Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase

J Biol Chem. 2022 Jul;298(7):102092. doi: 10.1016/j.jbc.2022.102092. Epub 2022 May 30.

Abstract

Homologous recombination repairs DNA breaks and sequence gaps via the production of joint DNA intermediates such as Holliday junctions. Dissolving Holliday junctions into linear DNA repair products requires the activity of the Sgs1 helicase in yeast and of its homologs in other organisms. Recent studies suggest that the functions of these conserved helicases are regulated by sumoylation; however, the mechanisms that promote their sumoylation are not well understood. Here, we employed in vitro sumoylation systems and cellular assays to determine the roles of DNA and the scaffold protein Esc2 in Sgs1 sumoylation. We show that DNA binding enhances Sgs1 sumoylation in vitro. In addition, we demonstrate the Esc2's midregion (MR) with DNA-binding activity is required for Sgs1 sumoylation. Unexpectedly, we found that the sumoylation-promoting effect of Esc2-MR is DNA independent, suggesting a second function for this domain. In agreement with our biochemical data, we found the Esc2-MR domain, like its SUMO E2-binding C-terminal domain characterized in previous studies, is required for proficient sumoylation of Sgs1 and its cofactors, Top3 and Rmi1, in cells. Taken together, these findings provide evidence that while DNA binding enhances Sgs1 sumoylation, Esc2-based stimulation of this modification is mediated by two distinct domains.

Keywords: Esc2; Holiday junction dissolution; Sgs1; Sgs1–Top3–Rmi1 complex; sumoylation.

MeSH terms

  • Cell Cycle Proteins* / genetics
  • Cell Cycle Proteins* / metabolism
  • DNA, Cruciform / metabolism
  • DNA-Binding Proteins / metabolism
  • RecQ Helicases* / genetics
  • RecQ Helicases* / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Sumoylation

Substances

  • Cell Cycle Proteins
  • DNA, Cruciform
  • DNA-Binding Proteins
  • Esc2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • SGS1 protein, S cerevisiae
  • RecQ Helicases