DNA polymerase α (swi7) and the flap endonuclease Fen1 (rad2) act together in the S-phase alkylation damage response in S. pombe.
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.
Division of Biomedical Cell Biology, Warwick Medical School, Gibbet Hill Campus, University of Warwick, Coventry, United Kingdom.
Milana
M
Koulintchenko
Sonya
S
Vengrova
Trevor
T
Eydmann
Prakash
P
Arumugam
Jacob Z
JZ
Dalgaard
D000477:Alkylating Agents / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects* / Q000528:radiation effects; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D042822:Genomic Instability; D006918:Hydroxyurea / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D000477:Alkylating Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C072981:SWI3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C513096:swi1 protein, S pombe; D008741:Methyl Methanesulfonate; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; C445562:Rad2 protein, S pombe; D006918:Hydroxyurea