DNA polymerase switching: II. Replication factor C abrogates primer synthesis by RNA polymerase alpha at a critical length

A crucial event in DNA replication is the polymerase switch from the synthe sis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?ce ssive elongation by DNA polymerase delta. In order to shed light on the rol e of replication factor C (RF-C) in this process, the effects of RF-C on DN A polymerase alpha were investigated. We show that RF-C stalls DNA polymera se alpha after synthesis of approximately 30 nucleotides, while not inhibit ing 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 sh ift experiments indicated that RF-C has a preference for 5' recessed ends a nd 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 necessa rily required for the loading of the clamp. Thus we propose a model where R F-C, upon binding to the RNA/DNA primer, influences primer synthesis and se ts the conditions for a polymerase switch after recruiting PCNA to DNA. (C) 2000 Academic Press.