SUMO
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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
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
Small ubiquitin-related modifier-2/3 (SUMO-2/3) is a member of the ubiquitin-like (Ubl) protein family. Conjugation of SUMO-2/3 to target proteins is influenced by various stress conditions and chemical inhibitors. SUMO-2/3 conjugation may serve as a neuroprotective mechanism and may play a role in protein quality control. A method for screening global changes in SUMO-2/3 conjugation would facilitate further research of SUMO-2/3 cellular function. Here we show that dot blot with immunochemical detection allows evaluation of changes in global cellular SUMO-2/3 conjugation and offers an alternative to more laborious Western blot analysis. The method is based on a change of SUMO-2/3 signal intensity upon its conjugation. The dot blot analysis presented here is a time-saving method that enables screening of large numbers of samples and easy statistical evaluation of the results.
- MeSH
- HEK293 buňky MeSH
- imunoblotting metody MeSH
- lidé MeSH
- malé modifikační proteiny související s ubikvitinem * analýza chemie metabolismus MeSH
- ubikvitiny * analýza chemie metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- 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
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
Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.
- MeSH
- benzochinony farmakologie MeSH
- biologické modely MeSH
- cykloheximid farmakologie MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- makrocyklické laktamy farmakologie MeSH
- malé modifikační proteiny související s ubikvitinem metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteosyntéza účinky léků MeSH
- puromycin farmakologie MeSH
- reakce na tepelný šok účinky léků MeSH
- sumoylace účinky léků MeSH
- ubikvitiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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
- 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
Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.
- MeSH
- bodová mutace MeSH
- chromatin metabolismus MeSH
- DNA-helikasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- poškození DNA genetika MeSH
- rekombinace genetická genetika MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikace DNA genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In vitro analysis of posttranslational modifications such as sumoylation provides a great tool to not only identify the target proteins but also to characterize the specific effects of this modification on the protein features and uncover possible regulatory mechanism. In this chapter, we will describe the purification of yeast SUMO machinery proteins and their use to identify SUMO modification of target proteins in vitro. Furthermore, we will show several examples characterizing the effect of sumoylation on the biochemical activities of various proteins involved in homologous recombination (HR) that helped to better understand the regulatory role of this modification.
- MeSH
- Escherichia coli genetika růst a vývoj metabolismus MeSH
- homologní rekombinace * MeSH
- komplexy ubikvitinligas metabolismus MeSH
- malé modifikační proteiny související s ubikvitinem metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- rekombinantní proteiny izolace a purifikace MeSH
- sumoylace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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