Development of methods for the study of the role of the Smc5-Smc6 complex in DNA stability

Abstract

The Structural Maintenance of Chromosomes (SMC) proteins play a number of crucial roles in the metabolism of chromosomes. The Smc5-Smc6 complex is the least well understood of the complexes formed by SMC proteins. Hitherto, the Smc5-Smc6 complex has been linked to protein post-translational modification by sumoylation and restart of collapsed replication forks by homologous recombination between sister chromatids (SCR). However, a detailed characterization of the roles of the Smc5-Smc6 complex is missing. The objective of this study is to characterize the function of the Smc5-Smc6 complex in DNA repair by SCR, and to identify sumoylation substrates of MMS21, a E3-sumoligase subunit of the Smc5-Smc6 complex. Recent studies suggest that DNA single-strand nicks are transformed to doublestrand breaks in a replication-dependent manner, and this triggers SCR. I developed an assay for the activation of SCR based on the expression of a site-specific nickase. Unfortunately, a stable site-specific nick was observed in only 30% of the population. This percentage was insufficient for the study of the molecular role of the Smc5-Smc6 complex during SCR. However, this assay could be used to confirm and further characterize the activation of SCR upon replication-induced DNA damage. To study the role of sumoylation within the Smc5-Smc6 activity, I have developed a proteome-wide approach for the in vivo identification of sumoylation-sites by mass spectrometry. This technique can be used for the identification of MMS21 substrates and for the mapping of their sumo-acceptor lysines. The mapping of sumo-acceptor sites allows the generation of sumo-specific mutant proteins that can be used to study the function of sumoylation. More than 360 sumo-acceptor lysines, belonging to 245 different proteins, were identified. In vivo sumoylation at these lysines was verified by MS-independent methods. In addition, I developed a SILAC-based mass spectrometry assay for the quantitative study of site-specific sumoylation.