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.     

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.     

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.     

The novel gene mus7(+) is involved in the repair of replication-associated DNA damage in fission yeast

The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Deltamus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Deltamus7 and Deltamus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Deltamus7 and Deltamus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Deltamus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

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.     

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.     

Tel2 is required for activation of the Mrc1-mediated replication checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.     

Smc5/6 maintains stalled replication forks in a recombination‐competent conformation

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis‐segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination‐dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome‐wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage‐induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination‐competent conformation primed for replication restart.     

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11^Rpa1 and Rad22^Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.     

Activation of Fanconi Anemia Pathway in Cells with Re-Replicated DNA

To maintain genome stability, the cell has to limit initiation of DNA replication to once per cell cycle. Loss of this control leads to DNA re-replication with repeated firing of replication origins in the same cell cycle. Re-replication generates both ssDNA and double strand breaks, as well as activation of the DNA damage checkpoint. In re-replicated cells, activation of the checkpoint is critical to arrest cells in G2 resulting in accumulation of cells with re-replicated DNA. Abrogation of this checkpoint suppresses the progressive accumulation of cells with excess DNA and causes apoptosis. Recently, the Fanconi Anemia pathway was reported to be activated in re-replicating cells. Interestingly, FA core complexes but not FANCD2, is required for checkpoint activation in re-replicated cells, suggesting that the pathway to checkpoint activation requires the ubiquitination of substrates other than FANCD2. In addition, FANCD2 is required for recruitment of Rad51 to foci in re-replicated cells, so that the repair pathways activated after small degrees of re-replication are expected to be compromised in cells with mutations in the FA pathway.     

Expanded CAG/CTG repeat DNA induces a checkpoint response that impacts cell proliferation in Saccharomyces cerevisiae

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.     

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Delta mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Delta cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.     

Mms22 preserves genomic integrity during DNA replication in Schizosaccharomyces pombe

The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important but poorly understood role in preservation of genome integrity. Here we describe a novel gene in Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22 functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability. There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to maintain genomic integrity in a pathway involving Mus81-Eme1.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Temporal separation of replication and recombination requires the intra-S checkpoint

In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block.     

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.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.