Chronic
inflammation involving constant generation of
nitric oxide (*NO) by macrophages has been recognized as
a factor related to
carcinogenesis. At the site of
inflammation, nitrosatively deaminated
DNA adducts such as
2'-deoxyinosine (dI) and
2'-deoxyxanthosine are primarily formed by *NO and may be associated with the development of
cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family
DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An
oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human
DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol alpha was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol eta and pol kappaDeltaC (a truncated form of pol kappa) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol alpha, pol eta, and pol kappaDeltaC, promoted preferential incorporation of 2'-deoxycytidine monophosphate (
dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of
2'-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol alpha, pol eta, and pol kappaDeltaC indicated that
dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating
dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A-->G transition. This ()NO-induced adduct may play an important role in initiating
inflammation-driven
carcinogenesis.