DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death. Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination. We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle. We show that cells arrested at the G 1 phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ. Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.

No abstract

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.

Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. We have screened to isolate yeast mutants exhibiting increased levels of recombination between repeated sequences. Here we characterize one of these mutants, elg1. Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats. These strains also exhibit increased levels of chromosome loss. The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18. Elg1p forms a complex with the Rfc2-5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18. Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses.T he processes of DNA replication, repair, and recombination are intimately linked. During DNA replication, the activity of the DNA polymerases may be impaired by the presence of secondary structures, bound proteins, or DNA lesions. This may lead to stalling and even collapse of replication forks. In response, cellular mechanisms are activated that arrest cell cycle progression, induce DNA repair, and restore replication (1).Replication factor C (RFC), a five-subunit protein complex, associates with the DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) and loads it onto DNA. PCNA tethers the polymerase to the DNA template and serves as a central platform to load many enzymes involved in the replication, repair, and modification of DNA. Recently, two alternative RFC-like protein complexes (RLCs) have been described in the yeast Saccharomyces cerevisiae and in other organisms, which include the Rfc2-5 subunits but not the Rfc1 protein. In one of these RLCs, the large RFC subunit is replaced by the checkpoint protein Rad24 (Rad17 in Schizosaccharomyces pombe and hRad17 humans) (2, 3). This complex is predicted to load an alternative DNA sliding clamp (4) composed of checkpoint proteins (5). In the second RLC, the large subunit of RFC is replaced by the Ctf18͞Chl12 protein, implicated in sister chromatid cohesion (6-8). It is not yet clear whether this alternative complex interacts with PCNA or with a yet-to-befound alternative clamp.The yeast S. cerevisiae has been extensively used to identify and dissect the response to DNA damage. These studies have demonstrated that a network of overlapping pathways operates to maintain genomic stability (9, 10). Checkpoint control failure and elevated levels of genomic instability are a hallmark of cancer cells (1).Homologous recombination is one of the main mechanisms able to restore replication competence to cells with stalled replication forks. Accordingly, mutations th...

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5 and 3, are rapidly degraded. This is not due to the inability to recombine, since the 3 single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.The process of recombination plays an essential role during meiosis and in DNA repair during vegetative growth. Doublestrand breaks (DSBs) arise frequently as a consequence of exposure to external insults or as a direct result of natural cell metabolism. Recombinational repair of DSBs is important in solving collapsed replication forks during DNA replication (20). If left unrepaired, DSBs result in broken chromosomes, genetic alterations, or cell death. Repair of DSBs (DSBR) takes place in eukaryotes by two competing processes: nonhomologous end joining and homologous recombination. In yeast cells, homologous recombination is the prevalent mechanism used (reviewed in reference 36).In a classic model for DSB-initiated recombination (53) (Fig. 1A), single-stranded degradation of the broken DNA molecule generates protruding 3Ј-OH ends that can invade homologous regions, creating a D-loop. The invasion process yields regions of heteroduplex DNA (hDNA) that may contain mismatches. The invading 3Ј end is then used to prime DNA synthesis. Eventually, the displaced donor strand pairs with single-stranded DNA (ssDNA) from the opposing end of the DSB, also serving as a template for DNA synthesis. Ligation results in the formation of a structure containing two Holliday junctions, which can be resolved to yield either crossover or noncrossover products. Mismatch repair of the hDNA may result in gene conversion events (Fig. 1A, left).An alternative model for gene conversion, termed the synthesis-dependent strand-annealing (SDSA) model (32), proposes that after DSB formation and resection, a single 3Ј single-stranded end invades the intact homologous template. DNA synthesis is followed by reannealing of the newly synthesized DNA with the opposite broken arm. In the basic version of this model (Fig. 1A, center), only gene conversion, and not crossover, can be obtained, although variations allowing crossing-over have been also proposed (reviewed in reference 36).Homologous recombination is catalyzed by a number of proteins encoded mostly by genes of the RAD52 epistasis group (36, 51). ...

No abstract

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.

Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.Homologous recombination is a universal process that plays a role in generating diversity during meiosis and is an important DNA repair mechanism in vegetative cells. Recombination results in the transfer of genetic information from one DNA molecule to a homologous one (gene conversion) and in the reciprocal exchange of DNA fragments between chromosomes (crossing over). The association between gene conversion and crossing-over events has led to the assumption that they are mechanistically related (Refs. 1-5; Fig. 1). One of the characteristic features of most eukaryotic genomes is the presence of large amounts of repetitive DNA. Reciprocal recombination between dispersed repeats may result in chromosomal aberrations, such as deletions, translocations, etc., that can affect the reproductive fitness of an organism or lead to cancer. Therefore, to maintain the genome integrity, crossing over must be prevented during recombinational repair of DNA lesions involving dispersed repeats. Double-strand breaks (DSBs) 1 in the DNA of living organisms occur as a consequence of the natural cell metabolism, or they can be created by exogenous sources such as chemical agents or radiation. If left unrepaired, DSBs result in broken chromosomes and cell death (6). Mitotic recombination plays an important role in the repair of this damage. In addition, DSBs are generated during certain developmental processes such as meiosis (7) and mating-type switch in yeast (8). In different experimental systems, it was found that the level of association between gene conversion and crossing over varies, from no coupling (e.g. mating-type switch (8 -10) or recombination between direct repeats (11)) to a level of association of 70% (5). In two of the currently held models of recombination, the synthesisdependent strand annealing (SDSA) model (12) and the DSB repair model (4), recombination is initiated by the creation of a DSB in one of the two participating DNA duplexes (Fig. 1). Although the mechanism suggested by the SDS...