Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

• MeSH
• chromozomální nestabilita MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• helikasy RecQ * metabolismus   genetika   MeSH
• homeostáza telomer MeSH
• lidé MeSH
• mutace MeSH
• replikace DNA * MeSH
• Rothmundův-Thomsonův syndrom * genetika   metabolismus   MeSH
• segregace chromozomů MeSH
• telomery * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• endonukleasy * MeSH
• helikasy RecQ * MeSH
• MUS81 protein, human MeSH Prohlížeč
• RECQL4 protein, human MeSH Prohlížeč

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 (genetika) * MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• homologní rekombinace MeSH
• resolvasy Hollidayova spojení metabolismus   genetika   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• endonukleasy * MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• resolvasy Hollidayova spojení MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Yen1 protein, S cerevisiae MeSH Prohlížeč

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

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.

• Klíčová slova
• Brca2, DNA replication, Mre11, Rad51, Xenopus laevis, fork protection,
• MeSH
• časové faktory MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA-polymerasa I metabolismus   MeSH
• DNA-polymerasa III metabolismus   MeSH
• DNA biosyntéza   genetika   MeSH
• endodeoxyribonukleasy genetika   metabolismus   MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• homologní protein MRE11 MeSH
• lidé MeSH
• mutace MeSH
• nestabilita genomu MeSH
• protein BRCA2 genetika   metabolismus   MeSH
• proteiny Xenopus genetika   metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikace DNA * MeSH
• replikační počátek MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Xenopus laevis genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• DNA-polymerasa I MeSH
• DNA-polymerasa III MeSH
• DNA MeSH
• endodeoxyribonukleasy MeSH
• exodeoxyribonukleasy MeSH
• homologní protein MRE11 MeSH
• MRE11 protein, human MeSH Prohlížeč
• MRE11 protein, S cerevisiae MeSH Prohlížeč
• protein BRCA2 MeSH
• proteiny Xenopus MeSH
• RAD51 protein, human MeSH Prohlížeč
• RAD51 protein, Xenopus MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SMARCAL1 protein, human MeSH Prohlížeč

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
• chromatidy chemie   metabolismus   MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• jaderné proteiny chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• malé modifikační proteiny související s ubikvitinem chemie   genetika   metabolismus   MeSH
• nestabilita genomu MeSH
• poškození DNA MeSH
• proteinové domény MeSH
• proteiny buněčného cyklu MeSH
• regulace genové exprese u hub * MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• replikace DNA MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA fungální MeSH
• DNA vazebné proteiny MeSH
• endonukleasy MeSH
• Esc2 protein, S cerevisiae MeSH Prohlížeč
• jaderné proteiny MeSH
• křížová struktura DNA MeSH
• malé modifikační proteiny související s ubikvitinem MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny 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.

• Klíčová slova
• DNA repair, homologous recombination, proliferating cell nuclear antigen (PCNA), protein-protein interaction, small ubiquitin-like modifier (SUMO),
• MeSH
• aminokyselinové motivy MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• oprava DNA fyziologie   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• protein SUMO-1 genetika   metabolismus   MeSH
• rekombinace genetická fyziologie   MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• sumoylace fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• DNA-helikasy MeSH
• proliferační antigen buněčného jádra MeSH
• protein SUMO-1 MeSH
• ribozomální DNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč