Rad52 and Ku bind to different DNA structures produced early in double-strand break repair

DNA double-strand breaks are repaired by one of two main pathways, non-homologous end joining or homologous recombination. A competition for binding to DNA ends by Ku and Rad52, proteins required for non-homologous end joining and homologous recombination, respectively, has been proposed to determine the choice of repair pathway. In order to test this idea directly, we compared Ku and human Rad52 binding to different DNA substrates. How ever, we found no evidence that these proteins would compete for binding to the same broken DNA ends. Ku bound preferentially to DNA with free ends. Under the same conditions, Rad52 did not bind preferentially to DNA ends. Using a series of defined substrates we showed that it is single-stranded DNA and not DNA ends that were preferentially bound by Rad52. In addition, Rad52 aggregated DNA, bringing different single-stranded DNAs in close proximity. This activity was independent of the presence of DNA ends and of the ability of the single-stranded sequences to form extensive base pairs. Based on these DNA binding characteristics it is unlikely that Rad52 and Ku compete as ‘gatekeepers’ of different DNA double-strand break repair pathways. Rather, they interact with different DNA substrates produced early in DNA double-strand break repair.     

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Ku86 deficiency leads to reduced intrachromosomal homologous recombination in vivo in mice

Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable (pun) allele results in reversion to the wildtype pink-eyed dilution (p) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo.     

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae

Yeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5'-end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was co-inactivated. Defective recombinational repair of DSBs in mrx cells thus arises from at least two separate processes: loss of Mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.     

Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination

To investigate the DNA damage response, we undertook a genome-wide study in Saccharomyces cerevisiae and identified 86 gene deletions that lead to increased levels of spontaneous Rad52 foci in proliferating diploid cells. More than half of the genes are conserved across species ranging from yeast to humans. Along with genes involved in DNA replication, repair, and chromatin remodeling, we found 22 previously uncharacterized open reading frames. Analysis of recombination rates and synthetic genetic interactions with rad52Δ suggests that multiple mechanisms are responsible for elevated levels of spontaneous Rad52 foci, including increased production of recombinogenic lesions, sister chromatid recombination defects, and improper focus assembly/disassembly. Our cell biological approach demonstrates the diversity of processes that converge on homologous recombination, protect against spontaneous DNA damage, and facilitate efficient repair.     

Rad52 and Rad59 exhibit both overlapping and distinct functions

Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination.     

Effect of Wortmannin on the repair profiles of DNA double-strand breaks in the whole genome and in interstitial telomeric sequences of Chinese hamster cells

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process.     

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.     

Mutational analysis of Brh2 reveals requirements for compensating mediator functions

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.     

Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.     

Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast

In mammalian and budding yeast cells treated with genotoxic agents, different proteins implicated in detecting, signalling or repairing DNA lesions form nuclear foci. We studied foci formed by proteins involved in these processes in living fission yeast cells, which is amenable to genetic and molecular analysis. Using fluorescent tags, we analysed subnuclear localisations of the DNA damage checkpoint protein Rad9, of the homologous recombination protein Rad22 and of PCNA, which are implicated in many aspects of DNA metabolism. After inducing double strand breaks (DSBs) with ionising radiations, Rad22, Rad9 and PCNA form a low number of nuclear foci. Rad9 recruitment to foci depends on the presence of Rad1, Hus1 and Rad17, but is independent of downstream checkpoint effectors and of homologous recombination proteins. Likewise, Rad22 and PCNA form foci despite inactive homologous recombination repair and impaired DNA damage checkpoint. Rad22 and Rad9 foci co-localise completely, whereas PCNA co-localises with Rad22 and Rad9 only partially. Foci do not disassemble in cells unable to repair DNA by homologous recombination. Thus, in fission yeast, DSBs are detected by the DNA damage checkpoint and are repaired by homologous recombination at a few spatially confined subnuclear compartments where Rad22, Rad9 and PCNA concentrate independently.     

Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity

Homologous recombination (HR) is a major DNA repair pathway and therefore essential for maintaining the integrity of the genome. HR is catalyzed by proteins encoded by genes of the RAD52 epistasis group, including the recombinase Rad51 and its mediator Rad52. HR proteins fused with green fluorescent protein form foci at damaged DNA reflecting the assembly of repair centers that harbor a high concentration of repair proteins. Rad52 mediates the recruitment of Rad51 and other HR proteins to DNA damage. To understand the mechanism for the assembly of Rad52-dependent DNA repair centers, we used a mutational strategy to identify a Rad52 domain essential for its recruitment to DNA repair foci. We present evidence to implicate an acidic domain in Rad52 in DNA repair focus formation. Mutations in this domain confer marked DNA damage sensitivity and recombination deficiency. Importantly, these Rad52 mutants are specifically compromised for interaction with the single-stranded DNA-binding factor RPA. Based on these findings, we propose a model where Rad52 displaces RPA from single-stranded DNA using the acidic domain as a molecular lever.     

Radiation-induced recombination is dependent on Ku80

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.     

Rad52 promotes postinvasion steps of meiotic double-strand-break repair

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.     

Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous end-joining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.     

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.