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.

• Klíčová slova
• DNA-protein cross-links repair, MUS81, TDP1, Topoisomerase 1,
• MeSH
• DNA vazebné proteiny * genetika   metabolismus   MeSH
• DNA-topoisomerasy I genetika   metabolismus   MeSH
• endonukleasy * genetika   metabolismus   MeSH
• fosfodiesterasy * genetika   metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• DNA-topoisomerasy I MeSH
• endonukleasy * MeSH
• fosfodiesterasy * MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny * MeSH
• Tdp1 protein, S cerevisiae MeSH Prohlížeč
• TOP1 protein, S cerevisiae MeSH Prohlížeč

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.

• Klíčová slova
• Mus81 complex, Proliferating cell nuclear antigen, Recombination, Replication, Replication factor C,
• MeSH
• "flap" endonukleasy genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• rekombinace genetická MeSH
• replikace DNA MeSH
• replikační protein C genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DNA vazebné proteiny MeSH
• endonukleasy MeSH
• MMS4 protein, S cerevisiae MeSH Prohlížeč
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• POL30 protein, S cerevisiae MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• replikační protein C MeSH
• Saccharomyces cerevisiae - proteiny MeSH

The Saccharomyces cerevisiae Mus81.Mms4 protein complex, a DNA structure-specific endonuclease, helps preserve genomic integrity by resolving pathological DNA structures that arise from damaged or aborted replication forks and may also play a role in the resolution of DNA intermediates arising through homologous recombination. Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes. However, the functional significance of this novel interaction remains unknown. Here, using highly purified S. cerevisiae proteins, we show that Rad54 strongly stimulates the Mus81.Mms4 nuclease activity on a broad range of DNA substrates. This nuclease enhancement does not require ATP binding nor its hydrolysis by Rad54. We present evidence that Rad54 acts by targeting the Mus81.Mms4 complex to its DNA substrates. In addition, we demonstrate that the Rad54-mediated enhancement of the Mus81.Mms4 (Eme1) nuclease function is evolutionarily conserved. We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3. These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

• MeSH
• "flap" endonukleasy MeSH
• adenosintrifosfatasy MeSH
• DNA fungální biosyntéza   genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy MeSH
• endonukleasy genetika   metabolismus   MeSH
• enzymy opravy DNA MeSH
• genom fungální fyziologie   MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• nestabilita genomu fyziologie   MeSH
• rekombinace genetická fyziologie   MeSH
• replikace DNA fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• publikace stažené z tisku MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• adenosintrifosfatasy MeSH
• DNA fungální MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• endonukleasy MeSH
• enzymy opravy DNA MeSH
• helikasy RecQ MeSH
• MMS4 protein, S cerevisiae MeSH Prohlížeč
• multienzymové komplexy MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• RAD54 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SGS1 protein, S cerevisiae MeSH Prohlížeč
• SNF2 protein, S cerevisiae MeSH Prohlížeč
• TOP3 protein, S cerevisiae MeSH Prohlížeč
• trans-aktivátory MeSH
• transkripční faktory MeSH