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
• 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
• 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

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• "flap" endonukleasy metabolismus   genetika   MeSH
• DEAD-box RNA-helikasy * metabolismus   genetika   MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• DNA-helikasy * metabolismus   genetika   MeSH
• DNA-ligasa ATP metabolismus   genetika   MeSH
• DNA metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• lidé MeSH
• multifunkční enzymy * metabolismus   genetika   MeSH
• R-smyčka * MeSH
• replikace DNA * MeSH
• RNA-helikasy * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DEAD-box RNA-helikasy * MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy * MeSH
• DNA-ligasa ATP MeSH
• DNA MeSH
• endonukleasy * MeSH
• multifunkční enzymy * MeSH
• MUS81 protein, human MeSH Prohlížeč
• RNA-helikasy * MeSH
• SETX 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 spoje 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 spoje 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

R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.

• MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA metabolismus   MeSH
• endonukleasy metabolismus   MeSH
• lidé MeSH
• R-smyčka * MeSH
• replikace DNA * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDX17 protein, human MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endonukleasy MeSH

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• bodová mutace MeSH
• helikasy RecQ genetika   metabolismus   ultrastruktura   MeSH
• homologní rekombinace * MeSH
• hydrolýza MeSH
• jednovláknová DNA metabolismus   ultrastruktura   MeSH
• kinetika MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• missense mutace MeSH
• molekulární motory metabolismus   ultrastruktura   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikační protein A metabolismus   MeSH
• substrátová specifita MeSH
• zobrazení jednotlivé molekuly * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfát MeSH
• helikasy RecQ MeSH
• jednovláknová DNA MeSH
• molekulární motory MeSH
• RAD51 protein, human MeSH Prohlížeč
• RECQL5 protein, human MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• RPA1 protein, human MeSH Prohlížeč

RECQ5 belongs to the RecQ family of DNA helicases. It is conserved from Drosophila to humans and its deficiency results in genomic instability and cancer susceptibility in mice. Human RECQ5 is known for its ability to regulate homologous recombination by disrupting RAD51 nucleoprotein filaments. It also binds to RNA polymerase II (RNAPII) and negatively regulates transcript elongation by RNAPII. Here, we summarize recent studies implicating RECQ5 in the prevention and resolution of transcription-replication conflicts, a major intrinsic source of genomic instability during cancer development.

• Klíčová slova
• DNA repair, R-loops, RECQ5, genomic instability, replication stress, transcription-replication conflicts,
• MeSH
• DNA genetika   metabolismus   MeSH
• genetická transkripce genetika   MeSH
• helikasy RecQ genetika   metabolismus   fyziologie   MeSH
• lidé MeSH
• nestabilita genomu MeSH
• replikace DNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• helikasy RecQ MeSH