title: The human lagging strand DNA polymerase δ holoenzyme is distributive.
abstract: Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5'-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ(AA). The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Zhenxin
authors.initials: Z
authors.lastname: Hu
authors.forename: Senthil K
authors.initials: SK
authors.lastname: Perumal
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yue
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003198:Computer Simulation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D020035:Holoenzymes / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009010:Monte Carlo Method; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000378:metabolism
chemical_list: D020035:Holoenzymes; D009711:Nucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III