DNA polymerase-mediated nucleotide incorporation adjacent to hydrocarbon-deoxyadenosine and hydrocarbon-deoxyguanosine adducts

Abstract

To examine the effect of DNA adducts on nucleotide incorporation by DNA polymerase at 3′ neighboring bases, synthetic oligonucleotides (16mers) containing a purine at position 13 from the 3′ end and any one of the four possible bases at psition 12 were prepared and readed with 7-bromomethylbenz[a]anthracene. Using HPLC, unmodified oligonucleotide was separated fhm oligonucleotide containtng a single adduct, at either an adenine or a guanine residue. These products were annealed with a ³²P 5′-end labeled primer (llmer) and incubated with modified T7 DNA polymerase (Sequenase, version 2.0) in the presence of deoxyribonucleoside 5′-triphosphates. Analysis by gel electrophoresis showed that unmodified oligonucleotide template allowed the primer to be rapidly extended to the entire length of the template. However, the presence of an adduct caused primer extension to stop at the base 3′ to the adduct. While correct base pairing occurred at this termination site with most adducted templates, there was a high frequency of misincorporation of guanine opposite a thymine located 3′ to an adenine adducts. This result suggests that some bulky carcinogen-DNA adducts may lead to base mismatches at neighboring bases.