Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

• Klíčová slova
• Genetics, Molecular biology, Molecular interaction, Properties of biomolecules,
• Publikační typ
• časopisecké články 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

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" endonukleasy fyziologie   MeSH
• DNA primery MeSH
• DNA vazebné proteiny fyziologie   MeSH
• DNA-helikasy fyziologie   MeSH
• endonukleasy fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• polymerázová řetězová reakce MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence nukleotidů MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DNA primery MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• endonukleasy MeSH
• MMS4 protein, S cerevisiae MeSH Prohlížeč
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation--sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.

• MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lysin metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• protein SUMO-1 metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• sekvence aminokyselin MeSH
• sumoylace * MeSH
• ubikvitinligasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA-helikasy MeSH
• lysin MeSH
• proliferační antigen buněčného jádra MeSH
• protein SUMO-1 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Siz1 protein, S cerevisiae MeSH Prohlížeč
• Siz2 protein, S cerevisiae MeSH Prohlížeč
• SRS2 protein, S cerevisiae MeSH Prohlížeč
• ubikvitinligasy MeSH

Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed.

• MeSH
• DNA fungální metabolismus   MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• lidé MeSH
• nestabilita genomu MeSH
• oprava DNA * MeSH
• replikace DNA MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA fungální MeSH
• DNA-helikasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

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