Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

Chembiochem : a European Journal of Chemical Biology
Janina CramerTobias Restle

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 bindi...Continue Reading

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Citations

Jun 22, 2012·Journal of Nucleic Acids·Jason M Walsh, Penny J Beuning
Jun 23, 2009·Chembiochem : a European Journal of Chemical Biology·Frank StreckenbachAndreas Marx
Jun 24, 2010·Angewandte Chemie·Karin BetzAndreas Marx
Jul 17, 2008·Journal of the American Chemical Society·Francesca Di PasqualeAndreas Marx
May 1, 2021·RSC Chemical Biology·Adeline EspinasseErin E Carlson

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