BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

• Keywords
• DNA-protein cross-links repair, MUS81, TDP1, Topoisomerase 1,
• MeSH
• DNA-Binding Proteins * genetics   metabolism   MeSH
• DNA Topoisomerases, Type I genetics   metabolism   MeSH
• Endonucleases * genetics   metabolism   MeSH
• Phosphoric Diester Hydrolases * genetics   metabolism   MeSH
• DNA Repair MeSH
• DNA Damage MeSH
• Saccharomyces cerevisiae Proteins * genetics   metabolism   MeSH
• Saccharomyces cerevisiae MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• DNA Topoisomerases, Type I MeSH
• Endonucleases * MeSH
• Phosphoric Diester Hydrolases * MeSH
• MUS81 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins * MeSH
• Tdp1 protein, S cerevisiae MeSH Browser
• TOP1 protein, S cerevisiae MeSH Browser

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

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

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres.

• MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Candida genetics   metabolism   MeSH
• DNA, Fungal genetics   metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression MeSH
• Genome, Fungal * MeSH
• Genome, Mitochondrial * MeSH
• Telomere Homeostasis MeSH
• Cloning, Molecular MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Protein Multimerization MeSH
• Mutation MeSH
• Gene Expression Regulation, Fungal * MeSH
• Recombination, Genetic MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Telomere chemistry   metabolism   MeSH
• Genetic Complementation Test MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA-Binding Proteins MeSH
• MGM101 protein, S cerevisiae MeSH Browser
• Mitochondrial Proteins MeSH
• Recombinant Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.

• MeSH
• Survival Analysis MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Endonucleases genetics   metabolism   MeSH
• DNA Repair * MeSH
• DNA Damage * MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae cytology   genetics   metabolism   MeSH
• Sumoylation 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-Binding Proteins MeSH
• Endonucleases MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Saw1 protein, S cerevisiae MeSH Browser

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

• MeSH
• DNA metabolism   MeSH
• Endonucleases chemistry   genetics   metabolism   MeSH
• DNA Repair Enzymes chemistry   genetics   metabolism   MeSH
• Intracellular Signaling Peptides and Proteins physiology   MeSH
• Lysine metabolism   MeSH
• Mutation MeSH
• DNA Repair * MeSH
• DNA Damage MeSH
• Protein Serine-Threonine Kinases physiology   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   physiology   MeSH
• Sumoylation * MeSH
• Ubiquitin-Protein Ligases physiology   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 MeSH
• Endonucleases MeSH
• DNA Repair Enzymes MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Lysine MeSH
• MEC1 protein, S cerevisiae MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• RAD1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Siz1 protein, S cerevisiae MeSH Browser
• Siz2 protein, S cerevisiae MeSH Browser
• Ubiquitin-Protein Ligases MeSH

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

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

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