CDC2 FAMILY KINASES PHOSPHORYLATE A HUMAN CELL-DNA REPLICATION FACTOR, RPA, AND ACTIVATE DNA-REPLICATION
CDC2 FAMILY KINASES PHOSPHORYLATE A HUMAN CELL-DNA REPLICATION FACTOR, RPA, AND ACTIVATE DNA-REPLICATION
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DOI：
10.1002/j.1460-2075.1992.tb05278.x
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发表时间：
1992-06-01
期刊：
影响因子：
11.4
通讯作者：
STILLMAN, B
中科院分区：
文献类型：
--
作者：
DUTTA, A;STILLMAN, B
RPA是从人细胞中纯化的单链DNA结合蛋白复合物，在体外SV 40 DNA复制的起始和延伸阶段至关重要。在人类和酵母细胞中，RPA的34 kDa多肽亚基在细胞周期的S和G2期磷酸化，而不是在G1期。人细胞提取物中存在的主要RPA激酶之一被纯化并显示为细胞周期蛋白B-cdc 2复合物。这种纯化的激酶，和一个密切相关的细胞周期蛋白A相关的cdc 2样激酶，磷酸化RPA p34的胰凝乳蛋白酶肽的一个子集，在体内磷酸化的G1-S转换。RPA p34的N-末端附近的两个丝氨酸被鉴定为cdc 2激酶磷酸化的可能位点。这些相同的丝氨酸是RPA在体内磷酸化所必需的。纯化的cdc 2激酶在体外刺激SV 40 DNA复制时，加入到G1细胞提取物。激酶还刺激解旋在复制起点，在DNA复制的最早的步骤之一，需要RPA，但只有在存在一个额外的因素存在于G1细胞提取物。因此，细胞周期蛋白-cdc 2激酶家族的一个或多个成员可能是启动和维持S期所必需的，部分原因是它们能够磷酸化和激活细胞DNA复制因子RPA。
RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.