Schizosaccharomyces pombe Dss1p Is a DNA Damage Checkpoint Protein That Recruits Rad24p, Cdc25p, and Rae1p to DNA Double-strand Breaks (original) (raw)

2010, Journal of Biological Chemistry

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 ⌬dss1 cells entered mitosis prematurely with indistinguishable timing from ⌬rad24 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. Eukaryotic cells respond to double-strand breaks (DSBs) 3 within DNA by activating DNA damage checkpoint proteins that send signals to the cells, ultimately resulting in cell cycle arrest. The arrest allows DNA damage to be repaired by the proteins of the DNA repair pathway before cells can enter mitosis (1, 2). In Schizosaccharomyces pombe, entry into mitosis at the G 2 /M boundary is regulated by the phosphorylation status of the mitotic regulator Cdc2p at the tyrosine 15 residue (3). The cells are maintained in G 2 by phosphorylation of Cdc2p by Wee1p and Mik1p kinases, whereas their entry into mitosis is triggered by dephosphorylation of Cdc2p by the Cdc25p phosphatase (3). Dss1p, or its Saccharomyces cerevisiae homolog Sem1p, is a small acidic protein that is required for efficient DNA repair and the nuclear export of messenger RNA (mRNA) (4-7). Dss1p is a co-factor for human breast cancer susceptibility protein BRCA2 (8). In Ustilago maydis, a Dss1p homolog is a cofactor for Brh2p, a homolog of the human BRCA2 (9). The association between Dss1p and BRCA2 regulates the function of recombination-repair protein Rad51p (a homolog of the bacterial RecA; Rhp51p in S. pombe) (10, 11). So far the homologs of BRCA2/Brh2p have not been reported in either S. cerevisiae or in S. pombe. S. cerevisiae Sem1p was shown to recruit to DSB sites following a pattern similar to Rad51p, with high enrichment around the break site and gradually decreasing away from the break in both directions (12). Both Dss1p and Sem1p were shown to associate with the 19 S subunit of the 26 S proteasomes (12-14). It was suggested that the function of Sem1p involves regulating the function of the proteasome complex in DNA repair (12). So far the corresponding role of S. pombe Dss1p in DNA recombination-repair has not been studied. S. pombe Rad24p belongs to the 14-3-3 family of proteins that play a significant role as checkpoint factors in monitoring DNA damage in the G 2 phase of the cell cycle (15-18). Their role in the cell cycle was first demonstrated in S. pombe, where the products of the rad24 and rad25 genes were shown to possess a DNA damage checkpoint function (19). Neither the rad24 nor the rad25 gene is essential for growth, but simultaneous loss of both genes is lethal (19). The loss of rad24, but not rad25, leads to premature entry of cells into mitosis, resulting in small, round cells. In addition, the loss of rad24 renders cells highly sensitive to DNA-damaging agents, whereas a rad25 null strain is only modestly sensitive (19). In response to DNA damage in the G 2 stage, activated Chk1p kinase phosphorylates Cdc25p. Recently Mek1p was shown to phosphorylate Cdc25p independent of Chk1p (20). Rad24p binds phosphorylated Cdc25p and apparently blocks a nuclear localization signal within Cdc25p. The Rad24p-Cdc25p complex exits the nucleus by using a dedicated nuclear export pathway. It was originally suggested that the "nuclear exclusion" of Cdc25p prevents the