Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability.

• Keywords
• DNA, Genomic instability,
• MeSH
• DNA, Fungal genetics   metabolism   MeSH
• DNA Helicases chemistry   genetics   metabolism   MeSH
• Phosphorylation MeSH
• Kinetics MeSH
• Metabolic Networks and Pathways MeSH
• DNA Damage MeSH
• Protein Domains MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• DNA Replication MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   genetics   growth & development   MeSH
• Enzyme Stability MeSH
• Sumoylation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA Helicases MeSH
• DNA2 protein, S cerevisiae MeSH Browser
• Recombinant Fusion Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Siz2 protein, S cerevisiae MeSH Browser

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

Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.

• Keywords
• Homologous recombination, Rad51, Rad52, Rad59, SUMOylation, Srs2,
• MeSH
• Chromosomes, Fungal genetics   MeSH
• Rad52 DNA Repair and Recombination Protein chemistry   metabolism   MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• Homologous Recombination * MeSH
• Lysine metabolism   MeSH
• Mitosis genetics   MeSH
• DNA Damage MeSH
• Protein Domains MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae cytology   genetics   metabolism   MeSH
• Sumoylation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Rad52 DNA Repair and Recombination Protein MeSH
• DNA-Binding Proteins MeSH
• Lysine MeSH
• RAD52 protein, S cerevisiae MeSH Browser
• RAD59 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins 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

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

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

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