KIAA0101在細胞週期及DNA修復之角色

Abstract

經由DNA微陣列的分析，我們發現KIAA0101 (p15PAF) 過度表現在肝細胞癌中。KIAA0101是一個與增殖細胞核抗原(PCNA)結合的蛋白質，但其功能目前尚未明瞭。在本篇論文裡，我們發現KIAA0101只表現在細胞週期中的S期。降低細胞中KIAA0101的表現會抑制細胞週期由G1期進入S期的進行，並且抑制BrdU的嵌入。降低KIAA0101的表現會誘導p21的過度表現。為了進一步的了解KIAA0101功能的分子機制，我們利用酵母菌雙雜交系統，尋找與之進行交互作用的蛋白質。我們發現Rad23A可與KIAA0101結合。經由免疫沈澱分析法我們證明EGFP-tagged KIAA0101確實能夠與Rad23A交互結合。Rad23A是一個在核苷酸切除修復擔任要角的蛋白質，也暗示KIAA0101可能參與DNA修復。結果如同預期，細胞群落分析的結果顯示，KIAA0101表現的降低會使細胞對紫外光敏感增高。在紫外光照射之下也觀察到KIAA0101表現降低的細胞其DNA合成會減少。這些結果顯示，KIAA0101是一個多功能的與增殖細胞核抗原結合的蛋白質，並參與調節DNA合成與修復。

By microarray analysis, we identified KIAA0101 (p15PAF) as a gene overexpressed in hepatocellular carcinoma. KIAA0101 is a proliferating cell nuclear antigen (PCNA)-binding protein of unknown function. In this study, we found that KIAA0101 was expressed only in S phase of the cell cycle. Knockdown of KIAA0101 inhibited cell cycle progression from G1 to S phase and suppressed BrdU incorporation. Knockdown of KIAA0101 induced p21 overexpression. To further delineate the molecular mechanism for the function of KIAA0101 protein, the yeast two hybrid assay was used to identify the interacting proteins. Rad23A was identified as a putative KIAA0101-binding protein. Using the immunoprecipitation assay, we demonstrated that EGFP-tagged KIAA0101 interacted with Rad23A, confirming the interaction of KIAA0101 and Rad23A in vivo. Rad23A is a major player in nucleotide excision repair. This result suggests that KIAA0101 play a role in DNA damage repair. Consistent with this suggestion, colony formation assay showed that KIAA0101 depletion sensitized cells to UV. Moreover, DNA synthesis was decreased in cells with KIAA0101 knockdown under UV irradiation. These results suggest that KIAA0101 is a multifunctional PCNA-interacting protein, and play important roles in regulating DNA synthesis and repair.