Dornreiter I, Höss A, Arthur A K, Fanning E
Institute for Biochemistry, Munich, FRG.
EMBO J. 1990 Oct;9(10):3329-36. doi: 10.1002/j.1460-2075.1990.tb07533.x.
Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.
纯化的SV40大T抗原与纯化的DNA聚合酶α-引发酶形成一种可通过ELISA和改良免疫印迹技术检测到的复合物。这种相互作用对聚合酶α的大催化亚基具有特异性。T抗原的氨基末端83个氨基酸对于与聚合酶结合既是必需的也是充分的。然而，位于T抗原羧基末端ATP酶结构域的抗体表位在聚合酶-T抗原复合物中被掩盖，并且针对T抗原羧基末端的抗体可抑制复合物的形成，这表明T抗原的该区域虽然不是结合所必需的，但与结合的聚合酶紧密相邻。人DNA聚合酶α对T抗原的亲和力比对来自小牛胸腺的聚合酶α的亲和力大约高10倍，这与以下解释一致，即聚合酶α至少部分负责SV40 DNA在体内和体外的灵长类特异性复制。结果表明，DNA聚合酶α与T抗原之间的特异性蛋白质-蛋白质相互作用在病毒DNA复制中起重要作用。
