Repair of psoralen interstrand cross-links in Xenopus laevis egg extracts is highly mutagenic

The recognition and removal of interstrand cross-links is perhaps the least understood of all repair pathways in eukaryotic cells. We have shown previously that uncoupling of cross-links occurs in mammalian cell extracts and have identified a number of factors that mediate this process. However, we have not observed complete repair of the substrate in this system. Here, we show that uncoupling of interstrand cross-links also occurs in Xenopus laevis egg extracts, and that the initial products of this reaction are identical to the products observed in mammalian cell extracts suggesting a common mechanism. However in contrast to mammalian cell extracts, we observe repair of the cross-linked substrate in the Xenopus extracts presumably by a translesion bypass mechanism that allows replication past the uncoupled monoadduct, and its likely subsequent removal by nucleotide excision repair. This repair process is shown to be highly mutagenic consistent with bypass synthesis.     

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.     

DNA alkylation and formation of DNA interstrand cross-links by potential antitumour 2,5-bis(1-aziridinyl)-1,4-benzoquinones

A series of 3,6-substituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives was shown to alkylate calf thymus DNA and to form DNA interstrand cross-links. Alkylation and cross-link formation were enhanced after electrochemical reduction of the compounds and increased with lower pH in the pH range from 4.5 to 8.0. Reduction especially shifts the pH at which cross-linking and alkylation occurs to higher values, which are more physiologically relevant. This shift is probably caused by the increase in pK_a value of the aziridine ring after reduction of the quinone moiety. The inactivation of single-stranded bacteriophage M13mp19 DNA to form phages in an E. coli host, by the 3,6-unsubstituted parent compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone (TW13) was dependent upon reduction and pH in a similar way as was alkylation. The compound in our series with the least bulky, 3,6-substitutents, TW13, caused a high amount of cross-link formation. Compounds with methyl-substituted aziridine rings showed low cross-linking ability. Our results support the concept that the protonated reduced compound is the reactive species that alkylates DNA, and that steric factors play an important role in the reactivity towards DNA. A correlation is observed between the ability to induce DNA interstrand cross-links and inactivation of M13mp19 bacteriophage DNA. Cross-link formation was also demonstrated in E. coli K12 cells, where the compounds are reduced endogenously by bacterial reductases.     

DNA interstrand cross-link repair in Saccharomyces cerevisiae

DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.     

Constitutively active Artemis nuclease recognizes structures containing single-stranded DNA configurations

The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure.     

ATP-driven Rad50 conformations regulate DNA tethering,end resection,and ATM checkpoint signaling

The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5′ strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

Ubiquitin and the DNA damage response

Comment on: Gatti M, et al. Cell Cycle 2012; 11:2538-44.     

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.     

Semiautomated autoradiographic measurement of DNA repair in normal and xeroderma pigmentosum cultured human fibroblasts

Summary Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semiautomated grain counting instruments. The instrument-determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (to 100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semiautomated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.     

Mapping the genetic landscape of DNA double-strand break repair

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Some properties of the interstrand crosslinks in depurinated DNA

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.     

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.Abbreviations HU hydroxyurea - dA deoxyadenosine - TSC pyridine-2-carboxaldehyde thiosemicarbazone - IMPY pyrazoloimidazole - THBA 3,4,5-trihydroxy benzohydroxamic acid - DHBA 3,4-dihydroxy benzohydroxamic acid - UDS unscheduled DNA synthesis - dT thymidine - dNTP deoxynucleoside triphosphate     

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo- or radiotherapy by pharmacologically modulating NHEJ cellular responses.     

Partial reconstitution of human interstrand cross-link repair in vitro: characterization of the roles of RPA and PCNA

The repair of DNA interstrand cross-links (ICLs) remains largely ill-defined in higher eukaryotic cells. Previously, we have developed assays that can be used to monitor the early stages of processing of ICLs in vitro. Here, we have used P11 phosphocellulose chromatography to fractionate HeLa nuclear extracts and have subsequently reconstituted these assays with the resulting fractions. RPA and PCNA were found in a single fraction, and were the only factors in this fraction required for the reconstitution of these assays. The roles of RPA and PCNA in the formation of incisions at ICLs and in the subsequent DNA synthesis step were assessed. RPA was found to be essential for both stages of ICL processing indicating that it is required for lesion recognition and/or for the subsequent endonucleolytic processing. PCNA is required for the DNA synthesis stage and although it is not critical for the incision stage of the reaction it does enhance this step presumably by a stimulation of lesion recognition by MutSbeta. These findings define novel roles for RPA and PCNA in the processing of ICLs in mammalian cells.     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。