DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length.
A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta. 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 alpha were investigated. We show that RF-C stalls DNA polymerase alpha 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.
Mossi
R
R
Department of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Keller
R C
RC
Ferrari
E
E
Hübscher
U
U
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013698:Templates, Genetic
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D019915:DNA Primase; D004256:DNA Polymerase I; D051818:Replication Protein C