title: Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase.
abstract: We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.
authors.affiliation: Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Cramer
authors.forename: Gopinath
authors.initials: G
authors.lastname: Rangam
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
mesh_terms: D001665:Binding Sites; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry; D013997:Time Factors
chemical_list: D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase