DNA polymerase α (swi7) and the flap endonuclease Fen1 (rad2) act together in the S-phase alkylation damage response in S. pombe

PLoS One. 2012;7(10):e47091. doi: 10.1371/journal.pone.0047091. Epub 2012 Oct 11.

Abstract

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylating Agents / pharmacology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • DNA Damage / drug effects*
  • DNA Damage / radiation effects
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA Repair / genetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Genomic Instability
  • Hydroxyurea / pharmacology
  • Methyl Methanesulfonate / pharmacology
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • S Phase / genetics
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Schizosaccharomyces / cytology
  • Schizosaccharomyces / drug effects
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Ultraviolet Rays

Substances

  • Alkylating Agents
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Nuclear Proteins
  • SWI3 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • swi1 protein, S pombe
  • Methyl Methanesulfonate
  • DNA Polymerase I
  • Endodeoxyribonucleases
  • Rad2 protein, S pombe
  • Hydroxyurea

Grant support

This work was supported by Marie Curie Cancer Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.