Shikata K, Ohta S, Yamada K, Obuse C, Yoshikawa H, Tsurimoto T
Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan.
J Biochem. 2001 May;129(5):699-708. doi: 10.1093/oxfordjournals.jbchem.a002909.
An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta. Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.
一种重要的真核生物DNA聚合酶，即DNA聚合酶δ（pol δ），在增殖细胞核抗原（PCNA）存在的情况下持续合成DNA。最近，一种66 kDa的多肽（p66）在其PCNA结合结构域内与裂殖酵母cdc27的PCNA结合结构域显示出显著同源性，被鉴定为小鼠和小牛胸腺pol δ的一个组成部分。我们的研究表明，在人细胞提取物的尺寸分级分离过程中，p66与pol δ的其他亚基紧密相互作用，并与这些亚基以及PCNA依赖性聚合酶活性一起进行共免疫沉淀。通过共表达p125、p50和p66重组杆状病毒可以重建有活性的人pol δ，但单独共表达p125和p50则不能。相互作用研究表明，p66稳定了p125和p50之间的结合。用与PCNA相连的珠子进行的下拉试验表明，p66增加了pol δ对PCNA的总体亲和力。这些结果表明，p66是人类pol δ的一个功能重要亚基，它稳定了pol δ复合物并增加了pol δ对PCNA的亲和力。
