Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.
Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.
Tsurimoto
T
T
Cold Spring Harbor Laboratory, New York 11724.
Stillman
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000494:pharmacology; D011067:Poly dA-dT / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D013698:Templates, Genetic; D013997:Time Factors; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011067:Poly dA-dT; D000255:Adenosine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase