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

DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.

• Keywords
• DNA damage tolerance, double-strand break repair, genomic instability, homologous recombination, replication fork maintenance,
• MeSH
• Exodeoxyribonucleases genetics   metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Mice MeSH
• Rad51 Recombinase biosynthesis   genetics   metabolism   MeSH
• DNA Replication * MeSH
• Transfection MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Exodeoxyribonucleases MeSH
• Phosphoproteins MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH
• TREX2 protein, human MeSH Browser

BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.

• Keywords
• Mus81 complex, Proliferating cell nuclear antigen, Recombination, Replication, Replication factor C,
• MeSH
• Flap Endonucleases genetics   metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Endonucleases genetics   metabolism   MeSH
• Proliferating Cell Nuclear Antigen genetics   metabolism   MeSH
• Recombination, Genetic MeSH
• DNA Replication MeSH
• Replication Protein C genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flap Endonucleases MeSH
• DNA-Binding Proteins MeSH
• Endonucleases MeSH
• MMS4 protein, S cerevisiae MeSH Browser
• MUS81 protein, S cerevisiae MeSH Browser
• POL30 protein, S cerevisiae MeSH Browser
• Proliferating Cell Nuclear Antigen MeSH
• Replication Protein C MeSH
• Saccharomyces cerevisiae Proteins MeSH

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

• Keywords
• Brca2, DNA replication, Mre11, Rad51, Xenopus laevis, fork protection,
• MeSH
• Time Factors MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• DNA Helicases genetics   metabolism   MeSH
• DNA Polymerase I metabolism   MeSH
• DNA Polymerase III metabolism   MeSH
• DNA biosynthesis   genetics   MeSH
• Endodeoxyribonucleases genetics   metabolism   MeSH
• Exodeoxyribonucleases genetics   metabolism   MeSH
• MRE11 Homologue Protein MeSH
• Humans MeSH
• Mutation MeSH
• Genomic Instability MeSH
• BRCA2 Protein genetics   metabolism   MeSH
• Xenopus Proteins genetics   metabolism   MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• DNA Replication * MeSH
• Replication Origin MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Xenopus laevis genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA Helicases MeSH
• DNA Polymerase I MeSH
• DNA Polymerase III MeSH
• DNA MeSH
• Endodeoxyribonucleases MeSH
• Exodeoxyribonucleases MeSH
• MRE11 Homologue Protein MeSH
• MRE11 protein, human MeSH Browser
• MRE11 protein, S cerevisiae MeSH Browser
• BRCA2 Protein MeSH
• Xenopus Proteins MeSH
• RAD51 protein, human MeSH Browser
• RAD51 protein, Xenopus MeSH Browser
• Rad51 Recombinase MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SMARCAL1 protein, human MeSH Browser

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

• Keywords
• DNA restriction enzymes, Domain interactions, E. coli, Molecular modeling, Multisubunit enzyme complex,
• Publication type
• Journal Article 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

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.

• Keywords
• DNA compaction, DNA-binding protein, HMG-box containing protein, Holliday junction, mitochondrial DNA (mtDNA), mitochondrial nucleoid,
• MeSH
• Candida genetics   metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Evolution, Molecular * MeSH
• High Mobility Group Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Yarrowia genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• High Mobility Group Proteins 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

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

• MeSH
• Adenosine Triphosphate chemistry   metabolism   MeSH
• DNA, Bacterial MeSH
• Escherichia coli genetics   metabolism   MeSH
• Exodeoxyribonuclease V chemistry   genetics   metabolism   MeSH
• Gene Expression MeSH
• Nucleic Acid Conformation MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Plasmids chemistry   metabolism   MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Protein Sorting Signals MeSH
• Escherichia coli Proteins chemistry   genetics   metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   metabolism   MeSH
• Signal Transduction MeSH
• DNA Cleavage MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA, Bacterial MeSH
• exodeoxyribonuclease V, E coli MeSH Browser
• Exodeoxyribonuclease V MeSH
• HsdR protein, E coli MeSH Browser
• Protein Subunits MeSH
• Protein Sorting Signals MeSH
• Escherichia coli Proteins MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH

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