Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.

• Klíčová slova
• Homologous recombination, Rad51, Rad52, Rad59, SUMOylation, Srs2,
• MeSH
• chromozomy hub genetika   MeSH
• DNA opravný a rekombinační protein Rad52 chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• homologní rekombinace * MeSH
• lysin metabolismus   MeSH
• mitóza genetika   MeSH
• poškození DNA MeSH
• proteinové domény MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• sumoylace * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA vazebné proteiny MeSH
• lysin MeSH
• RAD52 protein, S cerevisiae MeSH Prohlížeč
• RAD59 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

• MeSH
• adenosintrifosfatasy chemie   MeSH
• aminokyselinové motivy MeSH
• DNA primery metabolismus   MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• DNA metabolismus   MeSH
• enzymy opravy DNA chemie   metabolismus   MeSH
• konzervovaná sekvence MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny metabolismus   MeSH
• nestabilita genomu MeSH
• oprava DNA * MeSH
• párování chromozomů MeSH
• poškození DNA MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA primery MeSH
• DNA-helikasy MeSH
• DNA MeSH
• enzymy opravy DNA MeSH
• mutantní proteiny MeSH
• proliferační antigen buněčného jádra MeSH
• RAD54 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).

• MeSH
• delece genu MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• exodeoxyribonukleasy metabolismus   MeSH
• homologní rekombinace * MeSH
• jednovláknová DNA chemie   metabolismus   MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• replikační protein A metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• endodeoxyribonukleasy MeSH
• exodeoxyribonukleasy MeSH
• jednovláknová DNA MeSH
• křížová struktura DNA MeSH
• MRE11 protein, S cerevisiae MeSH Prohlížeč
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• RFA1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

DNA double-strand breaks (DSBs) comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR) constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO) peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

• Publikační typ
• časopisecké články MeSH

Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy.

• MeSH
• homologní rekombinace * MeSH
• lidé MeSH
• meióza MeSH
• nádory diagnóza   terapie   MeSH
• nemoc genetika   MeSH
• posttranslační úpravy proteinů MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikační protein A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• rekombinasa Rad51 MeSH
• replikační protein A MeSH

The error-free repair of double-strand DNA breaks by homologous recombination (HR) ensures genomic stability using undamaged homologous sequence to copy genetic information. While some of the aspects of the initial steps of HR are understood, the molecular mechanisms underlying events downstream of the D-loop formation remain unclear. Therefore, we have reconstituted D-loop-based in vitro recombination-associated DNA repair synthesis assay and tested the efficacy of polymerases Pol δ and Pol η to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol δ and Pol η extended up to 50% of the D-loop substrate, but differed in product length and dependency on proliferating cell nuclear antigen (PCNA). Mph1, but not Srs2 or Sgs1, displaced the extended primer very efficiently, supporting putative role of Mph1 in promoting the synthesis-dependent strand-annealing pathway. The experimental system described here can be employed to increase our understanding of HR events following D-loop formation, as well as the regulatory mechanisms involved.

• MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA-polymerasa III metabolismus   MeSH
• oprava DNA * MeSH
• rekombinace genetická * MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• DNA-helikasy MeSH
• DNA-polymerasa III MeSH

Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by the small ubiquitin-like modifier (SUMO) protein plays an important role in mitotic and meiotic recombination. However, the precise role of SUMOylation during recombination is still unclear. Here, we characterize the effect of SUMOylation on the biochemical properties of the Saccharomyces cerevisiae recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad52 foci in vivo and a shift in spontaneous mitotic recombination from single-strand annealing to gene conversion events in the SUMO-deficient Rad52 mutants. Taken together, our results highlight the importance of Rad52 SUMOylation as part of a 'quality control' mechanism regulating the efficiency of recombination and DNA repair.

• MeSH
• DNA opravný a rekombinační protein Rad52 chemie   metabolismus   MeSH
• jednovláknová DNA metabolismus   MeSH
• lysin metabolismus   MeSH
• oprava DNA * MeSH
• protein SUMO-1 metabolismus   MeSH
• rekombinace genetická * MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikační protein A metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA opravný a rekombinační protein Rad52 MeSH
• jednovláknová DNA MeSH
• lysin MeSH
• protein SUMO-1 MeSH
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• RAD52 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• replikační protein A 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
• odvolaná publikace MeSH
• práce podpořená grantem 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

The RAD51 gene was disrupted in three different parental wild-type strains to yield three rad51 null strains with different genetic background. The rad51 mutation sensitizes yeast cells to the toxic and mutagenic effects of H2O2, suggesting that Rad51-mediated repair, similarly to that of RecA-mediated, is relevant to the repair of oxidative damage in S. cerevisiae. Moreover, pulsed-field gel electrophoresis analysis demonstrated that increased sensitivity of the rad51 mutant to H2O2 is accompanied by its decreased ability to repair double-strand breaks induced by this agent. Our results show that ScRad51 protects yeast cells from H2O2-induced DNA double-strand breakage.

• MeSH
• DNA vazebné proteiny fyziologie   MeSH
• oprava DNA účinky léků   MeSH
• peroxid vodíku farmakologie   MeSH
• poškození DNA účinky léků   MeSH
• pulzní gelová elektroforéza MeSH
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Saccharomyces cerevisiae účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• peroxid vodíku MeSH
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny MeSH