Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway

Saccharomyces cerevisiae-based system for studying clustered DNA damages

DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status

This work was funded by the BBSRC, grant BB/H088071/1 (www.bbsrc.ac.uk), MRC, grant G0700760 (www.mrc.ac.uk), Wellcome Trust, grant 095645/Z/11/Z (www.wellcome.ac.uk) and the EU through EpiGeneSys (www.epigenesys.eu) and Blueprint (www.blueprint-epigenome.eu). C.K. was funded by the DFG, personal fellowship KR 3317/2-1 (www.dfg.de) and CTR, personal short term fellowship (www.trophoblast.cam.ac.uk). M.J.H. received funding through grant NCI/NIH 2RO1 CA089426 (www.nih.gov)

Pch2 Acts through Xrs2 and Tel1/ATM to Modulate Interhomolog Bias and Checkpoint Function during Meiosis

Proper segregation of chromosomes during meiosis requires the formation and repair of double-strand breaks (DSBs) to form crossovers. Repair is biased toward using the homolog as a substrate rather than the sister chromatid. Pch2 is a conserved member of the AAA+-ATPase family of proteins and is implicated in a wide range of meiosis-specific processes including the recombination checkpoint, maturation of the chromosome axis, crossover control, and synapsis. We demonstrate a role for Pch2 in promoting and regulating interhomolog bias and the meiotic recombination checkpoint in response to unprocessed DSBs through the activation of axial proteins Hop1 and Mek1 in budding yeast. We show that Pch2 physically interacts with the putative BRCT repeats in the N-terminal region of Xrs2, a member of the MRX complex that acts at sites of unprocessed DSBs. Pch2, Xrs2, and the ATM ortholog Tel1 function in the same pathway leading to the phosphorylation of Hop1, independent of Rad17 and the ATR ortholog Mec1, which respond to the presence of single-stranded DNA. An N-terminal deletion of Xrs2 recapitulates the pch2Δ phenotypes for signaling unresected breaks. We propose that interaction with Xrs2 may enable Pch2 to remodel chromosome structure adjacent to the site of a DSB and thereby promote accessibility of Hop1 to the Tel1 kinase. In addition, Xrs2, like Pch2, is required for checkpoint-mediated delay conferred by the failure to synapse chromosomes

Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between Homologous Chromosomes

Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle

Replicative Age Induces Mitotic Recombination in the Ribosomal RNA Gene Cluster of Saccharomyces cerevisiae

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent

Structural Maintenance of Chromosomes (SMC) Proteins Promote Homolog-Independent Recombination Repair in Meiosis Crucial for Germ Cell Genomic Stability

In meiosis, programmed DNA breaks repaired by homologous recombination (HR) can be processed into inter-homolog crossovers that promote the accurate segregation of chromosomes. In general, more programmed DNA double-strand breaks (DSBs) are formed than the number of inter-homolog crossovers, and the excess DSBs must be repaired to maintain genomic stability. Sister-chromatid (inter-sister) recombination is postulated to be important for the completion of meiotic DSB repair. However, this hypothesis is difficult to test because of limited experimental means to disrupt inter-sister and not inter-homolog HR in meiosis. We find that the conserved Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins in Caenorhabditis elegans are required for the successful completion of meiotic homologous recombination repair, yet they appeared to be dispensable for accurate chromosome segregation in meiosis. Mutations in the smc-5 and smc-6 genes induced chromosome fragments and dismorphology. Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover. Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers. The mutant fragmentation defect appeared to be preferentially enhanced by the disruptions of inter-homolog recombination but not by the disruptions of inter-sister recombination. Based on these findings, we propose that the C. elegans SMC-5/6 proteins are required in meiosis for the processing of homolog-independent, presumably sister-chromatid-mediated, recombination repair. Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability