title: Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli.
abstract: The polymerase and 5'-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5'-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5'-nuclease. The preferred substrate of the 5'-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5'-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5'-nuclease activities are coupled such that the majority of molecules cleaved by the 5'-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5'-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: N D
authors.initials: ND
authors.lastname: Grindley
authors.forename: C M
authors.initials: CM
authors.lastname: Joyce
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004256:DNA Polymerase I