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

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

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

• MeSH
• DNA Helicases genetics   metabolism   MeSH
• DNA Polymerase II genetics   metabolism   MeSH
• DNA Polymerase III genetics   metabolism   MeSH
• Homologous Recombination * MeSH
• Mutation genetics   MeSH
• Genomic Instability MeSH
• DNA Repair genetics   radiation effects   MeSH
• DNA Damage genetics   radiation effects   MeSH
• Proliferating Cell Nuclear Antigen genetics   metabolism   MeSH
• SUMO-1 Protein genetics   metabolism   MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• DNA Replication genetics   radiation effects   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Sumoylation MeSH
• Ultraviolet Rays adverse effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• DNA Polymerase II MeSH
• DNA Polymerase III MeSH
• Proliferating Cell Nuclear Antigen MeSH
• SUMO-1 Protein MeSH
• RAD51 protein, S cerevisiae MeSH Browser
• Rad51 Recombinase MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SRS2 protein, S cerevisiae MeSH Browser

The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).

• MeSH
• Gene Deletion MeSH
• DNA Helicases genetics   metabolism   MeSH
• Endodeoxyribonucleases metabolism   MeSH
• Exodeoxyribonucleases metabolism   MeSH
• Homologous Recombination * MeSH
• DNA, Single-Stranded chemistry   metabolism   MeSH
• DNA, Cruciform chemistry   metabolism   MeSH
• Rad51 Recombinase metabolism   MeSH
• DNA Replication * MeSH
• Replication Protein A metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• Endodeoxyribonucleases MeSH
• Exodeoxyribonucleases MeSH
• DNA, Single-Stranded MeSH
• DNA, Cruciform MeSH
• MRE11 protein, S cerevisiae MeSH Browser
• RAD51 protein, S cerevisiae MeSH Browser
• Rad51 Recombinase MeSH
• Replication Protein A MeSH
• RFA1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• SRS2 protein, S cerevisiae MeSH Browser

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation--sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.

• MeSH
• DNA Helicases chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Lysine metabolism   MeSH
• Molecular Sequence Data MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• SUMO-1 Protein metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   MeSH
• Amino Acid Sequence MeSH
• Sumoylation * MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA Helicases MeSH
• Lysine MeSH
• Proliferating Cell Nuclear Antigen MeSH
• SUMO-1 Protein MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Siz1 protein, S cerevisiae MeSH Browser
• Siz2 protein, S cerevisiae MeSH Browser
• SRS2 protein, S cerevisiae MeSH Browser
• Ubiquitin-Protein Ligases MeSH

Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy.

• MeSH
• Homologous Recombination * MeSH
• Humans MeSH
• Meiosis MeSH
• Neoplasms diagnosis   therapy   MeSH
• Disease genetics   MeSH
• Protein Processing, Post-Translational MeSH
• Rad51 Recombinase metabolism   MeSH
• Replication Protein A metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Rad51 Recombinase MeSH
• Replication Protein A MeSH

The error-free repair of double-strand DNA breaks by homologous recombination (HR) ensures genomic stability using undamaged homologous sequence to copy genetic information. While some of the aspects of the initial steps of HR are understood, the molecular mechanisms underlying events downstream of the D-loop formation remain unclear. Therefore, we have reconstituted D-loop-based in vitro recombination-associated DNA repair synthesis assay and tested the efficacy of polymerases Pol δ and Pol η to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol δ and Pol η extended up to 50% of the D-loop substrate, but differed in product length and dependency on proliferating cell nuclear antigen (PCNA). Mph1, but not Srs2 or Sgs1, displaced the extended primer very efficiently, supporting putative role of Mph1 in promoting the synthesis-dependent strand-annealing pathway. The experimental system described here can be employed to increase our understanding of HR events following D-loop formation, as well as the regulatory mechanisms involved.

• MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA Helicases metabolism   MeSH
• DNA Polymerase III metabolism   MeSH
• DNA Repair * MeSH
• Recombination, Genetic * MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• DNA Helicases MeSH
• DNA Polymerase III MeSH

Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by the small ubiquitin-like modifier (SUMO) protein plays an important role in mitotic and meiotic recombination. However, the precise role of SUMOylation during recombination is still unclear. Here, we characterize the effect of SUMOylation on the biochemical properties of the Saccharomyces cerevisiae recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad52 foci in vivo and a shift in spontaneous mitotic recombination from single-strand annealing to gene conversion events in the SUMO-deficient Rad52 mutants. Taken together, our results highlight the importance of Rad52 SUMOylation as part of a 'quality control' mechanism regulating the efficiency of recombination and DNA repair.

• MeSH
• Rad52 DNA Repair and Recombination Protein chemistry   metabolism   MeSH
• DNA, Single-Stranded metabolism   MeSH
• Lysine metabolism   MeSH
• DNA Repair * MeSH
• SUMO-1 Protein metabolism   MeSH
• Recombination, Genetic * MeSH
• Rad51 Recombinase metabolism   MeSH
• Replication Protein A metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Rad52 DNA Repair and Recombination Protein MeSH
• DNA, Single-Stranded MeSH
• Lysine MeSH
• SUMO-1 Protein MeSH
• RAD51 protein, S cerevisiae MeSH Browser
• RAD52 protein, S cerevisiae MeSH Browser
• Rad51 Recombinase MeSH
• Replication Protein A MeSH
• Saccharomyces cerevisiae Proteins MeSH