Abstract
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).
Highlights
Eukaryotic DNA polymerase 6 and its accessorypro- 1985;Wobbe et al, 1985;reviewedby Stillman, 1989 and teins are essential forSV40 DNA replication in uitro. Challberg and Kelly, 1989)
Subsequent characcomplex has significant single-strandedDNA binding terization of PCNA and another replicationfactor,RF-C, activity in addition to binding to a primer-template suggested that both DNA polymerases a and 6 functiona t the junction
Additioonf another replication fac- replication forkas a dimeric polymerase complex(Tsurimoto tor, RF-A, completely blocked the nonspecific, singie- and Stillman, 1989b, 1990).Genetic and biochemical studies stranded DNA binding by the RF-C-PCNA complex
Summary
RF-A-RF-A from human cells was purified by a published method (Tsurimoto et al, 1989). The digestion was stopped by the addition of 20 pl of stop solution (200 pg/ml tRNA, 10 mM EDTA, 2.5 mM EGTA, and 0.3 M sodium acetate) and DNA in the reaction was extracted with phenol/chloroform ( l : l ) , precipitated wit.h ethanol, dissolved with 2 pl of formamide-dye, and subconcentration of RF-C, more than 90% of input DNA was shifted (Fig. 2, lane 6) This suggested that the interaction between RF-C and ATP increased the affinity of RF-C for the hairpin DNA, but once the ATP was hydrolyzed by the DNA-dependent RF-C ATPase activity, RF-C rapidly dissojected to electrophoresisin a 10% polyacrylamide gel with T B E buffer ciated from the DNA. RF-C covered both single-stranded and double-strandDeNd A, abut-
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