Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.
To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.
Guo
M J
MJ
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK.
Hildbrand
S
S
Leumann
C J
CJ
McLaughlin
L W
LW
Waring
M J
MJ
D001483:Base Sequence; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D017931:DNA Primers; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D015394:Molecular Structure; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction; D011725:Pyridines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011728:Pyridones / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011742:Pyrimidine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D019914:Taq Polymerase / Q000037:antagonists & inhibitors; D013942:Thymine Nucleotides
C112207:2-amino-5-(2'-deoxyribofuranosyl)pyridine-5'-triphosphate; C112208:5-(2'-deoxyribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate; D017931:DNA Primers; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D007202:Indicators and Reagents; D019384:Nucleic Acid Synthesis Inhibitors; D011725:Pyridines; D011728:Pyridones; D011742:Pyrimidine Nucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D019914:Taq Polymerase; D004256:DNA Polymerase I; C024157:thymidine 5'-triphosphate