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DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N -methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions., Competing Interests: MD, SS, JA, JU, JH, GP, JW, PM No competing interests declared, (© 2022, Duan et al.)

Spermatogenesis is threatened by DNA alkylating agents, one major category of DNA damaging agents. Currently, little is known about the alterations in transcriptome profiling of the mouse spermatogenic cells in response to DNA alkylation at distinct stages of spermatogenesis. In this study, RNA sequencing (RNA-seq) was performed in pachytene spermatocytes (PS) and round spermatids (RS) at 0 or 30min following Methyl Methanesulfonate (MMS) treatment and with untreated controls. A large number of differentially expressed genes (DEGs) were identified by comparison of the three groups in PS and RS, respectively. Functional analyses of all DEGs highlighted the protein ubiquitination pathway and DNA damage response (DDR) network being the two main biological processes in common in the two cell types. Further analyses of the DEGs with 2-fold or more changes between 30min repair and control group indicated that several cytokine signaling pathways were the most strongly affected in PS and DDR related pathways in RS, respectively. Gene ontology (GO) analyses directly showed differential biological process (BP) affected between PS and RS, with "regulation of transcription" being most overrepresented in PS and "cellular response to stress" in RS, respectively. Moreover, 374 DDR-related genes in PS and 158 in RS among all DEGs were filtered and clustered, which showed dynamic expression patterns in PS and RS. Our analyses provide a transcriptional landscape for male germ cells in response to MMS during spermatogenesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Faithful transmission of genetic material is essential for cell viability and organism health. The occurrence of DNA damage, due to either spontaneous events or environmental agents, threatens the integrity of the genome. The consequences of these insults, if allowed to perpetuate and accumulate over time, are mutations that can lead to the development of diseases such as cancer. Alkylation is a relevant DNA lesion produced endogenously as well as by exogenous agents including certain chemotherapeutics. We sought to better understand the cellular response to this form of DNA damage using mass spectrometry-based proteomics. For this purpose, we performed sub-cellular fractionation to monitor the effect of methyl methanesulfonate (MMS) treatment on protein localization to chromatin. The levels of over 500 proteins were increased in the chromatin-enriched nuclear lysate including histone chaperones. Levels of ubiquitin and subunits of the proteasome were also increased within this fraction, suggesting that ubiquitin-mediated degradation by the proteasome has an important role in the chromatin response to MMS treatment. Finally, the levels of some proteins were decreased within the chromatin-enriched lysate including components of the nuclear pore complex. Our spatial proteomics data demonstrate that many proteins that influence chromatin organization are regulated in response to MMS treatment, presumably to open the DNA to allow access by other DNA damage response proteins. To gain further insight into the cellular response to MMS-induced DNA damage, we also performed phosphorylation enrichment on total cell lysates to identify proteins regulated via post-translational modification. Phosphoproteomic analysis demonstrated that many nuclear phosphorylation events were decreased in response to MMS treatment. This reflected changes in protein kinase and/or phosphatase activity in response to DNA damage rather than changes in total protein abundance. Using these two mass spectrometry-based approaches, we have identified a novel set of MMS-responsive proteins that will expand our understanding of DNA damage signaling., (Copyright © 2014 Elsevier B.V. All rights reserved.)

DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3 mA) and N7-methylguanine (7 mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK (OK), nucleotide excision repair mutant mus201, dmPolQ protein mutant mus308, and the double mutant mus201;mus308. Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that mus201, mus308, and the double mutant mus201;mus308 strains presented, respectively, 21.5%, 52.9%, and 14.8% of OK strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways. [ABSTRACT FROM AUTHOR]

Arabidopsis methylation elevated mutant 1 (mem1) mutants have elevated levels of global DNA methylation. In this study, such mutant alleles showed increased sensitivity to methyl methanesulfonate (MMS). In mem1 mutants, an assortment of genes engaged in DNA damage response (DDR), especially DNA‐repair‐associated genes, were largely upregulated without MMS treatment, suggestive of activation of the DDR pathway in them. Following MMS treatment, expression levels of multiple DNA‐repair‐associated genes in mem1 mutants were generally lower than in Col‐0 plants, which accounted for the MMS‐sensitive phenotype of the mem1 mutants. A group of DNA methylation pathway genes were upregulated in mem1 mutants under non‐MMS‐treated conditions, causing elevated global DNA methylation, especially in RNA‐directed DNA methylation (RdDM)‐targeted regions. Moreover, MEM1 seemed to help ATAXIA‐TELANGIECTASIA MUTATED (ATM) and/or SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) to fully activate/suppress transcription of a subset of genes regulated simultaneously by MEM1 and ATM and/or SOG1, because expression of such genes decreased/increased consistently in mem1 and atm and/or sog1 mutants, but the decreases/increases in the mem1 mutants were not as dramatic as in the atm and/or sog1 mutants. Thus, our studies reveals roles of MEM1 in safeguarding genome, and interrelationships among DNA damage, activation of DDR, DNA methylation/demethylation, and DNA repair. [ABSTRACT FROM AUTHOR]

During the growth and evolution of plants, genomic DNA is subject to constant assault from endogenous and environmental DNA damage compounds, which will result in mutagenic or genotoxic covalent adducts. Whether for prokaryotes, eukaryotes or even viruses, maintaining genome integrity is critical for the continuation of life. Escherichia coli and mammals have evolved the AlkB family of Fe(II)/alpha-ketoglutarate-dependent dioxygenases that repair DNA alkylation damage. We identified a functional homologue with EsAlkB and HsALKBH2 in tomatoes, and named it SlALKBH2. In our study, the SlALKBH2 knockout mutant showed hypersensitivity to the DNA mutagen MMS and displayed more severe growth abnormalities than wild-type plants under mutagen treatment, such as slow growth, leaf deformation and early senescence. Additionally, genes with high transcriptional activity, such as rDNA, have increased methylation under MMS treatment. In conclusion, this study shows that the tomato SlALKBH2 gene may play an important role in ensuring the integrity of the genome. • Knockout of SlALKBH2 weakens the DNA damage repair ability of tomato. • The repair pathway of this gene is different in animals and tomatoes. • It was found for the first time that this gene was related to rDNA demethylation in the presence of exogenous methylating agents. • Knockout of SlALKBH2 will lead to insufficient DNA repair under exogenous pressure, and then lead to leaf senescence and death. [ABSTRACT FROM AUTHOR]

Highlights: [•] We compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. [•] Me-lex efficiently and selectively generates 3-mA (>90%) in contrast to MMS. [•] Polζ is involved in the mutagenic bypass of MMS and Me-lex induced lesions. [•] Polη contributes towards the error-free bypass of Me-lex induced lesions. [•] AP sites are often more toxic than the initiating DNA lesion (3-mA). [Copyright &y& Elsevier]

The HRDC (helicase and RNase D C-terminal) domain at the C-terminal of WRNp (Werner protein) (1150-1229 amino acids) and BLMp (Bloom protein) (1212-1292 amino acids) recognize laser microirradiation-induced DNA dsbs (double-strand breaks). However, their role in the recognition of DNA damage other than dsbs has not been reported. In this work, we show that HRDC domain of both the proteins can be recruited to the DNA damage induced by MMS (methyl methanesulfonate) and MMC (methyl mitomycin C). GFP (green fluorescent protein)-tagged HRDC domain produces distinct foci-like respective wild-types after DNA damage induced by the said agents and co-localize with α-H2AX. However, in time course experiment, we observed that the foci of HRDC domain exist after 24 h of removal of the damaging agents, while the foci of full-length protein disappear completely. This indicates that the repair events are not completed by the presence of protein corresponding to only the HRDC domain. Consequently, cells overexpressing the HRDC domain fail to survive after DNA damage, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide] assay. Moreover, 24 h after removal of damaging agents, the extent of DNA damage is greater in cells overexpressing HRDC domain compared with corresponding wild-types, as observed by comet assay. Thus, our observations suggest that HRDC domain of both WRN and BLM can also recognize different types of DNA damages, but for the successful repair they fail to respond to subsequent repair events. [ABSTRACT FROM AUTHOR]

Abstract: Wild-type p53-induced phosphatase 1 (Wip1, PPM1D) is induced by p53 in response to various stressors and dephosphorylates cellular target proteins involved in DNA repair and cell cycle checkpoint pathways. The Wip1 gene is frequently amplified or overexpressed in human cancers, promoting tumor growth by switching off major checkpoint kinases and p53. To explore wild-type p53-independent Wip1 induction, Wip1 promoter activity and its transcript level were evaluated by luciferase assay and real-time PCR, after methylmethane sulfonate (MMS) treatment in breast cancer cell lines and p53-null cell lines. Wip1 promoter activities in response to UV irradiation and various anti-cancer agents were compared between wild-type and a p53-response element (p53RE) mutated construct. Wip1 expression and its effects were examined in primary non-small cell lung cancer (NSCLC) and colon tumor cells by using Wip1-specific siRNA. MMS induced Wip1 promoter activity in Hs578T, MDA-MB-231, and SK-BR-3 cells expressing DNA binding-deficient p53 mutants. A549-E6 and HCT116 (p53−/−) cells retained substantial Wip1 induction. Wip1 promoter activity was reduced, but not eliminated, in cells expressing a promoter containing a mutated p53-response element. Wip1 induction was not blocked by SB202190 or SP600125. MMS increased Wip1 expression in primary non-small cell lung cancer cells expressing a p53 R175H mutant. Our data indicate that Wip1 is induced in the absence of functional p53, like p38 MAPK and JNK, as a stress response terminator. [Copyright &y& Elsevier]

Abstract: In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), α-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3 →Arg+ revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB −) strain in comparison with the AB1157 (alkB +) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd +, and no repair in BS87 mfd − cells. Determination of specificity of Arg+ revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT→TA transversions by supL suppressor formation (1meA) and GC→AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB − mfd − bacteria but not in the alkB + mfd − counterpart. [Copyright &y& Elsevier]

Abstract: DNA damage from exogenous and endogenous sources can promote mutations and cell death. Fortunately, cells contain DNA repair and damage signaling pathways to reduce the mutagenic and cytotoxic effects of DNA damage. The identification of specific DNA repair proteins and the coordination of DNA repair pathways after damage has been a central theme to the field of genetic toxicology and we have developed a tool for use in this area. We have produced 99 molecular bar-coded Escherichia coli gene-deletion mutants specific to DNA repair and damage signaling pathways, and each bar-coded mutant can be tracked in pooled format using bar-code specific microarrays. Our design adapted bar-codes developed for the Saccharomyces cerevisiae gene-deletion project, which allowed us to utilize an available microarray product for pooled gene-exposure studies. Microarray-based screens were used for en masse identification of individual mutants sensitive to methyl methanesulfonate (MMS). As expected, gene-deletion mutants specific to direct, base excision, and recombinational DNA repair pathways were identified as MMS-sensitive in our pooled assay, thus validating our resource. We have demonstrated that molecular bar-codes designed for S. cerevisiae are transferable to E. coli, and that they can be used with pre-existing microarrays to perform competitive growth experiments. Further, when comparing microarray to traditional plate-based screens both overlapping and distinct results were obtained, which is a novel technical finding, with discrepancies between the two approaches explained by differences in output measurements (DNA content versus cell mass). The microarray-based classification of Δtag and ΔdinG cells as depleted after MMS exposure, contrary to plate-based methods, led to the discovery that Δtag and ΔdinG cells show a filamentation phenotype after MMS exposure, thus accounting for the discrepancy. A novel biological finding is the observation that while ΔdinG cells filament in response to MMS they exhibit wild-type sulA expression after exposure. This decoupling of filamentation from SulA levels suggests that DinG is associated with the SulA-independent filamentation pathway. [Copyright &y& Elsevier]

Abstract: In Saccharomyces cerevisiae, the linker histone HHO1 is involved in DNA repair. In higher eukaryotes, multiple variants of linker histone H1 exist but their involvement in the DNA damage response is unknown. To address this issue, we examined sensitivity to genotoxic agents in chicken DT40 cells lacking specific H1 variants. Among the six H1 variant mutants, only H1R −/− DT40 cells exhibited increased sensitivity to the alkylating agent methyl-methanesulfonate (MMS). The MMS sensitivity of H1R −/− cells was not enhanced by inactivation of Rad54. H1R −/− DT40 cells also exhibited: (i) a reduction in gene targeting efficiencies, (ii) impaired sister chromatid exchange, and (iii) an accumulation of IR-induced chromosomal aberrations at the G2 phase, all of which indicate the involvement of H1R in the Rad54-mediated homologous recombination (HR) pathway. The mobility of H1R but not H1L in the nucleus decreased after MMS treatment and the repair of double-stranded breaks generated by I-SceI was unaffected in H1R −/− cells, suggesting that H1R integrates into HR-mediated repair pathways at the chromosome structure level. Together, these findings provide the first genetic evidence that a specific H1 variant plays a unique and important role in the DNA damage response in vertebrates. [Copyright &y& Elsevier]

Abstract: BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein γ-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1–304) and the C-terminal sequence aa1078–1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage. [Copyright &y& Elsevier]

Abstract: The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7 + , that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Δmus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Δmus7 and Δmus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Δmus7 and Δmus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Δmus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81. [Copyright &y& Elsevier]

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1−/− DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1−/− cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR. [ABSTRACT FROM AUTHOR]

Abstract: Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24h but not after 48h, indicating repair. Double treatment with VitC (at 0 and 24h) induced a cumulative genotoxic response at 48h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate “effective” strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC. [Copyright &y& Elsevier]

DNA treated with alkylating agents is incised at sites of damage by cell extracts. A key component of this DNA repair function was shown by Verly and co-workers to be an endonuclease acting at secondary lesions, apurinic sites, rather than directly at alkylated nucleotide residues. [Copyright &y& Elsevier]

A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Δ mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Δ mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Δ mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse. [Copyright &y& Elsevier]

XRCC1 is known to be involved in base excision repair (BER)/single-strand break repair (SSBR) through interaction with other BER enzymes. Hypersensitivity of XRCC1-deficient cells against alkylating agents has been explained by loss of interaction with BER proteins. XRCC1 is a unique DNA repair protein containing two BRCT motifs, recently identified in several DNA repair and cell cycle regulating proteins. To study the function(s) of the two BRCT motifs of the XRCC1 protein, we established CHO EM9 (XRCC1-null) cells expressing XRCC1 protein altered in either one of the two BRCT motifs. Colony-forming ability after methyl methanesulfonate (MMS) treatment was dependent on the BRCT-a motif, but not on the BRCT-b motif. Surprisingly, reduced BER/SSBR rate in vivo, measured by an alkaline comet assay, was observed in the BRCT-b motif-deficient cells, while the BRCT-a motif-deficient cells showed the repair rate comparable with the wild-type (WT) cells. The BRCT-a motif-mutated cells, instead, showed deficiency in initiation of DNA replications after MMS treatment. Furthermore, we found that XRCC1 is multiply phosphorylated in vivo and hyperphosphorylation of XRCC1 after MMS treatment is dependent on the BRCT-a motif. These data suggest a new function dependent on the integrity of the BRCT-a motif of XRCC1 in recovery from MMS-induced damage. [Copyright &y& Elsevier]