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.

• 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

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

• MeSH
• buněčná diferenciace genetika   MeSH
• Caenorhabditis elegans genetika   růst a vývoj   MeSH
• DNA vazebné proteiny biosyntéza   genetika   MeSH
• mapy interakcí proteinů MeSH
• protein SUMO-1 genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce genetika   MeSH
• sumoylace * MeSH
• transkripční faktory biosyntéza   genetika   MeSH
• vulva cytologie   růst a vývoj   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

• MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA-polymerasa II genetika   metabolismus   MeSH
• DNA-polymerasa III genetika   metabolismus   MeSH
• homologní rekombinace * MeSH
• mutace genetika   MeSH
• nestabilita genomu MeSH
• oprava DNA genetika   účinky záření   MeSH
• poškození DNA genetika   účinky záření   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• protein SUMO-1 genetika   metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikace DNA genetika   účinky záření   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sumoylace MeSH
• ultrafialové záření škodlivé účinky   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

Cells exposed to genotoxic insults such as ionizing radiation activate a signaling cascade to repair the damaged DNA. Two recent articles published in Nature show that such genome maintenance requires modifications of tumor suppressor proteins BRCA1 and 53BP1 by the small ubiquitin-like modifier SUMO. Copyright (c) 2010 Elsevier Inc. All rights reserved.

• MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• nádory * genetika   metabolismus   MeSH
• oprava DNA * fyziologie   MeSH
• poškození DNA * fyziologie   MeSH
• protein BRCA1 metabolismus   MeSH
• protein SUMO-1 * metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata 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

• MeSH
• aktivní transport - buněčné jádro MeSH
• ATM protein MeSH
• biologické modely MeSH
• buněčná smrt MeSH
• buněčné jádro metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• kinasa I-kappa B * metabolismus   MeSH
• lidé MeSH
• nádorové supresorové proteiny * metabolismus   MeSH
• nádory patofyziologie   patologie   MeSH
• NF-kappa B metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA * MeSH
• protein SUMO-1 metabolismus   MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• ubikvitin metabolismus   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH