Brush G S, Morrow D M, Hieter P, Kelly T J
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15075-80. doi: 10.1073/pnas.93.26.15075.
Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.
复制蛋白A（RPA）是一种高度保守的单链DNA结合蛋白，是细胞DNA复制、修复和重组所必需的。在人类细胞中，RPA在细胞周期的S期和G2期以及对电离辐射或紫外线辐射的反应中会发生磷酸化。酿酒酵母表现出类似的细胞周期调节的RPA磷酸化模式，并且我们的研究表明辐射诱导的反应在酵母中也会发生。我们已经研究了正常细胞周期中以及对环境损伤作出反应时酵母RPA的磷酸化情况，并证明在所有测试条件下，检查点基因MEC1对于该反应是必需的。通过对几个检查点突变体的研究，我们将RPA磷酸化置于DNA损伤反应的一条新途径中。MEC1在序列上与人类ATM相似，ATM是共济失调毛细血管扩张症（A-T）患者中发生突变的基因。A-T细胞在多个检查点途径中存在缺陷，并且对电离辐射杀伤高度敏感。由于A-T细胞在电离辐射诱导的RPA磷酸化方面表现出延迟，我们的结果表明MEC1和ATM之间存在功能相似性，并表明RPA磷酸化参与了A-T中存在缺陷的保守真核DNA损伤反应途径。
