title: DNA polymerase beta fidelity: halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal differences at the chemical transition state.
abstract: The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Sucato
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Upton
authors.forename: Boris A
authors.initials: BA
authors.lastname: Kashemirov
authors.forename: Jorge
authors.initials: J
authors.lastname: Osuna
authors.forename: Keriann
authors.initials: K
authors.lastname: Oertell
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Jan
authors.initials: J
authors.lastname: Florián
authors.forename: Arieh
authors.initials: A
authors.lastname: Warshel
authors.forename: Charles E
authors.initials: CE
authors.lastname: McKenna
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
mesh_terms: D020137:Base Pair Mismatch; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D017666:Deuterium Oxide / Q000737:chemistry; D004164:Diphosphonates / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D006219:Halogens / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D003848:Deoxyguanine Nucleotides; D004164:Diphosphonates; D006219:Halogens; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; D019951:DNA Polymerase beta; D017666:Deuterium Oxide