Involvement of p29 in DNA replication and DNA damage response

• Main Authors: Po-Chen Chu, 朱伯振
• Other Authors: Mau-Sun Chang
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/65284301917900492500

博士 === 臺灣大學 === 生化科學研究所 === 98 === Human p29 protein was initially identified as a GCIP (Grab2 cyclin D interacting protein)-interaction protein and its function is largely undetermined. In this study, we found that p29 associated with chromatin, interacted with MCM3, and localized to DNA replication foci in the S phase. Silencing of p29 by siRNAs (small interfering RNAs) reduced DNA synthesis accompanied with the decreased expression of MCM3 and DNA polymerase α. By contrast, an increased expression of p107, a member of Rb (retinoblastoma) family, and of cyclin-dependent kinase inhibitor p21 was detected after p29 knockdown. Lethal events consisting of premature chromatin condensation with a reduced Chk1 phosphorylation were also observed in p29-depleted cells in response to UV irradiation. Intriguingly, the phosphorylation of ATM (ataxia telangectasia-mutated) kinase at S1981 was suppressed in p29-depleted HeLa cells with UV irradiation, but not in hydroxyurea- and ionizing radiation-treated cells. In addition to defects in checkpoint signaling, depletion of p29 also compromises the monoubiquitination of FANCI and FANCD2 (FA ID complex) in Fanconi anemia (FA) pathway, which sensitize cells to DNA crosslinking drug treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. We found the increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11-Ra50-NBS1 and ATRIP in p29 stably expressing cells. The increased checkpoint activity preserves the genetic integrity and protects p29 stably expressing cells from DNA damage and apoptosis. Monoubiquitination of FA ID complex was restored with increased chromatin-associated FANCL in p29 stably expressing FA-G cells. However, the stable expression of p29 in Fanconi anemia complementation group D2 (FA-D2) cells did not complement the sensitivity to DNA crosslinking drug treatment, suggesting that p29 may play a role upstream of FA ID complex. Furthermore, lower tumor incidence was observed in mp29 transgenic mice after UV-irradiation, suggesting that mp29 transgene protected these mice from UV irradiation and supporting the results in p29 stably expressing cells. These results provide evidence that p29 not only participates in the replication reaction but also in the DNA damage response.