SACCHAROMYCES-CEREVISIAE REPLICATION FACTOR-C .2. FORMATION AND ACTIVITY OF COMPLEXES WITH THE PROLIFERATING CELL NUCLEAR ANTIGEN AND WITH DNA POLYMERASE-DELTA AND POLYMERASE-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-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 mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP-gamma-S. 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.