Liu Jen-Sing, Kuo Shu-Ru, Melendy Thomas
Department of Microbiology, University at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York 14214, USA.
J Cell Biochem. 2006 Dec 1;99(5):1452-62. doi: 10.1002/jcb.21066.
Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.
复制蛋白A（RPA）是主要的真核单链DNA结合蛋白，在DNA复制、修复和重组中起核心作用。与许多DNA修复蛋白一样，RPA在DNA损伤时会发生大量磷酸化（特别是在其32 kDa亚基上）。许多修复蛋白的磷酸化已被证明对它们募集到DNA损伤诱导的核内病灶很重要。此外，H2AX（γ-H2AX）的磷酸化已被证明对DNA修复蛋白募集到这些核内病灶或在其中稳定保留很重要。我们在此探讨DNA损伤诱导的RPA过度磷酸化与其核内聚焦之间的关系，以及γ-H2AX是否是RPA在这些病灶中存在所必需的。使用绿色荧光蛋白（GFP）偶联的RPA，我们证明了DNA损伤或停滞的复制叉诱导形成了抗提取的RPA病灶。强烈的DNA损伤诱导的RPA病灶在磷酸化组蛋白H2AX和Chk1之后出现，但早于过度磷酸化RPA的出现。我们证明，虽然磷酸肌醇-3-激酶相关蛋白激酶的功能对于DNA损伤诱导的H2AX磷酸化和RPA过度磷酸化至关重要，但它们对于诱导抗提取的RPA和RPA病灶是可有可无的。此外，在基因上缺乏H2AX的小鼠细胞中，DNA损伤诱导的抗提取RPA出现的动力学与正常小鼠细胞相同。这些结果表明，RPA过度磷酸化和H2AX都不是DNA损伤/复制应激响应中RPA核内病灶形成所必需的，这与RPA作为参与初始识别受损DNA或受阻复制叉的DNA损伤传感器的作用一致。
