meetei, Amom Ruhikanta; Medhurst, Annette L; Ling, Chen; Xue, Yutong; Singh, Thiyam Ramsing; Bier, Patrick; Steltenpool, Jurgen; Stone, Stacie; Dokal, Inderjeet; Mathew, Christopher G; Hoatlin, Maureen; Joenje, Hans; de Winter, Johan P; Wang, Weidong

2005-09-01

340

Mouse DNA Polymerase Kappa Has A Functional Role In the Repair of DNA Strand Breaks  

PubMed Central

The Y-family of DNA polymerases support of translesion DNA synthesis (TLS) associated with stalled DNA replication by DNA damage. Recently, a number of studies suggest that some specialized TLS polymerases also support other aspects of DNA metabolism beyond TLS in vivo. Here we show that mouse polymerase kappa (Pol?) could accumulate at laser-induced sites of damage in vivo resembling polymerases eta and iota. The recruitment was mediated through Pol? C-terminus which contains the PCNA-interacting peptide, ubiquitin zinc finger motif 2 and nuclear localization signal. Interestingly, this recruitment was significantly reduced in MSH2-deficient LoVo cells and Rad18-depleted cells. We further observed that Pol?-deficient mouse embryo fibroblasts were abnormally sensitive to H2O2 treatment and displayed defects in both single-strand break repair and double-strand break repair. We speculate that Pol? may have an important role in strand break repair following oxidative stress in vivo. PMID:23522793

Zhang, Xiuli; Lv, Lingna; Chen, Qian; Yuan, Fenghua; Zhang, Ting; Yang, Yeran; Zhang, Hui; Wang, Yun; Jia, Yan; Qian, Liangyue; Chen, Benjamin; Zhang, Yanbin; Friedberg, Errol C.; Tang, Tie-Shan; Guo, Caixia

2013-01-01

341

In TFIIH, XPD Helicase Is Exclusively Devoted to DNA Repair  

PubMed Central

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription. PMID:25268380

Kuper, Jochen; Braun, Cathy; Elias, Agnes; Michels, Gudrun; Sauer, Florian; Schmitt, Dominik R.; Poterszman, Arnaud; Egly, Jean-Marc; Kisker, Caroline

2014-01-01

342

DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-methylpurine DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose.  

National Technical Information Service (NTIS)

To ensure that a cell's genome is not compromised, elaborate mechanisms of DNA repair are essential for both nuclear and mitochondrial DNA. The DNA Base Excision Repair (BER) pathway is responsible for the repair of alkylation and oxidative DNA damage. Th...

M. Limp-Foster, M. R. Kelly

2001-01-01

343

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

PubMed Central

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

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

2013-01-01

344

Genetic and molecular analysis of dna damage repair and tolerance pathways.  

National Technical Information Service (NTIS)

Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts ...

Sutherland

2001-01-01

345

DNA-damage repair; the good, the bad, and the ugly  

PubMed Central

Organisms have developed several DNA-repair pathways as well as DNA-damage checkpoints to cope with the frequent challenge of endogenous and exogenous DNA insults. In the absence or impairment of such repair or checkpoint mechanisms, the genomic integrity of the organism is often compromised. This review will focus on the functional consequences of impaired DNA-repair pathways. Although each pathway is addressed individually, it is essential to note that cross talk exists between repair pathways, and that there are instances in which a DNA-repair protein is involved in more than one pathway. It is also important to integrate DNA-repair process with DNA-damage checkpoints and cell survival, to gain a better understanding of the consequences of compromised DNA repair at both cellular and organismic levels. Functional consequences associated with impaired DNA repair include embryonic lethality, shortened life span, rapid ageing, impaired growth, and a variety of syndromes, including a pronounced manifestation of cancer. PMID:18285820

Hakem, Razqallah

2008-01-01

346

Mechanism of Cluster DNA Damage Repair in Response to High-Atomic Number and Energy Particles Radiation  

PubMed Central

Low-linear energy transfer (LET) radiation (i.e., ?- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles are slowly repaired or are irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure. PMID:21126526

Asaithamby, Aroumougame; Chen, David J.

2012-01-01

347

Repair, Revision, and Complexity in Syntactic Analysis: An Electrophysiological Differentiation  

Microsoft Academic Search

One of the core aspects of human sentence processing is the ability to detect errors and to recover from erroneous analysis through revision of ambiguous sentences and repair of ungrammatical sentences. In the present study, we used event-related potentials (ERPs) to help identify the nature of these processes by directly comparing ERPs to complex ambiguous sentence structures with and without

Edith Kaan; Tamara Y. Swaab

2003-01-01

348

Repair of uv damaged DNA: Genes and proteins of yeast and human  

SciTech Connect

Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

Prakash, L.

1992-04-01

349

Characterization of the alternative excision repair pathway of UV damaged DNA in Schizosaccharomyces pombe  

Microsoft Academic Search

Schizosaccharomyces pombe cells deficient in nucleo- tide excision repair (NER) are still able to remove photoproducts from cellular DNA, showing that there is a second pathway for repair of UV damage in this organism. We have characterized this repair pathway by cloning and disruption of the genomic gene encoding UV damage endonuclease (UVDE). Although uvde gene disruptant cells are only

Rie Yonemasu; Shirley J. McCready; Johanne M. Murray; Fikret Osman; Masashi Takao; Kazuo Yamamoto; Alan R. Lehmann; Akira Yasui

1997-01-01

350

Topical liposomal DNA-repair enzymes in polymorphic light eruption.  

PubMed

Polymorphic light eruption (PLE) is a very frequent photodermatosis in Europe whose pathogenesis may involve resistance to UV-induced immune suppression and simultaneous immune reactions against skin photoneoantigens. We performed a randomized, double-blind, placebo-controlled intra-individual half-body trial to investigate the protective effect of an after-sun (AS) lotion containing DNA-repair enzymes (photolyase from Anacystis nidulans and Micrococcus luteus extract with endonuclease activity). Fourteen PLE patients were exposed to suberythemal doses of solar-simulated UV radiation on 4 consecutive days at 4 symmetrically located PLE-prone test fields per patient. The test fields were treated with (i) active AS lotion or (ii) a placebo lotion immediately after each UV exposure, or (iii) an SPF30 sunscreen before UV exposure or left untreated. All test fields were exposed to photoactivating blue light 1 h after each UV exposure. As shown by a newly established specific PLE test score (AA + SI + 0.4P [range, 0-12], where AA is affected area score [range, 0-4], SI is skin infiltration score [range, 0-4], and P is pruritus score on a visual analogue scale [range, 0-10]), PLE symptoms were significantly fewer on test sites treated with active AS lotion than on untreated (P = 0.00049) or placebo-treated test sites (P = 0.024). At 144 h after first UV exposure (the time point of maximal PLE symptoms), the mean test scores for untreated, active AS lotion-treated, and placebo-treated test fields were 4.39, 1.73 (61% reduction; 95% confidence interval (CI), 36% to 85%), and 3.20 (27% reduction; 95% CI, 3% to 51%), respectively. Pretreatment with SPF30 sunscreen completely prevented PLE symptoms in all patients. The present results indicate that DNA damage may trigger PLE and that the application of topical liposomes containing DNA repair enzymes to increase DNA repair may effectively prevent PLE. PMID:21437317

Hofer, Angelika; Legat, Franz J; Gruber-Wackernagel, Alexandra; Quehenberger, Franz; Wolf, Peter

2011-07-01

351

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells  

Microsoft Academic Search

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridi- zation analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed

Seisuke Kimura; Yasue Tahira; Toyotaka Ishibashi; Yoko Mori; Toshio Mori; Junji Hashimoto; Kengo Sakaguchi

2004-01-01

352

Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes  

NASA Astrophysics Data System (ADS)

DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

Negureanu, Lacramioara; Salsbury, Freddie

2012-02-01

353

Oxidative DNA Damage and Repair in a Cell Lineage Model of Human Proliferative Breast Disease (PBD)  

Microsoft Academic Search

Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines—MCF-10A, MCF-10AT, and MCF-10ATG3B—with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and pres- ence of the DNA-repair inhibitors

Susan L. Starcevic; Nicole M. Diotte; Kim L. Zukowski; Mark J. Cameron; Raymond F. Novak

2003-01-01

354

Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors  

PubMed Central

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ?170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with ? photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. PMID:23382828

Kumala, Slawomir; Fujarewicz, Krzysztof; Jayaraju, Dheekollu; Rzeszowska-Wolny, Joanna; Hancock, Ronald

2013-01-01

355

The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress.  

PubMed

The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair. PMID:24107631

Kim, Ekaterina R; Selyutina, Anastasia A; Buldakov, Ilya A; Evdokimova, Valentina; Ovchinnikov, Lev P; Sorokin, Alexey V

2013-12-15

356

The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress  

PubMed Central

The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair. PMID:24107631

Kim, Ekaterina R; Selyutina, Anastasia A; Buldakov, Ilya A; Evdokimova, Valentina; Ovchinnikov, Lev P; Sorokin, Alexey V

2013-01-01

357

Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe  

Microsoft Academic Search

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similar- ity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation pro- ducts, the premutagenic deamination product hypo- xanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specifi- city.

Ingrun Alseth; Fikret Osman; Hanne Korvald; Irina Tsaneva; Matthew C. Whitby; Erling Seeberg

2005-01-01

358

Exonuclease 1 Preferentially Repairs Mismatches Generated by DNA Polymerase ?  

PubMed Central

The Saccharomyces cerevisiaeEXO1 gene encodes a 5? exonuclease that participates in mismatch repair (MMR) of DNA replication errors. Deleting EXO1 was previously shown to increase mutation rates to a greater extent when combined with a mutator variant (pol3-L612M) of the lagging strand replicase, DNA polymerase ? (Pol ?), than when combined with a mutator variant (pol2-M644G) of the leading strand replicase, DNA polymerase ? (Pol ?). Here we confirm that result, and extend the approach to examine the effect of deleting EXO1 in a mutator variant (pol1-L868M) of Pol ?, the proofreading-deficient and least accurate of the three nuclear replicases that is responsible for initiating Okazaki fragment synthesis. We find that deleting EXO1 increases the mutation rate in the Pol ? mutator strain to a significantly greater extent than in the Pol ? or Pol ? mutator strains, thereby preferentially reducing the efficiency of MMR of replication errors generated by Pol ?. Because these mismatches are closer to the 5? ends of Okazaki fragments than are mismatches made by Pol ? or Pol ?, the results not only support the previous suggestion that Exo1 preferentially excises lagging strand replication errors during mismatch repair, they further imply that the 5? ends serve as entry points for 5? excision of replication errors made by Pol ?, and possibly as strand discrimination signals for MMR. Nonetheless, mutation rates in the Pol ? mutator strain are 5- to 25-fold lower in an exo1? strain as compared to an msh2? strain completely lacking MMR, indicating that in the absence of Exo1, most replication errors made by Pol ? can still be removed in an Msh2-dependent manner by other nucleases and/or by strand displacement. PMID:23245696

Liberti, Sascha E.; Larrea, Andres A.; Kunkel, Thomas A.

2012-01-01

359

Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations  

NASA Astrophysics Data System (ADS)

We irradiated plateau phase normal human fibroblasts with 2 Gy X-rays 70 keV um carbon 290MeV n and 200 keV um iron ions 500 MeV n and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break DSB repair GammaH2AX foci a marker for DSBs were detected immediately after irradiation and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci In contrast phosphorylation of an NHEJ protein DNA-PKcs threonine 2609 was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX Furthermore in the case of ATM protein phosphorylation serine 1981 iron irradiation alone caused a significant initial delay but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions These results suggest that 1 high LET irradiation induces complex and or severe DNA DSB damage which affects the function of DSB repair proteins 2 Both ATM and DNA-PKcs may recognize the complexity of DSBs but ATM may be more sensitive to detecting the complexity of DSB damage 3 gamma-H2AX may

Okayasu, R.; Okabe, A.; Takakura, K.

360

RAD18-mediated ubiquitination of PCNA activates the Fanconi anemia DNA repair network.  

PubMed

The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)-FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage-activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation. PMID:20937699

Geng, Liyi; Huntoon, Catherine J; Karnitz, Larry M

2010-10-18

361

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality  

NASA Technical Reports Server (NTRS)

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (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 radiation induced higher 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 chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further investigate the sensitivity differences for low and low high doses, we performed chronic low dose-rate irradiation, and have begun studies with ATM and Nibrin inhibitors and siRNA knockout of these proteins. Results support the conclusion that for the endpoint of simple chromosomal aberrations (translocation or dicentrics), the increased radiation sensitivity of AT cells found at high doses (>1 Gy) does not carry over to low doses or doserates, while NBS cells show increased sensitivity for both high and low dose exposures.

George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

362

Identification of repairable radiation induced base modification in DNA  

SciTech Connect

Ionizing radiation causes formation of modified bases in DNA. A major derivative is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG). TG is also formed in DNA by uv radiation and in vitro by oxidants such as KMnO/sub 4/ and OsO/sub 4/. It has been suggested that TG is a prototype modification formed by free radical reactions. TG and other ring-saturated thymines are removed from DNA by enzymatic repair. Research in this area has been hampered by the absence of an assay which can identify all of the ring-saturated thymines in modified DNA. Our laboratory has developed high pressure liquid (HPLC) and reverse phase Sephadex LH-20 (LH-20) chromatographic techniques for this purpose. The DNA is enzymatically hydrolysed to 2'-deoxyribonucleosides leaving modified and unmodified bases intact. These hydrolysates are chromatographed with uv absorbing and (/sup 14/C)-labelled marker compounds. The cis-isomer of TG is further identified by LH-20 chromatography and several other acetyl-derivatives have been synthesized. DNA oxidized by OsO/sub 4/ has been shown to contain primarily TG and has been used as a substrate for in vitro enzyme assays. (/sup 3/H-methyl)-thymidine labelled DNA from control cells and cells exposed to gamma-irradiation has been analyzed. In addition to TG, many other thymine derivatives were present. The major thymine derivative was identified as 5-hydroxymethyl-2'-deoxyuridine (HMdU). The presence of this unusual derivative, a minor in vitro radiation product, led us to investigate the possibility that its formation was the result of (/sup 3/H)-transmutation. The DNA of irradiated and control cells labelled with (/sup 3/H-6)-thymidine contained TG and other derivatives seen in (/sup 3/H-methyl)-thymidine labelled DNA but no HMdU. Thus the transmutation of (/sup 3/H) to (/sup 3/He) in the methyl group probably led to the formation of the CH/sub 2//sup +/ carbonium ion which yielded HMdU.

Goldstein, M.S.

1983-01-01

363

A genetic screen identifies FAN1, a Fanconi anemia associated nuclease necessary for DNA interstrand crosslink repair  

PubMed Central

Summary The Fanconi Anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 and EXDL2. FAN1 co-localizes at sites of DNA damage with the ID complex in a manner dependent on FAN1’s ubiquitin binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5?-3? exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain. PMID:20603073

Smogorzewska, Agata; Desetty, Rohini; Saito, Takamune T.; Schlabach, Michael; Lach, Francis P.; Sowa, Mathew E.; Clark, Alan B.; Kunkel, Thomas A.; Harper, J. Wade; Colaiacovo, Monica P.; Elledge, Stephen J.

2010-01-01

364

Rapid and progressive pulmonary fibrosis in 2 families with DNA repair deficiencies of undetermined etiology.  

PubMed

Known genetic causes of pediatric interstitial lung disease include disorders of surfactant metabolism, telomerase, and DNA repair. We report 4 children from 2 families with rapidly progressive and fatal pulmonary fibrosis. A novel DNA repair defect unrelated to the ataxia-telangiectasia mutated gene was found in 1 child from each family. PMID:22240110

Vece, Timothy J; Schecter, Marc G; Gatti, Richard A; Tunuguntla, Rashmi; Garcia, Christine Kim; Langston, Claire; Dishop, Megan K; Moore, Robert H; Fan, Leland L

2012-04-01

365

Cell Cycle Checkpoints, DNA Damage\\/Repair, and Lung Cancer Risk  

Microsoft Academic Search

Given that defects in cell cycle control and DNA repair capacity may contribute to tumorigenesis, we hypothesized that patients with lung cancer would be more likely than healthy controls to exhibit deficiencies in cell cycle check- points and\\/or DNA repair capacity as gauged by cellular response to in vitro carcinogen exposure. In an ongoing case- control study of 155 patients

Xifeng Wu; Jack A. Roth; Hua Zhao; Sherry Luo; Yun-Ling Zheng; Silvia Chiang; Margaret R. Spitz

2005-01-01

366

The role of DNA damage repair in aging of adult stem cells  

Microsoft Academic Search

DNA repair maintains genomic stability and the loss of DNA repair capacity results in genetic instability that may lead to a decline of cellular function. Adult stem cells are extremely important in the long-term maintenance of tissues throughout life. They regen- erate and renew tissues in response to damage and replace senescent terminally differentiated cells that no longer function. Oxidative

Jonathan Kenyon; Stanton L. Gerson

2007-01-01

367

Role of SUMO\\/Ubc9 in DNA Damage Repair and Tumorigenesis  

Microsoft Academic Search

DNA damage repair is an important cell function for genome integrity and its deregulation can lead to genomic instability and development of malignancies. Sumoylation is an increasingly important ubiquitin-like modification of proteins affecting protein stability, enzymatic activity, nucleocytoplasmic trafficking, and protein-protein interactions. In particular, several important DNA repair enzymes are subject to sumoylation, which appears to play a role in

Stergios J. Moschos; Yin-Yuan Mo

2006-01-01

368

Genetic and Molecular Analysis of DNA Damage Repair and Tolerance Pathways  

Microsoft Academic Search

Alterations of the structural genes coding for DNA repair enzymes can have severe effects on the organisms, whether a simple bacterium, or a higher plant or person. Modern biochemical and molecular biological approaches allow determination of the molecular origin of radiation sensitivity, including those resulting from deficiencies in DNA repair.

B. M. SUTHERLAND

2001-01-01

369

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

Microsoft Academic Search

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

Allon Weiner; Nathan Zauberman; Abraham Minsky

2009-01-01

370

DNA repair gets physical: mapping a XPA binding site on ERCC1  

PubMed Central

Two recent reports provide new physical information on how the XPA protein recruits the ERCC1-XPF heterodimer to the site of damage during the process of mammalian nucleotide excision repair (NER). Using chemical shift perturbation NMR experiments, the contact sites between a central fragment of ERCC1 and a XPA fragment have been mapped. While both studies agree with regard to the XPA binding site, they differ on whether the ERCC1-XPA complex can simultaneously bind DNA. These studies have important implications for both the molecular process and the design of potential inhibitors of NER. PMID:18343204

Croteau, Deborah L.; Peng, Ye; Van Houten, Bennett

2008-01-01

371

Polymorphisms in XPC, XPD, XRCC1, and XRCC3 DNA repair genes and lung cancer risk in a population of Northern Spain  

Microsoft Academic Search

BACKGROUND: Polymorphisms in DNA repair genes have been associated to repair DNA lesions, and might contribute to the individual susceptibility to develop different types of cancer. Nucleotide excision repair (NER), base excision repair (BER), and double-strand break repair (DSBR) are the main DNA repair pathways. We investigated the relationship between polymorphisms in two NER genes, XPC (poly (AT) insertion\\/deletion: PAT-\\/+)

M Felicitas López-Cima; Patricia González-Arriaga; Laura García-Castro; Teresa Pascual; Manuel G Marrón; Xose S Puente; Adonina Tardón

2007-01-01

372

DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant  

Microsoft Academic Search

DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1)

1985-01-01

373

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes  

PubMed Central

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5? untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis. PMID:19451221

Powley, Ian R.; Kondrashov, Alexander; Young, Lucy A.; Dobbyn, Helen C.; Hill, Kirsti; Cannell, Ian G.; Stoneley, Mark; Kong, Yi-Wen; Cotes, Julia A.; Smith, Graeme C.M.; Wek, Ron; Hayes, Christopher; Gant, Timothy W.; Spriggs, Keith A.; Bushell, Martin; Willis, Anne E.

2009-01-01

374

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes.  

PubMed

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis. PMID:19451221

Powley, Ian R; Kondrashov, Alexander; Young, Lucy A; Dobbyn, Helen C; Hill, Kirsti; Cannell, Ian G; Stoneley, Mark; Kong, Yi-Wen; Cotes, Julia A; Smith, Graeme C M; Wek, Ron; Hayes, Christopher; Gant, Timothy W; Spriggs, Keith A; Bushell, Martin; Willis, Anne E

2009-05-15

375

Electron Tunneling Pathway and Role of Adenine in Repair of Damaged DNA by Photolyase  

NASA Astrophysics Data System (ADS)

Through electron tunneling, photolyase, a photoenzyme, restores damaged DNA into normal bases. Here, we report our systematic characterization and analyses of three electron transfer processes in thymine dimer restoration by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repairing and for the final electron return to restore the active cofactor and complete the repair photocycle. Significantly, we found that the adenine moiety of the unusual bent cofactor is essential to mediating all electron transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure these elementary dynamics in synergy in this complex photorepair machinery to achieve the maximum repair efficiency close to unity. Z. Liu, C. Tan, X. Guo, Y.-T. Kao, J. Li, L. Wang, A. Sancar, and D. Zhong, Proc. Natl. Acad. Sci. USA 108, 14831 (2011) J. Li, Z. Liu, C. Tan, X. Guo, L. Wang, A. Sancar, and D. Zhong, Nature 466, 887 (2010)

Liu, Zheyun; Tan, Chuang; Guo, Xunmin; Kao, Ya-Ting; Li, Jiang; Wang, Lijuan; Zhong, Dongping

2012-06-01

376

Laser microbeam - kinetic studies combined with molecule - structures reveal mechanisms of DNA repair  

NASA Astrophysics Data System (ADS)

Kinetic studies on double strand DNA damages induced by a laser microbeam have allowed a precise definition of the temporal order of recruitment of repair molecules. The order is KU70 / KU80 - XRCC4 --NBS1 -- RAD51. These kinetic studies are now complemented by studies on molecular structures of the repair proteins, using the program YASARA which does not only give molecular structures but also physicochemical details on forces involved in binding processes. It turns out that the earliest of these repair proteins, the KU70 / KU80 heterodimer, has a hole with high DNA affinity. The next molecule, XRCC4, has a body with two arms, the latter with extremely high DNA affinity at their ends and a binding site for Ligase 4. Together with the laser microbeam results this provides a detailed view on the early steps of DNA double strand break repair. The sequence of DNA repair events is presented as a movie.

Altenberg, B.; Greulich, K. O.

2011-10-01

377

Reduced local mutation density in regulatory DNA of cancer genomes is linked to DNA repair  

PubMed Central

Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes1, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in multiple nucleotide excision repair components. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation. PMID:24336318

Polak, Paz; Lawrence, Michael S.; Haugen, Eric; Stoletzki, Nina; Stojanov, Petar; Thurman, Robert E; Garraway, Levi A.; Mirkin, Sergei; Getz, Gad; Stamatoyannopoulos, John A.; Sunyaev, Shamil R.

2014-01-01

378

A conceptual modeling framework for the study of DNA mismatch repair pathway to improve therapeutic gain in cancer treatment  

Microsoft Academic Search

The DNA mismatch repair pathway is an important repair mechanism in the cell that ensures genomic stability. Mismatch repair deficiencies are shown to be associated with certain hereditary forms of cancer as well as many sporadic cancers. The loss of mismatch repair also leads to resistance to chemotherapeutic agents and other types of DNA stress including ionizing radiation. An alternative

Evren Gurkan; Jane E. Schupp; Timothy J. Kinsella; Kenneth A. Loparo

2007-01-01

379

Nascent DNA processing by RecJ favors lesion repair over translesion synthesis at arrested replication  

E-print Network

Nascent DNA processing by RecJ favors lesion repair over translesion synthesis at arrested, are required for the rapid recovery of DNA synthesis and prevent the potentially mutagenic bypass of UV lesions to remove the blocking lesion and DNA synthesis to resume. In the absence of nascent DNA processing

Courcelle, Justin

380

Unusual DNA binding modes for metal anticancer complexes  

PubMed Central

DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophibic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes. PMID:19344743

Pizarro, Ana M.; Sadler, Peter J.

2010-01-01

381

BRCA2: One Small Step for DNA Repair, One Giant Protein Purified  

PubMed Central

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherite