Characterization of human REV proteins which are involved in mutagenesis

在本研究中，我们检测了人REV蛋白在细胞中的定位，并分析了UV诱导的DNA损伤对REV蛋白定位的影响。制备GFP融合REV蛋白的表达载体并将其导入COS 7细胞。使用共聚焦激光显微镜分析GFP融合REV蛋白的细胞内定位。结果表明，REV 1蛋白定位于细胞核内，约3%的细胞内有大量的微小病灶。REV 7主要定位于细胞核，部分定位于细胞质，无病灶形成，REV 3由于在细胞中表达较低，无法进行详细分析。在此基础上，我们重点分析了REV 1的定位及其与紫外线诱导的DNA损伤的关系。对表达GFP-REV 1的细胞进行UV照射，并分析REV 1病灶形成。结果表明，在正常条件下，在约3%的细胞中观察到REV 1病灶形成，并且在8 J/m^2照射后8小时，病灶形成细胞的百分比增加至约25%。发现该百分比以UV剂量依赖性和时间依赖性方式增加。大多数REV 1病灶与PCNA共定位，PCNA是DNA复制叉的标志物。REV 1灶还与其他参与跨损伤DNA合成的蛋白Pol κ和Pol η共定位。还通过使用与GFP融合的REV 1的截短突变体分析了焦点形成所需的REV 1的结构域。结果发现，REV 1的C-末端区域，其中存在REV 7、Pol κ和Pol η的结合结构域，是焦点形成所必需的。这些结果表明，REV 1焦点的形成是由紫外线照射诱导，并可能与紫外线诱导的DNA损伤。这些病灶可能也参与了PCNA相关的DNA复制。REV 1在体外作为模板DNA上脱碱基损伤的末端脱氧胞苷酰转移酶发挥作用。我们的数据支持REV 1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.