title: Binding Affinities among DNA Helicase-Primase, DNA Polymerase, and Replication Intermediates in the Replisome of Bacteriophage T7.
abstract: The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase · DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.
authors.affiliation: From the Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boson, Massachusetts 02115 huidongzhang@tmmu.edu.cn.
authors.forename: Huidong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: From the Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China and.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.affiliation: the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boson, Massachusetts 02115.
authors.forename: Seung-Joo
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: From the Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China and.
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Wei
authors.affiliation: From the Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China and.
authors.forename: Jia
authors.initials: J
authors.lastname: Cao
authors.affiliation: the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boson, Massachusetts 02115 ccr@hms.harvard.edu.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D017123:Bacteriophage T7 / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D000099272:Replisomes
chemical_list: D004247:DNA; D019915:DNA Primase; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D013879:Thioredoxins; D014764:Viral Proteins; D000099272:Replisomes; C022262:Okazaki fragments; C517609:TrxA protein, E coli; C034970:bacteriophage T7 induced DNA polymerase
keywords: DNA replication; DNA-protein interaction; bacteriophage; enzyme kinetics; pre-steady-state kinetics