Lu Xiaoqing, Tan Cheng-Keat, Zhou Jin-Qiu, You Min, Carastro L Michael, Downey Kathleen M, So Antero G
Departments of Medicine and Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
J Biol Chem. 2002 Jul 5;277(27):24340-5. doi: 10.1074/jbc.M200065200. Epub 2002 May 1.
The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.
增殖细胞核抗原（PCNA）与DNA聚合酶δ之间的相互作用对于DNA复制/修复过程中的持续性DNA合成至关重要；然而，直到现在，DNA聚合酶δ中直接与PCNA相互作用的亚基的身份仍未明确。在本研究中，我们使用了相互免疫共沉淀实验来确定核心DNA聚合酶δ的两个亚基（125-kDa催化亚基或50-kDa小亚基）中哪一个直接与PCNA相互作用。我们发现PCNA与人p50以及小牛胸腺DNA聚合酶δ异二聚体进行了共免疫沉淀，但单独与p125没有共沉淀，这表明PCNA直接与p50相互作用而不与p125相互作用。在p50的N末端鉴定出一个与噬菌体RB69 DNA聚合酶的滑动夹结合基序相似的PCNA结合基序。通过远缘Western分析显示，含有该序列（MRPFL）的22个氨基酸的寡肽可结合PCNA，并在共免疫沉淀实验中与p50竞争结合PCNA。p21抑制了p50与PCNA的结合，这表明这两种蛋白质竞争PCNA上的相同结合位点。这些结果表明，PCNA与DNA聚合酶δ的相互作用是通过该酶的小亚基介导的。
