Recognition of the primers containing different modified nucleotide units by the Klenow fragment of DNA polymerase I from E coli

Abstract

Listen

Translate article

A comparison of K m values and maximal rates of extension (V max) for primers containing different modified bases or mismatches, and fully complementary primers of the same length catalyzed by the Klenow fragment of E coli DNA polymerase I was carried out. Base modifications include T-T dimers and apurinic sites. In the case of mismatch, the number of complementary bases from the 3′-terminus to the non-complementary nucleotide determines the efficiency of substrate incorporation, which is a measure of degree of interaction of the enzyme with its primer template. Differently, removal of one base in any position from the 3′-terminus of the primer is equivalent to shortening of the primer by one nucleotide unit, and decreases the affinity to the enzyme by 1.8-fold. Since apurinic sites fail to interfere with the efficiency of DNA synthesis, we suppose that the Klenow fragment of E coli DNA polymerase I does not participate in the correction of DNAs containing apurinic nucleotides units. Finally, the efficiency of elongation of the d(pT) 10 primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT) 10 primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8–18.