INHIBITION OF DNA-POLYMERASE REACTIONS BY PYRIMIDINE NUCLEOTIDE ANALOGS LACKING THE 2-KETO GROUP

To investigate the influence of the pyrimidine 2-keto group on selecti on of nucleotides for incorporation into DNA by polymerases, we have p repared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely eoxy-beta-D-ribofuranosyl)pyridine-5'-triphosphate (dCTP ) and -deoxy-beta-D-ribofuranosyl)-3-methyl-2-pyridone-5 (dTTP) respe ctively. Both proved strongly inhibitory to PCR catalysed by Tao polym erase; dTTP rather more so than d*CTP. In primer extension experiment s conducted with either Tao polymerase or the Klenow fragment of Esche richia coli DNA polymerase I, both nucleotides failed to substitute fo r their natural pyrimidine counterparts. Neither derivative was incorp orated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly fold ed form of the polymerase enzyme needed to stabilize the transition st ate and catalyse phosphodiester bond formation.