Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

Biochemistry. 1989 Feb 21;28(4):1478-83. doi: 10.1021/bi00430a008.

Abstract

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkylation
  • Base Sequence
  • DNA / biosynthesis
  • DNA Polymerase I / metabolism*
  • Indicators and Reagents
  • Kinetics
  • Methylation
  • Oligodeoxyribonucleotides / chemical synthesis*
  • Structure-Activity Relationship
  • Templates, Genetic
  • Thymine Nucleotides / chemical synthesis*

Substances

  • Indicators and Reagents
  • Oligodeoxyribonucleotides
  • Thymine Nucleotides
  • DNA
  • DNA Polymerase I
  • thymidine 5'-triphosphate