Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase a by multiple mechanisms
Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase a by multiple mechanisms
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DOI：
10.1021/bi970473r
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发表时间：
1997-07-15
期刊：
影响因子：
2.9
通讯作者：
Wold, MS
中科院分区：
文献类型：
--
作者：
Braun, KA;Lao, Y;Wold, MS
来自人类细胞的复制蛋白A（RPA）是70-、32-和14-kDa亚基的稳定复合物，其是DNA代谢中的多个过程所需的。RPA以高亲和力与单链DNA结合，并与多种蛋白质相互作用，包括启动SV 40 DNA复制所需的蛋白质、DNA聚合酶α和SV 40大T抗原。我们已经使用RPA的一系列突变衍生物来定位RPA的特定蛋白质-蛋白质相互作用所需的区域，并检查了这些相互作用在DNA复制T抗原中的作用，DNA聚合酶α和VP 16的活化结构域在RPA的70-kDa亚基的N-末端一半（残基1-327）中都具有相互作用的重叠位点。此外，DNA聚合酶α的相互作用位点由两个功能不同的区域组成，一个（残基1-类似于170）刺激聚合酶活性，第二个（残基类似于170-327）增加聚合酶持续合成能力。在后者中，RPA的直接蛋白质-蛋白质相互作用和ssDNA结合活性都需要RPA来调节聚合酶的持续合成能力。我们还发现，SV 40 T抗原抑制RPA增加DNA聚合酶α的持续合成能力的能力，这表明RPA的这种活性可能对延伸很重要，但在DNA复制的起始过程中并不重要，DNA聚合酶α，而不是T抗原也与RPA的32和/或14 kDa亚基相互作用，但这些相互作用似乎并不影响聚合酶活性。
Replication Protein A (RPA) from human cells is a stable complex of 70-, 32-, and 14-kDa subunits that is required for multiple processes in DNA metabolism. RPA binds with high affinity to single-stranded DNA and interacts with multiple proteins, including proteins required for the initiation of SV40 DNA replication, DNA polymerase alpha and SV40 large T antigen, We have used a series of mutant derivatives of RPA to map the regions of RPA required for specific protein-protein interactions and have examined the roles of these interactions in DNA replication T antigen, DNA polymerase alpha and the activation domain of VP16 all have overlapping sites of interaction in the N-terminal half (residues 1-327) of the 70-kDa subunit of RPA. In addition, the interaction site for DNA polymerase alpha is composed of two functionally distinct regions, one (residues 1-similar to 170) which stimulates polymerase activity and a second (residues similar to 170-327) which increases polymerase processivity. In the latter, both the direct protein-protein interaction and ssDNA-binding activities of RPA were needed for RPA to modulate polymerase processivity. We also found that SV40 T antigen inhibited the ability of RPA to increase processivity of DNA polymerase alpha, suggesting that this activity of RPA may be important for elongation but not during the initiation of DNA replication, DNA polymerase alpha, but not T antigen also interacted with the 32- and/or 14-kDa subunits of RPA, but these interactions did not seem to effect polymerase activity.