Schizosaccharomyces pombe Dss1p is a DNA damage checkpoint protein that recruits Rad24p, Cdc25p, and Rae1p to DNA double-strand breaks.
Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light Deltadss1 cells entered mitosis prematurely with indistinguishable timing from Deltarad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G(2)/M boundary in response to DNA damage.
Selvanathan
Saravana P
SP
Basic Research Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
Thakurta
Anjan G
AG
Dhakshnamoorthy
Jothy
J
Zhou
Ming
M
Veenstra
Timothy D
TD
Dhar
Ravi
R
D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D018816:Genes, cdc; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays; D020666:ras-GRF1 / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; C508690:Dss1 protein, S pombe; D047908:Intracellular Signaling Peptides and Proteins; D034521:Nuclear Matrix-Associated Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C501114:rad24 protein, S pombe; C092953:rae1 protein, S pombe; D020666:ras-GRF1