Helicases and endonucleases play crucial roles in genome maintenance by unwinding or cleaving various forms of DNA and RNA structures in order to facilitate essential biological processes, such as DNA replication and recombination. Here, we identified fission yeast Dbl2 as a potential interactor of several complexes that exhibit either helicase or endonuclease activity, namely Fml1-MHF, SCFFbh1, Rqh1-Top3-Rmi1, and Mus81-Eme1. In vitro, Dbl2 binds to DNA, with a preference for branched molecules, such as D-loops, mobile Holliday junctions, and fork structures, making it a good candidate to play a central role in modulating the activity of helicases and endonucleases during replication and recombination repair. Previously, we showed that Dbl2 recruits Fbh1 to the ongoing homologous recombination sites, affecting the Rad51-nucleofilament. In this study, we determined that deleting dbl2 in an fbh1Δ background did not increase sensitivity to DNA-damaging agents or the frequency of Tf2 ectopic recombination. Therefore, Dbl2 and Fbh1 might be involved in the same molecular pathway, maintaining genome integrity by hindering ectopic recombination at repetitive elements.

• Keywords
• Schizosaccharomyces pombe, DNA repair, Dbl2, Helicases, Homologous recombination,
• MeSH
• DNA, Fungal metabolism   genetics   MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA Helicases * metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• DNA Damage MeSH
• Repetitive Sequences, Nucleic Acid * MeSH
• DNA Replication MeSH
• Schizosaccharomyces pombe Proteins * metabolism   genetics   MeSH
• Schizosaccharomyces * genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA-Binding Proteins MeSH
• DNA Helicases * MeSH
• Endonucleases * MeSH
• Schizosaccharomyces pombe Proteins * MeSH

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

• MeSH
• Crossing Over, Genetic * MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• Homologous Recombination MeSH
• Holliday Junction Resolvases metabolism   genetics   MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• Endonucleases * MeSH
• MUS81 protein, S cerevisiae MeSH Browser
• Holliday Junction Resolvases MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Yen1 protein, S cerevisiae MeSH Browser

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

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids - E162 and D317 - in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

• Keywords
• Cell biology, Structural biology,
• Publication type
• Journal Article MeSH

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Escherichia coli drug effects   genetics   radiation effects   MeSH
• Hydrogen-Ion Concentration MeSH
• Nalidixic Acid pharmacology   MeSH
• Microbial Viability drug effects   radiation effects   MeSH
• Genomic Instability drug effects   genetics   radiation effects   MeSH
• DNA Damage drug effects   genetics   radiation effects   MeSH
• Propidium pharmacology   MeSH
• Flow Cytometry MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Rifampin pharmacology   MeSH
• Ultraviolet Rays MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Nalidixic Acid MeSH
• Propidium MeSH
• Rifampin MeSH

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

Mitochondrial nucleoids consist of several different groups of proteins, many of which are involved in essential cellular processes such as the replication, repair and transcription of the mitochondrial genome. The eukaryotic, ATP-dependent protease Lon is found within the central nucleoid region, though little is presently known about its role there. Aside from its association with mitochondrial nucleoids, human Lon also specifically interacts with RNA. Recently, Lon was shown to regulate TFAM, the most abundant mtDNA structural factor in human mitochondria. To determine whether Lon also regulates other mitochondrial nucleoid- or ribosome-associated proteins, we examined the in vitro digestion profiles of the Saccharomyces cerevisiae TFAM functional homologue Abf2, the yeast mtDNA maintenance protein Mgm101, and two human mitochondrial proteins, Twinkle helicase and the large ribosomal subunit protein MrpL32. Degradation of Mgm101 was also verified in vivo in yeast mitochondria. These experiments revealed that all four proteins are actively degraded by Lon, but that three of them are protected from it when bound to a nucleic acid; the Twinkle helicase is not. Such a regulatory mechanism might facilitate dynamic changes to the mitochondrial nucleoid, which are crucial for conducting mitochondrial functions and maintaining mitochondrial homeostasis.

• MeSH
• Enzyme Activation MeSH
• DNA-Binding Proteins metabolism   MeSH
• Humans MeSH
• DNA, Mitochondrial metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Protease La metabolism   MeSH
• Proteolysis MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Protein Transport MeSH
• Protein Binding 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, Mitochondrial MeSH
• Mitochondrial Proteins MeSH
• Protease La MeSH

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.

• MeSH
• Chromatids chemistry   metabolism   MeSH
• DNA, Fungal genetics   metabolism   MeSH
• DNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Endonucleases chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Nuclear Proteins chemistry   genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• DNA, Cruciform chemistry   metabolism   MeSH
• Small Ubiquitin-Related Modifier Proteins chemistry   genetics   metabolism   MeSH
• Genomic Instability MeSH
• DNA Damage MeSH
• Protein Domains MeSH
• Cell Cycle Proteins MeSH
• Gene Expression Regulation, Fungal * MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• DNA Replication MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA-Binding Proteins MeSH
• Endonucleases MeSH
• Esc2 protein, S cerevisiae MeSH Browser
• Nuclear Proteins MeSH
• DNA, Cruciform MeSH
• Small Ubiquitin-Related Modifier Proteins MeSH
• MUS81 protein, S cerevisiae MeSH Browser
• Cell Cycle Proteins MeSH
• Recombinant Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH

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

Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.

• Keywords
• DNA repair, Mph1, Mte1, genome integrity, homologous recombination, telomere maintenance,
• MeSH
• Crossing Over, Genetic genetics   MeSH
• DEAD-box RNA Helicases genetics   metabolism   MeSH
• Gene Deletion MeSH
• Stress, Physiological genetics   MeSH
• Telomere Homeostasis genetics   MeSH
• Telomere-Binding Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DEAD-box RNA Helicases MeSH
• MPH1 protein, S cerevisiae MeSH Browser
• Mte1 protein, S cerevisiae MeSH Browser
• Telomere-Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH