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.

Pivotal roles of PCNA loading and unloading in heterochromatin function.

In Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the proliferating cell nuclear antigen (PCNA) unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively, these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.

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.

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Alternative clamp loaders/unloaders.

Each time a cell duplicates, the whole genome must be accurately copied and distributed. The enormous amount of DNA in eukaryotic cells requires a high level of coordination between polymerases and other DNA and chromatin-interacting proteins to ensure timely and accurate DNA replication and chromatin formation. PCNA forms a ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and as a landing platform for different proteins that interact with DNA and chromatin. It thus serves as a signaling hub and influences the rate and accuracy of DNA replication, the r-formation of chromatin in the wake of the moving fork and the proper segregation of the sister chromatids. Four different, conserved, protein complexes are in charge of loading/unloading PCNA and similar molecules onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA, the replication clamp, during S-phase. The Rad24, Ctf18 and Elg1 proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast.

The cohesin complex of yeasts: sister chromatid cohesion and beyond.

Each time a cell divides, it needs to duplicate the genome and then separate the two copies. In eukaryotes, which usually have more than one linear chromosome, this entails tethering the two newly replicated DNA molecules, a phenomenon known as sister chromatid cohesion (SCC). Cohesion ensures proper chromosome segregation to separate poles during mitosis. SCC is achieved by the presence of the cohesin complex. Besides its canonical function, cohesin is essential for chromosome organization and DNA damage repair. Surprisingly, yeast cohesin is loaded in G1 before DNA replication starts but only acquires its binding activity during DNA replication. Work in microorganisms, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe has greatly contributed to the understanding of cohesin composition and functions. In the last few years, much progress has been made in elucidating the role of cohesin in chromosome organization and compaction. Here, we discuss the different functions of cohesin to ensure faithful chromosome segregation and genome stability during the mitotic cell division in yeast. We describe what is known about its composition and how DNA replication is coupled with SCC establishment. We also discuss current models for the role of cohesin in chromatin loop extrusion and delineate unanswered questions about the activity of this important, conserved complex.

S. cerevisiae Cells Can Grow without the Pds5 Cohesin Subunit.

During DNA replication, the newly created sister chromatids are held together until their separation at anaphase. The cohesin complex is in charge of creating and maintaining sister chromatid cohesion (SCC) in all eukaryotes. In Saccharomyces cerevisiae cells, cohesin is composed of two elongated proteins, Smc1 and Smc3, bridged by the kleisin Mcd1/Scc1. The latter also acts as a scaffold for three additional proteins, Scc3/Irr1, Wpl1/Rad61, and Pds5. Although the HEAT-repeat protein Pds5 is essential for cohesion, its precise function is still debated. Deletion of the ELG1 gene, encoding a PCNA unloader, can partially suppress the temperature-sensitive pds5-1 allele, but not a complete deletion of PDS5. We carried out a genetic screen for high-copy-number suppressors and another for spontaneously arising mutants, allowing the survival of a pds5Δ elg1Δ strain. Our results show that cells remain viable in the absence of Pds5 provided that there is both an elevation in the level of Mcd1 (which can be due to mutations in the CLN2 gene, encoding a G1 cyclin), and an increase in the level of SUMO-modified PCNA on chromatin (caused by lack of PCNA unloading in elg1Δ mutants). The elevated SUMO-PCNA levels increase the recruitment of the Srs2 helicase, which evicts Rad51 molecules from the moving fork, creating single-stranded DNA (ssDNA) regions that serve as sites for increased cohesin loading and SCC establishment. Thus, our results delineate a double role for Pds5 in protecting the cohesin ring and interacting with the DNA replication machinery. IMPORTANCE Sister chromatid cohesion is vital for faithful chromosome segregation, chromosome folding into loops, and gene expression. A multisubunit protein complex known as cohesin holds the sister chromatids from S phase until the anaphase stage. In this study, we explore the function of the essential cohesin subunit Pds5 in the regulation of sister chromatid cohesion. We performed two independent genetic screens to bypass the function of the Pds5 protein. We observe that Pds5 protein is a cohesin stabilizer, and elevating the levels of Mcd1 protein along with SUMO-PCNA accumulation on chromatin can compensate for the loss of the PDS5 gene. In addition, Pds5 plays a role in coordinating the DNA replication and sister chromatid cohesion establishment. This work elucidates the function of cohesin subunit Pds5, the G1 cyclin Cln2, and replication factors PCNA, Elg1, and Srs2 in the proper regulation of sister chromatid cohesion.

PCNA Loaders and Unloaders-One Ring That Rules Them All.

During each cell duplication, the entirety of the genomic DNA in every cell must be accurately and quickly copied. Given the short time available for the chore, the requirement of many proteins, and the daunting amount of DNA present, DNA replication poses a serious challenge to the cell. A high level of coordination between polymerases and other DNA and chromatin-interacting proteins is vital to complete this task. One of the most important proteins for maintaining such coordination is PCNA. PCNA is a multitasking protein that forms a homotrimeric ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and acts as a landing platform for different proteins interacting with DNA and chromatin. Therefore, PCNA is a signaling hub that influences the rate and accuracy of DNA replication, regulates DNA damage repair, controls chromatin formation during the replication, and the proper segregation of the sister chromatids. With so many essential roles, PCNA recruitment and turnover on the chromatin is of utmost importance. Three different, conserved protein complexes are in charge of loading/unloading PCNA onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA during the S-phase. The Ctf18 and Elg1 (ATAD5 in mammalian) proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast and mammals.

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.

PCNA promotes context-specific sister chromatid cohesion establishment separate from that of chromatin condensation.

Cellular genomes undergo various structural changes that include cis tethering (the tethering together of two loci within a single DNA molecule), which promotes chromosome condensation and transcriptional activation, and trans tethering (the tethering together of two DNA molecules), which promotes sister chromatid cohesion and DNA repair. The protein complex termed cohesin promotes both cis and trans forms of DNA tethering, but the extent to which these cohesin functions occur in temporally or spatially defined contexts remains largely unknown. Prior studies indicate that DNA polymerase sliding clamp PCNA recruits cohesin acetyltransferase Eco1, suggesting that sister chromatid cohesion is established in the context of the DNA replication fork. In support of this model, elevated levels of PCNA rescue the temperature growth and cohesion defects exhibited by eco1 mutant cells. Here, we test whether Eco1-dependent chromatin condensation is also promoted in the context of this DNA replication fork component. Our results reveal that overexpressed PCNA does not promote DNA condensation in eco1 mutant cells, even though Smc3 acetylation levels are increased. We further provide evidence that replication fork-associated E3 ligase impacts on Eco1 are more complex that previously described. In combination, the data suggests that Eco1 acetylates Smc3 and thus promotes sister chromatid cohesion in context of the DNA replication fork, whereas a distinct cohesin population participates in chromatin condensation outside the context of the DNA replication fork.

The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast.

Protein-DNA complexes can impede DNA replication and cause replication fork collapse. Whilst it is known that homologous recombination is deployed in such instances to restart replication, it is unclear how a stalled fork transitions into a collapsed fork at which recombination proteins can load. Previously we established assays in Schizosaccharomyces pombe for studying recombination induced by replication fork collapse at the site-specific protein-DNA barrier RTS1 (Nguyen et al., 2015). Here, we provide evidence that efficient recruitment/retention of two key recombination proteins (Rad51 and Rad52) to RTS1 depends on unloading of the polymerase sliding clamp PCNA from DNA by Elg1. We also show that, in the absence of Elg1, reduced recombination is partially suppressed by deleting fbh1 or, to a lesser extent, srs2, which encode known anti-recombinogenic DNA helicases. These findings suggest that PCNA unloading by Elg1 is necessary to limit Fbh1 and Srs2 activity, and thereby enable recombination to proceed.

Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

Is PCNA unloading the central function of the Elg1/ATAD5 replication factor C-like complex?

Maintaining genome stability is crucial for all cells. The budding yeast Elg1 protein, the major subunit of a replication factor C-like complex, is important for genome stability, since cells lacking Elg1 exhibit increased recombination and chromosomal rearrangements. This genome maintenance function of Elg1 seems to be conserved in higher eukaryotes, since removal of the human Elg1 homolog, encoded by the ATAD5 gene, also causes genome instability leading to tumorigenesis. The fundamental molecular function of the Elg1/ATAD5-replication factor C-like complex (RLC) was, until recently, elusive, although Elg1/ATAD5-RLC was known to interact with the replication sliding clamp PCNA. Two papers have now reported that following DNA replication, the Elg1/ATAD5-RLC is required to remove PCNA from chromatin in yeast and human cells. In this Review, we summarize the evidence that Elg1/ATAD5-RLC acts as a PCNA unloader and discuss the still enigmatic relationship between the function of Elg1/ATAD5-RLC in PCNA unloading and the role of Elg1/ATAD5 in maintaining genomic stability.

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.