Analysis of interactions between Proliferating Cell Nuclear Antigen (PGNA) and DNA replication / repair factor

增殖细胞核抗原（PCNA）是真核细胞DNA复制和修复机制的重要组成部分。酵母和人类PCNAs的晶体学研究表明，蛋白质形成同源三聚体环。其他一些研究提出，PCNA在DNA合成过程中起分子钳的作用。除了其在DNA复制和修复中的重要功能外，PCNA还通过与细胞周期蛋白和/或细胞周期蛋白依赖性蛋白激酶抑制剂p21相互作用参与细胞周期控制（CIP 1/WAF 1/SDI 1）。为了研究PCNA的结构-功能关系，已经通过定点突变引入覆盖整个PCNA基因的几个突变，并利用大肠杆菌基因表达系统纯化了突变蛋白。对这些蛋白的生化分析表明，PCNA的功能结构域是刺激复制因子C（RFC）ATP酶和pold DNA合成所必需的。瓣状核酸内切酶1（FEN-1）是一种结构特异性核酸内切酶，对5 ′-瓣状DNA结构具有核酸内切酶活性，对切口双链DNA具有S ′-外切酶活性。这种酶是完成滞后链DNA合成所必需的，也被认为对DNA修复很重要。近年来，有报道称PCNA与FEN-1结合后，可使FEN-1的核酸内切酶活性和核酸外切酶活性提高10-50倍。将突变型PCNA蛋白对重组人FEN-1的5 ′-内切核酸酶活性的刺激与野生型进行比较。凝胶位移分析检测FEN-1与突变型PCNA蛋白之间的直接相互作用。我们确定了人类PCNA的相互作用的关键氨基酸。

Proliferating Cell Nuclear Antigen (PCNA) is an essential component of the DNA replication and repair machinery in eukaryotic cells. The crystallographic studies of yeast and human PCNAs reveal that the protein forms a homotrimeric ring. Several other studies have proposed that PCNA would act as a molecular clamp during DNA synthesis. In addition to its important function in DNA replication and repair, PCNA is also involved in cell cycle control through interactions with the cyclins and/or the cyclin dependent protein kinase inhibitor, p21 (CIP1/WAFl/SDI1).In order to study structure-function relationships of PCNA, several mutations covering the whole PCNA gene have been introduced by site-directed mutagenesis, and the mutant proteins have been purified using E.coli gene expression system. Biochemical analyses of these proteins reveals that the functional domains of human PCNA required for stimulation of replication factor C (RFC) ATPase and pold DNA synthesis by biochemical analyzes.The flap endonuclease 1 (FEN-1) is a structure-specific endonuclease that has the endonucleolytic activity at 5'-flap DNA structures and the S'-exonucleolytic activity on nicked double-stranded DNA. This enzyme is essential for completion of lagging strand DNA synthesis and is also considered to be important for DNA repair. Recently, It was reported that PCNA binds to FEN-1 and stimulates both its endonucleolytic and exonucleolytic activities 10-50 fold.We carried out studies of the ternary interaction among PCNA, FEN-1 and 5'-flap DNA. Stimulation of 5'-endonuclease activity of recombinant human FEN-1 by mutant PCNA proteins was compared to the wild type. The direct interaction between FEN-1 and mutant PCNA protein was examined by gel shift analysis. We identified critical amino acids of human PCNA for the interaction.