Characterization of human REV proteins which are involved in mutagenesis

在本研究中，我们检查了人类REV蛋白在细胞中的定位，并分析了紫外线诱导的DNA损伤对REV蛋白定位的影响。制备了gfp融合REV蛋白的表达载体，并将其导入COS7细胞。用激光共聚焦显微镜分析gfp融合REV蛋白的细胞内定位。结果表明，在约3%的细胞中，REV1蛋白定位于细胞核内并形成许多小灶。发现REV7主要定位于细胞核，部分定位于细胞质，不形成病灶，REV3在细胞中表达较低，无法对其进行详细分析。随后，我们重点分析了REV1的定位及其与紫外线诱导DNA损伤的关系。对表达GFP-REV1的细胞进行紫外线照射，分析REV1病灶形成情况。结果表明，正常情况下约3%的细胞有REV1灶形成，8J/m^2辐照8 h后，形成灶的细胞比例增加至25%左右。发现该百分比以紫外线剂量依赖和时间依赖的方式增加。大多数REV1病灶与DNA复制叉的标记物PCNA共定位。REV1位点还与其他参与翻译DNA合成的蛋白Pol κ和Pol η共定位。利用与GFP融合的REV1截断突变体分析了REV1形成病灶所需的结构域。结果发现，在REV1的c端区域，存在REV7、Pol κ和Pol η的结合域，是形成焦点所必需的。这些结果表明，REV1的焦点形成是由紫外线照射诱导的，并且可能与紫外线诱导的DNA损伤有关。病灶也可能与pcna相关的DNA复制有关。REV1在体外模板DNA的基本损伤处作为末端脱氧胞基转移酶。我们的数据支持REV1在体内的功能。

In this study, we checked the localization of human REV proteins in cells, and we also analyzed the effect of UV-induced DNA damage, to the REV protein localization. The expression vectors for GFP-fusion REV proteins were produced and introduced into COS7 cells. The intracellular localization of the GFP-fusion REV proteins were analyzed using a confocal laser microscope. It was revealed that REV1 protein localizes in nucleus and forms a lot of tiny foci in about 3% of cells. REV7 was found to localize mainly in nucleus and partially in cytoplasm without focus formation, and REV3 could not be analyzed in detail because of its low expression in cells. Thereafter, we focused on the analysis of REV1 localization and its relation with UV-induced DNA damage. Cells with GFP-REV1 expression were UV-irradiated and REV1 focus formation was analyzed. It was revealed that REV1 focus formation was observed in about 3% of cells under normal condition and the percentage of the focus forming cells was increased up to about 25% 8 hours after 8J/m^2 irradiation. The percentage was found to increase in a UV dose-dependent and a time-dependent manner. Most of the REV1 foci were co-localized with PCNA, a marker of the DNA replication fork. And the REV1 foci were also co-localized with Pol κ and Pol η, the other proteins involved in translesion DNA synthesis. The domain of REV1 required for the foci formation was also analyzed by using truncation mutants of REV1 fused with GFP. It was found that C-terminal region of REV1,where the binding domain for REV7,Pol κ and Pol η exists, was required for the focus formation. These results indicate that REV1 focus formation is induced by UV irradiation and may be associated with UV-induced DNA damage. The foci may be also involved the PCNA-associated DNA replication. REV1 functions as a terminal deoxycytidyl transferase at abasic lesions on template DNA in vitro. Our data support the REV1 function in vivo.