Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).
The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.
Eger
B T
BT
Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
S J
SJ
D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D013698:Templates, Genetic
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D011756:Diphosphates; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D004256:DNA Polymerase I; C026600:2'-deoxyadenosine triphosphate