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