Brush G S, Anderson C W, Kelly T J
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12520-4. doi: 10.1073/pnas.91.26.12520.
The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.
复制蛋白A（RPA）的32 kDa亚基在体内细胞周期的S期以及体外猿猴病毒40 DNA复制过程中会发生磷酸化。为了探究这种修饰的功能意义，我们纯化了一种在单链DNA存在下能使RPA磷酸化的HeLa细胞蛋白激酶。通过多项标准，我们确定纯化的酶是DNA激活蛋白激酶（DNA-PK）的一种形式，DNA-PK是一种先前描述的高分子量蛋白激酶，能够使多种核DNA结合蛋白磷酸化。DNA-PK对RPA的磷酸化受到天然单链DNA的刺激，但不受缺乏二级结构的同聚物的刺激。对猿猴病毒40模型系统的研究表明，DNA-PK是DNA复制依赖性RPA磷酸化所必需的。然而，激酶活性的缺失对体外DNA复制的程度没有影响。我们的数据支持一种模型，即DNA-PK对RPA的磷酸化是由包含单链和双链区域的复制中间体的形成所激活的。这一事件可能参与了一种将DNA复制与细胞周期协调起来的信号机制。
