title: Kinetic mechanism of DNA polymerase I (Klenow).
abstract: The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: R D
authors.initials: RD
authors.lastname: Kuchta
authors.forename: V
authors.initials: V
authors.lastname: Mizrahi
authors.forename: P A
authors.initials: PA
authors.lastname: Benkovic
authors.forename: K A
authors.initials: KA
authors.lastname: Johnson
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I