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

Milana Koulintchenko; Sonya Vengrova; Trevor Eydmann; Prakash Arumugam; Jacob Z Dalgaard

doi:10.1371/journal.pone.0047091

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.

Authors

Milana Koulintchenko¹, Sonya Vengrova, Trevor Eydmann, Prakash Arumugam, Jacob Z Dalgaard

Affiliation

¹ Division of Biomedical Cell Biology, Warwick Medical School, Gibbet Hill Campus, University of Warwick, Coventry, United Kingdom.

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

Grants and funding

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.