Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate.
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.
Singer
B
B
Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Chavez
F
F
Spengler
S J
SJ
Kuśmierek
J T
JT
Mendelman
L
L
Goodman
M F
MF
D000478:Alkylation; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D008745:Methylation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000138:chemical synthesis*
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D013942:Thymine Nucleotides; D004247:DNA; D004256:DNA Polymerase I; C024157:thymidine 5'-triphosphate