The assembly and enzymatic ability of the replication DNA polymerase holoenzyme from Sulfolobus solfataricus (Sso) was investigated using presteady-state fluorescence resonance energy transfer assays coupled with functional and structural studies. Kinetic experiments reveal that ATP binding to replication factor C (RFC) is sufficient for loading the heterotrimeric PCNA123 [proliferating cell nuclear antigen (PCNA)] clamp onto DNA that includes a rate-limiting conformational rearrangement of the complex. ATP hydrolysis is required for favorable recruitment and interactions with the replication polymerase (PolB1) that most likely include clamp closing and RFC dissociation. Surprisingly, the assembled holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. We show that PolB1 repeatedly disengages from the DNA template, leaving PCNA123 behind. Interactions with a newly identified C-terminal PCNA-interacting peptide (PIP) motif on PolB1 specifically with PCNA2 are required for holoenzyme formation and continuous re-recruitment during synthesis. The extended tail-like structure of the C-terminal PIP motif in PolB1 is revealed alone and when bound to DNA using small-angle X-ray scattering allowing us to develop a model for the holoenzyme complex. This is the first detailed kinetic description of clamp loading and holoenzyme assembly in crenarchaea and has revealed a novel mode for dynamic processivity that occurs by a polymerase exchange mechanism. This work has important implications for processive DNA replication synthesis and also suggests a potential mechanism for polymerase switching to bypass lesions.
Keywords: DNA polymerase holoenzyme; EDTA; FRET; PCNA; PCNA-interacting peptide; PIP; PIP motif; RFC; SAXS; Sso; Sulfolobus solfataricus; archaeal replication; dynamic processivity; ethylenediaminetetraacetic acid; fluorescence resonance energy transfer; primer-template DNA; proliferating cell nuclear antigen; ptDNA; replication factor C; salmon sperm DNA; small-angle X-ray scattering; spDNA.
© 2013.