RECOGNITION OF THE PRIMERS CONTAINING DIFFERENT MODIFIED NUCLEOTIDE UNITS BY THE KLENOW FRAGMENT OF DNA-POLYMERASE-I FROM E-COLI

A comparison of K-m values and maximal rates of extension (V-max) for primers containing different modified bases or mismatches, and fully c omplementary primers of the same length catalyzed by the Klenow fragme nt of E coli DNA polymerase I was carried out. Base modifications incl ude 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 t emplate. Differently, removal of one base in any position from the 3'- terminus of the primer is equivalent to shortening of the primer by on e nucleotide unit, and decreases the affinity to the enzyme by 1.8-fol d. Since apurinic sites fail to interfere with the efficiency of DNA s ynthesis, we suppose that the Klenow fragment of E coil 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)lo primer contains about 2.6 T-T dimers per mol ecule, the efficiency of its elongation decreases by a factor of 8-18.