title: Insights into Okazaki fragment synthesis by the T4 replisome: the fate of lagging-strand holoenzyme components and their influence on Okazaki fragment size.
abstract: In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.
authors.affiliation: From 414, Wartik Laboratories, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Danqi
authors.initials: D
authors.lastname: Chen
authors.affiliation: From 414, Wartik Laboratories, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yue
authors.affiliation: From 414, Wartik Laboratories, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.affiliation: From 414, Wartik Laboratories, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802. Electronic address: sjb1@psu.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D017122:Bacteriophage T4 / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D015398:Signal Transduction; D014764:Viral Proteins / Q000378:metabolism; D000099272:Replisomes
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D020035:Holoenzymes; D009097:Multienzyme Complexes; D014764:Viral Proteins; D000099272:Replisomes; C022262:Okazaki fragments; C074855:gene 44 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4
keywords: DNA Binding Protein; DNA Enzymes; DNA Polymerase; DNA Replication; DNA Synthesis; Holoenzyme; Lagging-strand Polymerase Recycling; Okazaki Fragment; Sliding Clamp; T4 Replisome