Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.
Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).
Tsurimoto
T
T
Cold Spring Harbor Laboratory, New York 11724.
Stillman
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009687:Nuclear Proteins / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D018809:Proliferating Cell Nuclear Antigen; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D013698:Templates, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D000255:Adenosine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase