Abstract

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).

Highlights

  • Toshihiko Eki and Jerard Hurwitz From the Memorial Sloan-Kettering Cancer Center, Program in Molecular Biology, New York, New York 10021

  • The initiation of leading strand synthesis requires the ac-. These results suggest that the bindionfgPARP at the ends of nascent DNA chains can be displaced by the Theabbreviations usedare: SV40, simianvirus 40; (H)SSB, binding of A 1 and PCNA to primerends

  • PARP Effect oSnV40 DNA Synthesis tion of a system capable of interacting with 3"hydroxyl nally, we show that human singlestrand DNA-binding protein (HSSB) can be ADP-ribosylated both in primer ends

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Summary

Introduction

Toshihiko Eki and Jerard Hurwitz From the Memorial Sloan-Kettering Cancer Center, Program in Molecular Biology, New York, New York 10021. Preparations of PARP isolated by glycerol gradient centrifugation were used dipolyrnerase system containedpolcr-primase complex, (0.03 and 0.18 unit, respectively) 0.16 unit of polb, 0.06 unit of Al, and 0.35 pg of PCNA, in reaction mixtures as described under “Experimental Procedures;” both systems contained 0.8 pg of HSSR and 0.7 pg of T antigen.

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