DNA Polymerase Switching: II. Replication Factor C Abrogates Primer Synthesis by DNA Polymerase  at a Critical Length

Author: Mossi R.   Keller R.C.   Ferrari E.   Hübscher U.  

Publisher: Academic Press

ISSN: 0022-2836

Source: Journal of Molecular Biology, Vol.295, Iss.4, 2000-01, pp. : 803-814

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Abstract

A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase /primase to the pro?cessive elongation by DNA polymerase &dgr;. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase  were investigated. We show that RF-C stalls DNA polymerase  after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se. This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5′ recessed ends and double-stranded DNA over 3′ ends. Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.

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