Translesional bypass of a bulky DNA adduct: Insights into the mechanism of chemical carcinogenesis

Benzo[a]pyrene adducts are known to be strong blocks of DNA replication, allowing only the rare incorporation of a nucleotide across from the lesion. Further bypass is also inhibited for up to 5 nucleotides downstream from the adduct. A kinetic analysis of extension past either the (+)-trans- or (+)-cis-B[a]P-N 2-dG adducts positioned within two different sequence contexts by DNA Polymerase I Klenow Fragment (KF) provided the first in vitro evidence that may explain mutation spectra induced by these adducts in vivo. Interestingly, the (+)-cis adduct, which causes much stronger inhibition of the nucleotides insertion across from and extension past the adduct, affected the downstream replication much less than did the (+)-trans adduct. In the absence of sufficient structural data these findings provide the first evidence that the conformations of these stereoisomers within the polymerase active site are significantly different and differentially affect the minor groove interactions with KF, thereby leading to different replication trends.; During replication DNA polymerases incorporate a nucleotide through a multistep mechanism that involves a conformational change from an open to a closed complex. This step is believed to contribute to the high fidelity of DNA replication. Using a limited proteolysis method and gel-retardation assay, we found that this conformational change is affected by the presence of DNA damage or by a mispaired or altered dNTP. Consistent with steric exclusion model for nucleotide insertion, any deviation from Watson/Crick (W/C) geometry for the resulting base-pair had a destabilizing effect on the complex. The degree of destabilization varied and was correlated with the size of the mismatched base-pair and space available in the active site. We found that the B[ a]P adducts can be accommodated in the polymerase active site, but interfere with the conformational change to the catalytically active closed complex and cause a significant destabilization of this complex. A model is proposed in which the addition of a dNTP induces a conformational change but if the resulting geometry of the active site is different from a W/C base pair, the structure that forms is unstable resulting in dissociation.