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

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.

• MeSH
• DNA-Binding Proteins genetics   MeSH
• DNA Breaks, Double-Stranded MeSH
• Endodeoxyribonucleases genetics   MeSH
• Endonucleases genetics   MeSH
• Exodeoxyribonucleases genetics   MeSH
• Phosphorylation MeSH
• Humans MeSH
• DNA End-Joining Repair genetics   MeSH
• DNA Repair genetics   MeSH
• DNA Damage genetics   MeSH
• Solubility MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae MeSH
• Sumoylation genetics   MeSH
• Check Tag
• Humans 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
• Endodeoxyribonucleases MeSH
• Endonucleases MeSH
• Exodeoxyribonucleases MeSH
• MRE11 protein, S cerevisiae MeSH Browser
• RAD50 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• SAE2 protein, S cerevisiae MeSH Browser
• XRS2 protein, S cerevisiae MeSH Browser

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