核蛋白p29參與DNA複製及損害反應之探討

Abstract

人類p29蛋白質是一個與細胞週期調控蛋白質GCIP有作用，但功能仍舊未知的細胞核蛋白質。本論文主要是探討這一個新發現，且功能未知的細胞核蛋白質p29的細胞功能及所扮演的角色。從我們的研究中發現，p29蛋白屬於可與染色質(chromatin)結合的蛋白質，並可與MCM3蛋白質作用，並且在細胞週期的DNA合成期(S phase)時會與DNA複製的複合體聚集。為了進一步探究p29蛋白的細胞功能，我們利用siRNAs抑制p29蛋白的表現後，p107及p21兩個蛋白質表現增加，但卻也發現MCM3及DNA合成酶α (DNA polymerase α) 的表現量下降，因而導致細胞的DNA合成降低。若進一步使用紫外線(UV)照射p29蛋白被抑制的HeLa細胞時，細胞會因缺少p29蛋白而使檢查點(checkpoint)蛋白質Chk1的磷酸化及活性降低，而無法阻擋細胞進入有絲分裂(mitosis)，因此造成所謂PCC (premature chromatin condensation)而死亡。此外，利用紫外線(UV)照射缺少p29蛋白的細胞時，我們發現細胞因缺少p29蛋白的表現而影響了與DNA損害修復有關的ATM蛋白質在絲氨酸1981的磷酸化，也因此可能影響細胞DNA損害修復的活性。除了影響DNA損害檢查點的訊息傳遞外，p29蛋白被抑制時也造成FANCI及FANCD2蛋白質的monoubiquitination現象減少，因而增加細胞對DNA crosslinking藥物的敏感性。 為了進一步研究p29蛋白質的生理功能，我們也分別利用U2OS細胞及缺乏FANCG蛋白的細胞 (FA-G cell)，建立p29蛋白持續表現的細胞株。在p29蛋白持續表現的U2OS細胞株中，我們發現結合在染色質上的Mre11-Rad50-NBS1及ATRIP蛋白質皆有增多的情形，而且伴隨著檢查點蛋白質Chk1及Chk2的磷酸化明顯增加，代表提供細胞修復DNA損害及保護細胞染色體的能力大幅增加。另一方面，在p29蛋白持續表現的FA-G細胞株中，因p29蛋白的增加促使結合在染色質上的FANCL蛋白質增加，使細胞對FANCI及FANCD2蛋白質進行monoubiquitination的能力得以回復，因而降低對DNA crosslinking藥物的敏感性。但在缺乏FANCD2蛋白質的FA-D2細胞中，就算p29蛋白質持續表現，仍無法降低FA-D2細胞對DNA crosslinking藥物的敏感性，因此推測p29蛋白可能具有調控FANCI及FANCD2蛋白質進行monoubiquitination的功能。 我們也進一步從mp29基因轉殖鼠對紫外線所引發的皮膚腫瘤比非轉殖鼠有較低的發生率結果看來，說明p29蛋白在細胞中持續表現時，可以保護並修復細胞的DNA免於傷害。綜合我們的研究結果，證明了p29蛋白除了在DNA複製時的重要性外，也發現p29蛋白具有影響DNA受損時所引發訊息傳遞的功能。

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.