Abstract
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.
Highlights
Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA
We suggest that the recruitment of Cdc25p to the double-strand break (DSB) site could lead to cell cycle arrest at the G2/M boundary in S. pombe cells in response to DNA damage
Relative fold enrichment of Mts2p was observed in the vicinity of the DSB site similar to the recruitment pattern observed for Dss1p in the strain expressing the homothallic switching (HO)-endonuclease. These results demonstrate that Dss1p, Rhp51p, and Mts2p are recruited preferentially surrounding the region of a DSB site introduced into the S. pombe cells, consistent with observations made with their homologs [12]
Summary
Strains and Culture—Basic genetic and cell culture techniques have been described previously [30, 31]. The rad24-tap strain was constructed by techniques described previously [32, 33], in which TAP is fused to the rad gene at the C terminus by homologous recombination using the kanamycin gene as a selectable marker. Purification of TAP-tagged proteins was performed by published methods using IgG-Sepharose beads [34]. The cleaved fraction was bound to calmodulin beads, and the bound proteins were eluted by calmodulin elution buffer and precipitated by trichloroacetic acid [34]. Genotypes hϪ leu ura4-D18 hϪ leu ura4-D18 rae hϪ leu ura4-D18 ade704 rae rad24::ura4ϩ hϪ leu ura4-D18 ade704 rad24::ura4ϩ hϪ leu ura4-D18 rad24-TAP hϩ leuϩ ura4ϩ rad hϩ leu ura4-D18 adeϪ dss1::kan hϪ leu ura4-D18 cdc25-GFPint cdc25::ura4ϩ hϪ leu1-32::2xYFP-crb2ϩ -leu1ϩ ura4D18. Source Ref. 25 Ref. 23 This study Ref. 19 This study Ref. Ref. 5 Ref. Ref. 29
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