RecG Directs DNA Synthesis during Double-Strand Break Repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.     

A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation

Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.     

C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression

Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs) and repair of a subset of these breaks as interhomolog crossovers (COs). Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR). In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR)-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition.     

Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.     

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).     

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.     

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

SUMMARY

Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen.     

Detection of poly(ADP-ribose) synthesis in Drosophila testes upon γ-irradiation

Poly(ADP-ribose) polymerase (PARP) activity is widespread among eukaryotes. Upon DNA damage PARP binds to DNA strand breaks and transfers ADP-ribose residues from NAD⁺ to acceptor proteins and to ADP-ribosyl protein adducts. This leads to branched polymers of protein-coupled poly(ADP-ribose) (pADPr). Because the germline of Drosophila has recently become important in the study of DNA double-strand break repair (DSBR) as opposed to somatic DSBR we tested whether the catalytic activity of PARP can be stimulated by γ-irradiation during Drosophila spermatogenesis. Using antibodies against pADPr we detected a significant increase in PARP activity in male germline cells during spermatogenesis upon γ-irradiation. Different stages of spermatogenesis revealed different subnuclear localization patterns of pADPr. In premeiotic and postmeiotic cells pADPr localized in a pattern overlapping with lamin and topoisomerase II at the nuclear rim. In primary spermatocytes pADPr is associated with three loci corresponding to the chromosomes at the nuclear periphery. Received: 12 October 1998; in revised form: 21 December 1998 / Accepted: 23 December 1998     

Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these proces­ses is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.     

Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae

Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.     

Homologous recombination in the archaea: the means justify the ends

The process of information exchange between two homologous DNA duplexes is known as homologous recombination (HR) or double-strand break repair (DSBR), depending on the context. HR is the fundamental process underlying the genome shuffling that expands genetic diversity (for example during meiosis in eukaryotes). DSBR is an essential repair pathway in all three domains of life, and plays a major role in the rescue of stalled or collapsed replication forks, a phenomenon known as recombination-dependent replication (RDR). The process of HR in the archaea is gradually being elucidated, initially from structural and biochemical studies, but increasingly using new genetic systems. The present review focuses on our current understanding of the structures, functions and interactions of archaeal HR proteins, with an emphasis on recent advances. There are still many unknown aspects of archaeal HR, most notably the mechanism of branch migration of Holliday junctions, which is also an open question in eukarya.     

Coordination of DNA ends during double-strand-break repair in bacteriophage T4

The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model.     

Analysis of one-sided marker segregation patterns resulting from mammalian gene targeting

The double-strand break repair (DSBR) model is currently accepted as the paradigm for acts of double-strand break (DSB) repair that lead to crossing over between homologous sequences. The DSBR model predicts that asymmetric heteroduplex DNA (hDNA) will form on both sides of the DSB (two-sided events; 5:3/5:3 segregation). In contrast, in yeast and mammalian cells, a considerable fraction of recombinants are one sided: they display full conversion (6:2 segregation) or half-conversion (5:3 segregation) on one side of the DSB together with normal 4:4 segregation on the other side of the DSB. Two mechanisms have been proposed to account for these observations: (i) hDNA formation is restricted to one side of the DSB or the other, and (ii) recombination is initially two sided, but hDNA repair directed by Holliday junction cuts restores normal 4:4 segregation on that side of the DSB in which the mismatch is closest to the cut junction initiating repair. In this study, we exploited a well-characterized gene-targeting assay to test the predictions that these mechanisms make with respect to the frequency of recombinants displaying 4:4 marker segregation on one side of the DSB. Unexpectedly, the results do not support the predictions of either mechanism. We propose a derivation of mechanism (ii) in which the nicks arising from Holliday junction cleavage are not equivalent with respect to directing repair of adjacent hDNA, possibly as a result of asynchronous cleavage of the DSBR intermediate.     

Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR.     

Involvement of MRE11A and XPA gene polymorphisms in the modulation of DNA double-strand break repair activity: a genotype-phenotype correlation study

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend <0.0001) and γH2AX dephosphorylation at 3h (p-trend <0.0001). Our study revealed that in addition to SNPs of genes that are well-established players in DSBR, non-DSBR genes, such as the XPA gene that is mainly involved in the nucleotide excision repair pathway, can also influence DSBR in healthy subjects. This suggests that successful DSBR may require both DSBR and non-DSBR mechanisms.     

Genetic analysis of BRCA1 function in a defined tumor cell line

Retrovirally expressed, wild-type BRCA1 decreased the gamma radiation (IR) sensitivity and increased the efficiency of double-strand DNA break repair (DSBR) of the BRCA1-/- human breast cancer line, HCC1937. It also reduced its susceptibility to DSB generation by IR. By contrast, multiple, clinically validated, missense mutant BRCA1 products were nonfunctional in these assays. These data constitute the basis for a BRCA1 functional assay and suggest that efficient repair of double-strand DNA breaks is linked to BRCA1 tumor suppression function.     

Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.     

A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces Cerevisiae

We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARG4 locus. The ``canonical'''' DSBR model, in which only 5'' ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a ``trans'''' configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.     

Strand invasion and DNA synthesis from the two 3' ends of a double-strand break in Mammalian cells

Analysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion ("ends-in") gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3' vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3' ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.