DNA double strand breaks

In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3. We also observed cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor-proximal caspase-8 was not activated after methylation. Also, inhibition of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis. Overall, the data indicate that O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.

Various tumor-therapeutic drugs and environmental carcinogens alkylate DNA inducing O 6 -methylguanine (O 6 MeG) that provokes cell death by apoptosis. In rodent fibroblasts, apoptosis triggered by O 6 MeG is executed via the mitochondrial damage pathway. Conversion of O 6 MeG into critical downstream lesions requires mismatch repair (MMR). This is thought to signal apoptosis upon binding to O 6 MeG lesions mispaired with thymine. Alternatively, O 6 MeG lesions might be processed by MMR giving rise to DNA double-strand breaks (DSBs) during replication that finally provoke apoptosis. To test this, we examined apoptosis triggered by O 6 MeG in human peripheral lymphocytes in which O 6 -methylguanine-DNA methyltransferase (MGMT) had been inactivated by O 6 -benzylguanine (O 6 BG) and which were not proliferating or proliferating upon CD3/CD28 stimulation. Treatment with N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) or the anticancer drug temozolomide induced apoptosis only in proliferating, but not resting cells. With exceptional high alkylation doses (X15 lM of MNNG), apoptosis was also observed in resting lymphocytes, albeit at a lower level than in proliferating cells. This response was not affected by O 6 BG, suggesting that replication-independent apoptosis at high dose levels is caused by lesions other than O 6 MeG. O 6 MeG-triggered apoptosis in proliferating lymphocytes was preceded by a wave of DSBs, which coincided with p53 and Fas receptor upregulation, while Fas ligand, Bax and Bcl-2 expression was not altered. Treatment with anti-Fas neutralizing antibody attenuated MNNG-induced apoptosis in MGMT-depleted proliferating lymphocytes. The data suggest that O 6 MeG is converted by MMR and DNA replication into DSBs that trigger apoptosis by p53 stabilization and Fas/CD95/Apo-1 upregulation. This is supported by the finding that ionizing radiation, inducing DSBs on its own, provokes apoptosis in lymphocytes in a replication-independent way. The strict proliferation dependence of apoptosis triggered by O 6 MeG may explain the specific killing response of MGMT-deficient proliferating cells, including tumors, to O 6 MeG generating anticancer drugs and suggests that tumor proliferation rate, Fas responsiveness, MGMT and MMR status are important prognosis parameters.

The proteins of SMC family are characterised by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recN K35D . Reverse genetics was utilized to delete the entire recN gene (DrecN108) or introduce the recN K35D gene into the E. coli chromosomal DNA. The recN K35D cells showed decreasing in the frequency of excision of Tn10 from gal76::Tn10 after treatment with mitomycin C compared to recN + cells. The DrecN108 cells showed an uninduced increase frequency of Tn10 excision from gal76::Tn10 in rec + background. While, recBC sbcBC DrecN108 cells are completely deficient in Tn10 excision. The recombination proficiency is reduced in cells carrying recBC sbcBC cells in addition recN K35D mutation. We observed that the Walker A nucleotide binding site is important for the RecN protein. Strains that deleted recN gene are recombination deficient and more sensitive to mitomycin C than strains carrying recN K35D .

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catal...

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.

XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by A...

Aging tissues accumulate DNA damage, while genome instability is a defining feature of cancer. Despite this shared foundation, DNA damage is still largely viewed as a transient lesion that is either faithfully repaired or converted into a mutation. New evidence challenges this binary view, indicating that DNA double-strand breaks (DSBs), even when accurately repaired at the sequence level, can leave durable and heritable alterations in chromatin organization and gene regulation. Accordingly, DSB repair restores DNA integrity but does not necessarily re-establish the original regulatory architecture. The biological consequences of such post-repair regulatory memory remain largely underappreciated, progressively contributing to age-associated tissue dysfunction while simultaneously creating permissive states for malignant transformation and therapy resistance. In this commentary, we argue that reframing DNA damage as a source of heritable regulatory change, rather than solely as a mutational event, reshapes our understanding of aging trajectories and cancer risk.

HMG1 or 2, form a complex (the RAG complex) that binds at the heptamer/nonamer recombination signal sequences (designated 12-or 23-RSS, based on the spacer length) (Gellert, 1997). The RAG complex endonucleolytically nicks the DNA 5Ј of the heptamer, where

Glioblastoma multiforme (GBM) is the most lethal of brain tumors and is highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, epidermal growth factor receptor variant III (EGFRvIII), confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSB), the most lethal lesion inflicted by IR. The EGFR inhibitor gefitinib (Iressa) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated Akt-1 accelerates repair, implicating the PI3K/Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevat...

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 6 S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the antirecombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Constitutive expression of telomerase prevents senescence and crisis by maintaining telomere homeostasis. However, recent evidence suggests that telomerase is dynamically regulated in normal cells and also contributes to transformation independently of net telomere elongation. Here, we show that suppression of the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] expression abrogates the cellular response to DNA double strand breaks. Loss of hTERT does not alter short-term telomere integrity but instead affects the overall configuration of chromatin. Cells lacking hTERT exhibit increased radiosensitivity, diminished capacity for DNA repair, and fragmented chromosomes, demonstrating that loss of hTERT impairs the DNA damage response.

DNA double-strand breaks (DSBs) with protein covalently attached to 5′ strand termini are formed by Spo11 to initiate meiotic recombination 1,2 . The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal has been unclear 3 . We show here that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3′-OH. Surprisingly, two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-significantly earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs. We previously proposed that Spo11 might be removed from DSB ends by either of two mechanisms: direct hydrolysis of the covalent protein-DNA linkage, or single-stranded endonucleolytic cleavage releasing Spo11 covalently attached to a short oligonucleotide (Fig. ) 1,4 . These mechanisms are distinguished by the presence or absence of an oligonucleotide bound to Spo11. We identified this predicted protein-DNA complex using a direct biochemical approach in S. cerevisiae. A strain expressing HA epitope-tagged Spo11 was induced to enter meiosis, denaturing extracts were prepared, then Spo11-HA was immunoprecipitated and treated with 32 P-labelled nucleotide and terminal deoxynucleotidyl transferase (TdT), which catalyzes untemplated addition of nucleotides to a free 3′-OH DNA end. A chain terminating nucleotide was used to limit incorporation to a single residue. Four radiolabelled bands were observed between ~60 and 110 kDa (Fig. , lane 3). Two bands (asterisks) were non-specific because they were present when TdT and nucleotide were incubated alone (Fig. , lane 1). Two bands (solid arrows) were specific for Spo11-HA because they were not seen with mock immunoprecipitation (Fig. , lane 2), or untagged Spo11 (Fig. , lane 4). Labelling was not observed if DSBs were not formed, namely, when the catalytic tyrosine of Spo11 was mutated to phenylalanine 2 (Fig. , lane 5) or in a mei4 mutant 4 (Fig. , lane 6). Labelled Spo11 species were also not observed in rad50S or sae2Δ mutants (Fig. , lanes 7-8), in which Spo11 remains covalently attached to DSB ends 1,5 . Thus, Spo11-oligonucleotide complexes did not arise from non-physiological disruption of covalent Spo11-DSB complexes.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires resection of 5′-termini to generate 3′-single-strand DNA tails 1 . Key components of this reaction are Exonuclease 1 and the bifunctional endo/exonuclease, Mre11 2-4 . Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein-such as by the DNA end-joining complex 5 , topoisomerases 6 , or the meiotic nuclease, Spo11 7-13 -but a specific function for the Mre11 3′-5′ exonuclease activity has remained elusive. Here, we reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5′-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated resection to traverse unidirectionally 1 . Using a combination of physical assays for 5′-end-processing, our results suggest an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5′-terminus of the DSB-much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5′-3′ direction away from the DSB, and Mre11 in the 3′-5′ direction towards the DSB end. Finally, Mre11 exonuclease activity confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways. We sought to clarify Mre11-dependent processing reactions at DSBs with blocked termini. Blocked DSB ends are created in meiosis by the topoisomerase-like transesterase, Spo11, to initiate crossover recombination 14 (Fig. ). Aside from the importance of meiotic recombination to sexual reproduction and genetic diversity, we reasoned that the molecular reactions pertaining to repair of covalently blocked Spo11-DSBs would be informative to the repair of DNA lesions with other types of end blockage, such as failed topoisomerase reactions and DSB ends competitively bound by the nonhomologous end-joining (NHEJ) machinery. Mre11-dependent endonucleolytic processing (nicking) of Spo11-DSBs generates two classes of Spo11-oligonucleotide fragments originating from the DSB end 15 (Fig. ). Spo11-oligo complexes do not form in mre11 mutants completely abrogated for nuclease activity (mre11-D56N, and mre11-H125N; Fig. ) solidifying earlier conclusions that Mre11 directly processes Spo11-DSBs 7-13 . Recently, an allele of Mre11 that is exonuclease deficient, but mostly endonuclease proficient was described for the orthologous proteins in S. pombe and P. furiosus 16 . We generated the equivalent mutation in budding yeast Mre11 (Histidine 59 to Serine; Fig. ) and tested its function. During meiosis, Spo11-oligo Correspondence and requests for materials should be addressed to M.J.N.

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was ...

In most sexually reproducing organisms, meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. In budding yeast, nine other proteins are also required for DSB formation, but roles of these proteins and interactions among them are poorly understood. We report here further studies of the behaviors of these proteins. Consistent with other studies, we find that Mei4 and Rec114 bind to chromosomes from leptonema through early pachynema. Both proteins showed only limited colocalization with the meiotic cohesin subunit Rec8, suggesting that Mei4 and Rec114 associated preferentially with chromatin loops. Rec114 localization was independent of other DSB factors, but Mei4 localization was strongly dependent on Rec114 and Mer2. Systematic deletion analysis identified protein regions important for a previously described two-hybrid interaction between Mei4 and Rec114. We also report functional characterization of a previously misannotated 5′ coding exon of REC102. Sequences encoded in this exon are essential for DSB formation and for Rec102 interaction with Rec104, Spo11, Rec114, and Mei4. Finally, we also examined genetic requirements for a set of previously described two-hybrid interactions that can be detected only when the reporter strain is induced to enter meiosis. This analysis reveals new functional dependencies for interactions among the DSB proteins. Taken together, these studies support the view that Mei4, Rec114, and Mer2 make up a functional subgroup that is distinct from other subgroups of the DSB proteins: Spo11-Ski8, Rec102-Rec104, and Mre11-Rad50-Xrs2. These studies also suggest that an essential function of Rec102 and Rec104 is to connect Mei4 and Rec114 to Spo11.

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1D cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1D cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. DNA double-strand breaks (DSB) are one of the most severe DNA lesions caused directly and indirectly by benzene metabolites. DSB may lead to chromosome aberrations, apoptosis and hematopoietic progenitor cell suppression. We hypothesized that genetic polymorphisms in genes involved in DNA DSB repair may modify benzene-induced hematotoxicity. We analyzed one or more single nucleotide polymorphisms (SNPs) in each of seven candidate genes (WRN, TP53, NBS1, BRCA1, BRCA2, XRCC3 and XRCC4) in a study of 250 workers exposed to benzene and 140 controls in China. Four SNPs in WRN (Ex4 À16 G > A, Ex6 +9 C > T, Ex20 À88 G > T and Ex26 À12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with a statistically significant decrease in total white blood cell (WBC) counts among exposed workers. The SNPs in WRN and TP53 remained significant after accounting for multiple comparisons. One or more SNPs in WRN had broad effects on WBC subtypes, with significantly decreased granulocyte, total lymphocyte, CD4 + -T cell, CD8 + -T cell and monocyte counts. Haplotypes of WRN were associated with decreased WBC counts among benzene-exposed subjects. Likewise, subjects with TP53 Ex4 +119 C > G variant had reduced granulocyte, CD4 + -T cell and B cell counts. The effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. These results suggest that genetic polymorphisms in WRN, TP53 and BRCA2 that maintain genomic stability impact benzene-induced hematotoxicity.

Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C 2 H 2 zincfinger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zincfinger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rg gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMSderived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac + mutants, with and without evidence of descent from the HMS, have similar Lac + mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutationstimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac + mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.

Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light ⌬dss1 cells entered mitosis prematurely with indistinguishable timing from ⌬rad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G 2 /M boundary in response to DNA damage. Eukaryotic cells respond to double-strand breaks (DSBs) 3 within DNA by activating DNA damage checkpoint proteins that send signals to the cells, ultimately resulting in cell cycle arrest. The arrest allows DNA damage to be repaired by the proteins of the DNA repair pathway before cells can enter mitosis (1, 2). In Schizosaccharomyces pombe, entry into mitosis at the G 2 /M boundary is regulated by the phosphorylation status

The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen (a-values ranged from 0.12 to 0.54). After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding a-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.

Smm_ary Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t, j of rejoining ranged from 18.0 ± 1.4 to 36.4 ± 3.2 min (fast rejoining process) and from 1.5 ± 0.2 to 5.1 ± 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t, of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell. Keywords radiosensitivity; DNA double-strand breaks; dsb rejoining; pulsed field gel electrophoresis

Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ec...

The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double‐strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc‐1) during meiotic prophase. The null mutant in the brc‐1 gene is viable, fertile and shows the wild‐type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc‐1 mutants show an abnormal increase in apoptosis and RAD‐51 foci at pachytene that are abolished by loss of spo‐11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him‐14/MSH4 and syp‐2, loss of brc‐1 leads to chromosome fragmentation suggesting that brc‐1 is dispensable for crossing over but essential for DSB repair through in...