PCNA Unloading Is Crucial for the Bypass of DNA Lesions Using Homologous Recombination.

DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells to progress normally through the cell cycle in the face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex, plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitates an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyze the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that, in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair.

A Role for the Interactions between Polδ and PCNA Revealed by Analysis of pol3-01 Yeast Mutants.

Several DNA polymerases participate in DNA synthesis during genome replication and DNA repair. PCNA, a homotrimeric ring, acts as a processivity factor for DNA polymerases. PCNA also acts as a "landing pad" for proteins that interact with chromatin and DNA at the moving fork. The interaction between PCNA and polymerase delta (Polδ) is mediated by PIPs (PCNA-interacting peptides), in particular the one on Pol32, a regulatory subunit of Polδ. Here, we demonstrate that pol3-01, an exonuclease mutant of Polδ's catalytic subunit, exhibits a weak interaction with Pol30 compared to the WT DNA polymerase. The weak interaction activates DNA bypass pathways, leading to increased mutagenesis and sister chromatid recombination. Strengthening pol3-01's weak interaction with PCNA suppresses most of the phenotypes. Our results are consistent with a model in which Pol3-01 tends to detach from the chromatin, allowing an easier replacement of Polδ by the trans-lesion synthesis polymerase Zeta (Polz), thus leading to the increased mutagenic phenotype.

DNA damage bypass pathways and their effect on mutagenesis in yeast.

What is the origin of mutations? In contrast to the naïve notion that mutations are unfortunate accidents, genetic research in microorganisms has demonstrated that most mutations are created by genetically encoded error-prone repair mechanisms. However, error-free repair pathways also exist, and it is still unclear how cells decide when to use one repair method or the other. Here, we summarize what is known about the DNA damage tolerance mechanisms (also known as post-replication repair) for perhaps the best-studied organism, the yeast Saccharomyces cerevisiae. We describe the latest research, which has established the existence of at least two error-free and two error-prone inter-related mechanisms of damage tolerance that compete for the handling of spontaneous DNA damage. We explore what is known about the induction of mutations by DNA damage. We point to potential paradoxes and to open questions that still remain unanswered.

How yeast cells deal with stalled replication forks.

DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.

Telomeres and stress in yeast cells: When genes and environment interact.

Telomeres are structures composed of simple DNA repeats and specific proteins that protect the eukaryotic chromosomal ends from degradation, and facilitate the replication of the genome. They are central to the maintenance of the genome integrity, and play important roles in the development of cancer and in the process of aging in humans. The yeast Saccharomyces cerevisiae has greatly contributed to our understanding of basic telomere biology. Our laboratory has carried out systematic screen for mutants that affect telomere length, and identified ∼500 genes that, when mutated, affect telomere length. Remarkably, all ∼500 TLM (Telomere Length Maintenance) genes participate in a very tight homeostatic process, and it is enough to mutate one of them to change the steady-state telomere length. Despite this complex network of balances, it is also possible to change telomere length in yeast by applying several types of external stresses. We summarize our insights about the molecular mechanisms by which genes and environment interact to affect telomere length.

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae.

During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism ("salvage recombination") that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

The Yeast PCNA Unloader Elg1 RFC-Like Complex Plays a Role in Eliciting the DNA Damage Checkpoint.

The PCNA (proliferating cell nuclear antigen) ring plays central roles during DNA replication and repair. The yeast Elg1 RFC-like complex (RLC) is the principal unloader of chromatin-bound PCNA and thus plays a central role in maintaining genome stability. Here we identify a role for Elg1 in the unloading of PCNA during DNA damage. Using DNA damage checkpoint (DC)-inducible and replication checkpoint (RC)-inducible strains, we show that Elg1 is essential for eliciting the signal in the DC branch. In the absence of Elg1 activity, the Rad9 (53BP1) and Dpb11 (TopBP1) adaptor proteins are recruited but fail to be phosphorylated by Mec1 (ATR), resulting in a lack of checkpoint activation. The chromatin immunoprecipitation of PCNA at the Lac operator sites reveals that accumulated local PCNA influences the checkpoint activation process in elg1 mutants. Our data suggest that Elg1 participates in a mechanism that may coordinate PCNA unloading during DNA repair with DNA damage checkpoint induction.IMPORTANCE The Elg1protein forms an RFC-like complex in charge of unloading PCNA from chromatin during DNA replication and repair. Mutations in the ELG1 gene caused genomic instability in all organisms tested and cancer in mammals. Here we show that Elg1 plays a role in the induction of the DNA damage checkpoint, a cellular response to DNA damage. We show that this defect is due to a defect in the signal amplification process during induction. Thus, cells coordinate the cell's response and the PCNA unloading through the activity of Elg1.

Tight Regulation of Srs2 Helicase Activity Is Crucial for Proper Functioning of DNA Repair Mechanisms.

Proper DNA damage repair is one of the most vital and fundamental functions of every cell. Several different repair mechanisms exist to deal with various types of DNA damage, in various stages of the cell cycle and under different conditions. Homologous recombination is one of the most important repair mechanisms in all organisms. Srs2, a regulator of homologous recombination, is a DNA helicase involved in DNA repair, cell cycle progression and genome integrity. Srs2 can remove Rad51 from ssDNA, and is thought to inhibit unscheduled recombination. However, Srs2 has to be precisely regulated, as failure to do so is toxic and can lead to cell death. We noticed that a very slight elevation of the levels of Srs2 (by addition of a single extra copy of the SRS2 gene) leads to hyper-sensitivity of yeast cells to methyl methanesulfonate (MMS, a DNA damaging agent). This effect is seen in haploid, but not in diploid, cells. We analyzed the mechanism that controls haploid/diploid sensitivity and arrived to the conclusion that the sensitivity requires the activity of RAD59 and RDH54, whose expression in diploid cells is repressed. We carried out a mutational analysis of Srs2 to determine the regions of the protein required for the sensitization to genotoxins. Interestingly, Srs2 needs the HR machinery and its helicase activity for its toxicity, but does not need to dismantle Rad51. Our work underscores the tight regulation that is required on the levels of Srs2 activity, and the fact that Srs2 helicase activity plays a more central role in DNA repair than the ability of Srs2 to dismantle Rad51 filaments.

A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA.

A structure-function analysis of the yeast Elg1 protein reveals the importance of PCNA unloading in genome stability maintenance.

The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1-RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin.

Regulation of Elg1 activity by phosphorylation.

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.

Elg1, a central player in genome stability.

ELG1 is a conserved gene uncovered in a number of genetic screens in yeast aimed at identifying factors important in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice, acts as a tumor suppressor in mice and humans, exhibits physical interactions with components of the human Fanconi Anemia pathway and may be responsible for some of the phenotypes associated with neurofibromatosis. In this review, we summarize the information available on Elg1-related activities in yeast and mammals, and present models to explain how the different phenotypes observed in the absence of Elg1 activity are related.

Effect of nuclear architecture on the efficiency of double-strand break repair.

The most dangerous insults to the genome's integrity are those that break both strands of the DNA. Double-strand breaks can be repaired by homologous recombination; in this conserved mechanism, a global genomic homology search finds sequences similar to those near the break, and uses them as a template for DNA synthesis and ligation. Chromosomes occupy restricted territories within the nucleus. We show that yeast genomic regions whose nuclear territories overlap recombine more efficiently than sequences located in spatially distant territories. Tethering of telomeres and centromeres reduces the efficiency of recombination between distant genomic loci, lowering the chances of non-allelic recombination. Our results challenge present models that posit an active scanning of the whole nuclear volume by the broken chromosomal end; they demonstrate that the search for homology is a limiting step in homologous recombination, and emphasize the importance of nuclear organization in genome maintenance.

A genetic screen for high copy number suppressors of the synthetic lethality between elg1Δ and srs2Δ in yeast.

Elg1 and Srs2 are two proteins involved in maintaining genome stability in yeast. After DNA damage, the homotrimeric clamp PCNA, which provides stability and processivity to DNA polymerases and serves as a docking platform for DNA repair enzymes, undergoes modification by the ubiquitin-like molecule SUMO. PCNA SUMOylation helps recruit Srs2 and Elg1 to the replication fork. In the absence of Elg1, both SUMOylated PCNA and Srs2 accumulate at the chromatin fraction, indicating that Elg1 is required for removing SUMOylated PCNA and Srs2 from DNA. Despite this interaction, which suggests that the two proteins work together, double mutants elg1Δ srs2Δ have severely impaired growth as haploids and exhibit synergistic sensitivity to DNA damage and a synergistic increase in gene conversion. In addition, diploid elg1Δ srs2Δ double mutants are dead, which implies that an essential function in the cell requires at least one of the two gene products for survival. To gain information about this essential function, we have carried out a high copy number suppressor screen to search for genes that, when overexpressed, suppress the synthetic lethality between elg1Δ and srs2Δ. We report the identification of 36 such genes, which are enriched for functions related to DNA- and chromatin-binding, chromatin packaging and modification, and mRNA export from the nucleus.

Genetic and physical interactions between the yeast ELG1 gene and orthologs of the Fanconi anemia pathway.

Fanconi anemia (FA) is a human syndrome characterized by genomic instability and increased incidence of cancer. FA is a genetically heterogeneous disease caused by mutations in at least 15 different genes; several of these genes are conserved in the yeast Saccharomyces cerevisiae. Elg1 is also a conserved protein that forms an RFC-like complex, which interacts with SUMOylated PCNA. The mammalian Elg1 protein has been recently found to interact with the FA complex. Here we analyze the genetic interactions between elg1Δ and mutants of the yeast FA-like pathway. We show that Elg1 physically contacts the Mhf1/Mhf2 histone-like complex and genetically interacts with MPH1 (ortholog of the FANCM helicase) and CHL1 (ortholog of the FANCJ helicase) genes. We analyze the sensitivity of double, triple, quadruple and quintuple mutants to methylmethane sulfonate (MMS) and to hydroxyurea (HU). Our results show that genetic interactions depend on the type of DNA damaging agent used and show a hierarchy: Chl1 and Elg1 play major roles in the survival to these genotoxins and exhibit synthetic fitness reduction. Mph1 plays a lesser role, and the effect of the Mhf1/2 complex is seen only in the absence of Elg1 on HU-containing medium. Finally, we dissect the relationship between yeast FA-like mutants and the replication clamp, PCNA. Our results point to an intricate network of interactions rather than a single, linear repair pathway.

Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins.

PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent.

The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage.

DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.

Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA.

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.

Analysis of repair mechanism choice during homologous recombination.

Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.