title: Stepwise assembly of the human replicative polymerase holoenzyme.
abstract: In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication. DOI:http://dx.doi.org/10.7554/eLife.00278.001.
authors.affiliation: Department of Chemistry , The Pennsylvania State University , University Park , United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Hedglin
authors.forename: Senthil K
authors.initials: SK
authors.lastname: Perumal
authors.forename: Zhenxin
authors.initials: Z
authors.lastname: Hu
authors.forename: Stephen
authors.initials: S
authors.lastname: Benkovic
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis*; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D031541:Fluorescence Resonance Energy Transfer; D020035:Holoenzymes; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D020035:Holoenzymes; D018809:Proliferating Cell Nuclear Antigen; D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III
keywords: DNA polymerase holoenzyme assembly; Human; clamp loader; clamp loading; clamp unloading; sliding clamp