Tratner I, Piard K, Grenon M, Perderiset M, Baldacci G
IFC1, CNRS, UPR 9044, Nillejuif, France.
Biochem Biophys Res Commun. 1997 Feb 13;231(2):321-8. doi: 10.1006/bbrc.1997.6082.
DNA polymerase delta (pol delta) is constituted of at least two subunits: the catalytic subunit of about 125 kDa (p125), and a subunit of approximately 50 kDa (p50) of unknown function. Processivity of pol delta is dependent on its auxiliary protein PCNA (proliferating cell nuclear antigen). Contradictory data were reported regarding a direct interaction between p125 and PCNA. We investigated this matter further using the baculovirus system to overexpress p125 and PCNA from S. pombe. We show that the recombinant p125 is active for basal DNA polymerase activity and for 3'-->5' exonuclease activity but is not stimulated by PCNA. Interaction between p125 and PCNA was tested by: (i) co-immunoprecipitation assay using antibodies specific for one or other polypeptides after co-expression in insect cells, and (ii) a two-hybrid assay. In both cases, no direct interaction between the two proteins was detected. Taken together, our data show that p125 and PCNA do not interact directly.
DNA聚合酶δ（pol δ）至少由两个亚基组成：一个约125 kDa的催化亚基（p125）和一个功能未知的约50 kDa亚基（p50）。pol δ的持续合成能力取决于其辅助蛋白增殖细胞核抗原（PCNA）。关于p125与PCNA之间的直接相互作用，有相互矛盾的数据报道。我们利用杆状病毒系统进一步研究了这个问题，该系统可过表达来自粟酒裂殖酵母的p125和PCNA。我们发现，重组p125具有基础DNA聚合酶活性和3'→5'核酸外切酶活性，但不受PCNA刺激。通过以下方法检测了p125与PCNA之间的相互作用：（i）在昆虫细胞中共表达后，使用针对一种或另一种多肽的特异性抗体进行共免疫沉淀分析；（ii）双杂交分析。在这两种情况下，均未检测到这两种蛋白质之间的直接相互作用。综上所述，我们的数据表明p125与PCNA不直接相互作用。
