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

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

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• forkhead transkripční faktory metabolismus   MeSH
• genetické matrice MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• poškození DNA * MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• replikační protein A metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• DNA MeSH
• forkhead transkripční faktory MeSH
• FOXN2 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• RAD5 protein, S cerevisiae MeSH Prohlížeč
• RAD54L protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• 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

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

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