Tan C K, Sullivan K, Li X Y, Tan E M, Downey K M, So A G
Department of Medicine, University of Miami School of Medicine, FL 33101.
Nucleic Acids Res. 1987 Nov 25;15(22):9299-308. doi: 10.1093/nar/15.22.9299.
Two murine monoclonal antibodies to the proliferating cell nuclear antigen (PCNA), a rabbit anti-N-terminal peptide antibody and human auto-antibody to PCNA reacted with the auxiliary protein for DNA polymerase delta from fetal calf thymus following SDS-polyacrylamide gel electrophoresis, confirming the identity of PCNA and the auxiliary protein. Undenatured auxiliary protein was immunoprecipitated by the human autoantibody, but not by the monoclonal antibodies, which were raised to SDS-denatured PCNA, nor by the anti-N-terminal peptide antibody, suggesting that the epitopes recognized by both the monoclonal antibodies and the anti-peptide antibody are not exposed in the native protein. The human anti-PCNA autoantibody neutralized the activity of the auxiliary protein for DNA polymerase delta, but did not inhibit the activity of pol delta itself. The ability of pol delta to utilize template/primers containing long stretches of single-stranded template was inhibited by the anti-PCNA autoantibody, whereas the activity of pol alpha on such templates was not affected, confirming the specificity of the auxiliary protein for pol delta. The ability of PCNA, a cell cycle-regulated protein, to regulate the activity of pol delta suggests a central role for pol delta in cellular DNA replication.
两种针对增殖细胞核抗原（PCNA）的鼠单克隆抗体、一种兔抗N端肽抗体以及人抗PCNA自身抗体，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，与来自胎牛胸腺的DNA聚合酶δ辅助蛋白发生反应，从而证实了PCNA与该辅助蛋白的一致性。未变性的辅助蛋白可被人自身抗体免疫沉淀，但不能被针对SDS变性PCNA产生的单克隆抗体或抗N端肽抗体免疫沉淀，这表明单克隆抗体和抗肽抗体识别的表位在天然蛋白中未暴露。人抗PCNA自身抗体可中和DNA聚合酶δ辅助蛋白的活性，但不抑制DNA聚合酶δ本身的活性。抗PCNA自身抗体抑制了DNA聚合酶δ利用含有长链单链模板的模板/引物的能力，而DNA聚合酶α在此类模板上的活性未受影响，这证实了辅助蛋白对DNA聚合酶δ的特异性。PCNA作为一种细胞周期调节蛋白，具有调节DNA聚合酶δ活性的能力，这表明DNA聚合酶δ在细胞DNA复制中起核心作用。
