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.

• MeSH
• Chromatin metabolism   MeSH
• DNA Helicases genetics   MeSH
• DNA biosynthesis   MeSH
• Methyl Methanesulfonate toxicity   MeSH
• Mutation MeSH
• Genomic Instability * MeSH
• DNA Damage * MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• Recombination, Genetic MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Structural Homology, Protein MeSH
• Suppression, Genetic MeSH
• Carrier Proteins chemistry   genetics   metabolism   MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA Helicases MeSH
• DNA MeSH
• Elg1 protein, S cerevisiae MeSH Browser
• Methyl Methanesulfonate MeSH
• POL30 protein, S cerevisiae MeSH Browser
• Proliferating Cell Nuclear Antigen MeSH
• RAD5 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Carrier Proteins MeSH

Cells use homology-dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology-based mechanisms involves nuclease-dependent DNA end resection, which generates long tracts of single-stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re-synthesis of resected DNA We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re-synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single-stranded gap and terminate further resection.

• Keywords
• PCNA, DNA re‐synthesis, Rad51, Srs2, recombination machinery,
• MeSH
• Models, Biological MeSH
• DNA metabolism   MeSH
• DNA Repair Enzymes metabolism   MeSH
• Homologous Recombination * MeSH
• DNA Damage * MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• Recombinational DNA Repair * MeSH
• Recombinases metabolism   MeSH
• Saccharomyces cerevisiae enzymology   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• DNA Repair Enzymes MeSH
• Proliferating Cell Nuclear Antigen MeSH
• Recombinases MeSH

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

• MeSH
• Amino Acid Motifs MeSH
• DNA-Binding Proteins chemistry   metabolism   physiology   MeSH
• DNA Polymerase III antagonists & inhibitors   MeSH
• DNA biosynthesis   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Recombinational DNA Repair * MeSH
• Ubiquitin-Protein Ligases physiology   MeSH
• Ultraviolet Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA Polymerase III MeSH
• DNA MeSH
• PARPBP protein, human MeSH Browser
• RAD18 protein, human MeSH Browser
• Ubiquitin-Protein Ligases MeSH

Srs2 plays many roles in DNA repair, the proper regulation and coordination of which is essential. Post-translational modification by small ubiquitin-like modifier (SUMO) is one such possible mechanism. Here, we investigate the role of SUMO in Srs2 regulation and show that the SUMO-interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of SIM in asrs2ΔPIMstrain leads to a decrease in recombination, indicating a pro-recombination role of SUMO. Thus SIM has an ambivalent function in Srs2 regulation; it not only mediates interaction with SUMO-PCNA to promote the anti-recombination function but it also plays a PCNA-independent pro-recombination role, probably by stimulating the formation of recombination complexes. The fact that deletion of PIM suppresses the phenotypes of Srs2 lacking SIM suggests that proper balance between the anti-recombination PCNA-bound and pro-recombination pools of Srs2 is crucial. Notably, sumoylation of Srs2 itself specifically stimulates recombination at the rDNA locus.

• Keywords
• DNA repair, homologous recombination, proliferating cell nuclear antigen (PCNA), protein-protein interaction, small ubiquitin-like modifier (SUMO),
• MeSH
• Amino Acid Motifs MeSH
• DNA, Fungal genetics   metabolism   MeSH
• DNA Helicases genetics   metabolism   MeSH
• DNA Repair physiology   MeSH
• Proliferating Cell Nuclear Antigen genetics   metabolism   MeSH
• SUMO-1 Protein genetics   metabolism   MeSH
• Recombination, Genetic physiology   MeSH
• DNA, Ribosomal genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Sumoylation physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA Helicases MeSH
• Proliferating Cell Nuclear Antigen MeSH
• SUMO-1 Protein MeSH
• DNA, Ribosomal MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SRS2 protein, S cerevisiae MeSH Browser

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• Replication Protein C * MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Comment MeSH
• News MeSH
• Names of Substances
• Replication Protein C * MeSH
• Saccharomyces cerevisiae Proteins MeSH

Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.

• Keywords
• DNA damage tolerance, SUMO, genotoxic stress, recombination, replication,
• MeSH
• Point Mutation MeSH
• Chromatin metabolism   MeSH
• DNA Helicases genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• DNA Damage genetics   MeSH
• Cell Cycle Proteins MeSH
• Recombination, Genetic genetics   MeSH
• Rad51 Recombinase metabolism   MeSH
• DNA Replication genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   genetics   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA Helicases MeSH
• Esc2 protein, S cerevisiae MeSH Browser
• Nuclear Proteins MeSH
• Cell Cycle Proteins MeSH
• RAD51 protein, S cerevisiae MeSH Browser
• Rad51 Recombinase MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SRS2 protein, S cerevisiae MeSH Browser

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

• MeSH
• Flap Endonucleases physiology   MeSH
• DNA Primers MeSH
• DNA-Binding Proteins physiology   MeSH
• DNA Helicases physiology   MeSH
• Endonucleases physiology   MeSH
• Microscopy, Fluorescence MeSH
• Polymerase Chain Reaction MeSH
• Recombination, Genetic * MeSH
• Saccharomyces cerevisiae Proteins physiology   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Base Sequence MeSH
• Two-Hybrid System Techniques MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Flap Endonucleases MeSH
• DNA Primers MeSH
• DNA-Binding Proteins MeSH
• DNA Helicases MeSH
• Endonucleases MeSH
• MMS4 protein, S cerevisiae MeSH Browser
• MUS81 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• SRS2 protein, S cerevisiae MeSH Browser

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

• MeSH
• Adenosine Triphosphatases chemistry   MeSH
• Amino Acid Motifs MeSH
• DNA Primers metabolism   MeSH
• DNA Helicases chemistry   metabolism   MeSH
• DNA metabolism   MeSH
• DNA Repair Enzymes chemistry   metabolism   MeSH
• Conserved Sequence MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Mutation genetics   MeSH
• DNA Mutational Analysis MeSH
• Mutant Proteins metabolism   MeSH
• Genomic Instability MeSH
• DNA Repair * MeSH
• Chromosome Pairing MeSH
• DNA Damage MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• Recombination, Genetic * MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• DNA Primers MeSH
• DNA Helicases MeSH
• DNA MeSH
• DNA Repair Enzymes MeSH
• Mutant Proteins MeSH
• Proliferating Cell Nuclear Antigen MeSH
• RAD54 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.

• Keywords
• D-loop, DNA repair synthesis, Homologous recombination, Reconstitution, TLS polymerases,
• MeSH
• DNA-Directed DNA Polymerase chemistry   physiology   MeSH
• DNA Polymerase III chemistry   physiology   MeSH
• DNA Polymerase iota MeSH
• HeLa Cells MeSH
• Homologous Recombination * MeSH
• DNA, Single-Stranded biosynthesis   MeSH
• Humans MeSH
• Osmolar Concentration MeSH
• DNA Damage MeSH
• Proliferating Cell Nuclear Antigen chemistry   physiology   MeSH
• RNA-Binding Protein FUS chemistry   physiology   MeSH
• Rad51 Recombinase chemistry   MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• DNA Polymerase III MeSH
• DNA Polymerase iota MeSH
• DNA, Single-Stranded MeSH
• POLI protein, human MeSH Browser
• POLK protein, human MeSH Browser
• Proliferating Cell Nuclear Antigen MeSH
• RNA-Binding Protein FUS MeSH
• Rad30 protein MeSH Browser
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH

DNA double-strand breaks (DSBs) comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR) constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO) peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

• Publication type
• Journal Article MeSH