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

A comparison of Km values and maximal rates of extension (Vmax) 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(p primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10m primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.