Eki T, Hurwitz J
Memorial Sloan-Kettering Cancer Center, Program in Molecular Biology, New York, New York 10021.
J Biol Chem. 1991 Feb 15;266(5):3087-100.
The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).
研究了聚（ADP - 核糖）聚合酶（PARP）对含有SV40复制起点的DNA复制的影响。在T抗原、拓扑异构酶I、多聚体人单链DNA结合蛋白（HSSB）以及DNA聚合酶α - DNA引物酶（polα - 引物酶）复合物（单聚合酶系统）存在的情况下，SV40 DNA能够进行广泛的复制。在单聚合酶系统中，会形成来自滞后链合成的小产物（冈崎片段）以及来自前导链合成的长产物。长产物的合成需要相对较高水平的polα - 引物酶复合物。在PARP存在的情况下，长产物的合成被阻断，仅积累了来自滞后链模板复制产生的小冈崎片段。通过向单聚合酶系统中补充多聚体激活剂1蛋白（A1）、增殖细胞核抗原（PCNA）以及PCNA依赖性DNA聚合酶δ（双聚合酶系统），PARP对前导链合成的抑制作用能够被有效逆转。单聚合酶系统中前导链合成的抑制是由于PARP与DNA链末端结合，从而阻止了polα对其进一步延伸。冈崎片段的选择性积累被证明是由于DNA引物酶对引物的偶联合成以及与引物酶复合的polα对其的立即延伸。PARP对这种偶联反应影响很小，但确实抑制了产物随后的延伸，推测是在polα从DNA片段的3'末端解离之后。PARP抑制了其他几种需要DNA链自由末端的酶促反应。PARP抑制了核酸外切酶III、DNA连接酶、5'至3'核酸外切酶以及polα对带引物DNA模板的延伸。相比之下，PARP仅部分与需要SSB、A1和PCNA的polδ延伸系统对带引物DNA模板的延伸竞争。这些结果表明，PARP在新生DNA链末端的结合可以被A1和PCNA与引物末端的结合所取代。HSSB在体内和体外都可以被聚（ADP - 核糖基）化。然而，PARP在单聚合酶系统中阻断前导链合成的选择性作用主要取决于其DNA结合特性，而非其合成聚（ADP - 核糖）的能力。
