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Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon
Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-...
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Published in: | The Journal of biological chemistry 1991-11, Vol.266 (33), p.22698-22706 |
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Main Author: | |
Format: | Article |
Language: | English |
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Online Access: | Get full text |
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Summary: | Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase. The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration. A stable complex of RF-C and PCNA on primed single-stranded mpl8 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATPgammaS. These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex. Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement. DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis. The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast. A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)54625-1 |