Te. Spratt et De. Levy, STRUCTURE OF THE HYDROGEN-BONDING COMPLEX OF O-6-METHYLGUANINE WITH CYTOSINE AND THYMINE DURING DNA-REPLICATION, Nucleic acids research, 25(16), 1997, pp. 3354-3361
During DNA replication, mutations occur when an incorrect dNTP is inco
rporated opposite a carcinogen-modified nucleotide. We have probed the
structures of the interaction between O-6-methylguanine (O(6)mG) and
cytosine and thymine during replication by kinetic means in order to e
xamine the structure during the rate determining step. The kinetics of
incorporation of dCTP and dTTP opposite O(6)mG and three analogs, S-G
-methyl-6-thioguanine, O-6-methyl-1-deazaguanine and O-6-methylhypoxan
thine, have been measured with four polymerases, the Klenow fragment o
f DNA polymerase I, the Klenow fragment with the proof-reading exonucl
ease inactivated, Tag and Tth polymerases. In the insertion of dTTP op
posite O(6)mG, a large decrease in V-max/K-m was observed only upon mo
dification of the N1 position. This result is consistent with a Watson
-Crick type configuration. For the incorporation of dCTP, the V-max/K-
m was significantly decreased only with removal of the exocyclic amino
group at the 2 position. The pH dependence of the ratio of incorporat
ion of dCTP and dTTP was independent of pH at physiological pH. This r
esult suggests that dCTP is incorporated via an uncharged complex such
as the wobble configuration.