[摘要]：In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC . PCNA . DNA complex and its progression to holoenzyme upon addition of polymerase delta (pol delta). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC . PCNA . DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC . PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of pol delta only one species converts to the RFC . PCNA . DNA . pol delta holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.
