Klenow fragment-DNA interaction required for the incorporation of nucleotides opposite guanine and O⁶-methylguanine

A mechanism by which the Klenow fragment of DNA polymerase I monitors the geometry of the base pairs may involve hydrogen bonds between the polymerase and the minor groove of the nascent base pair. The involvement of the 3-position of guanine in the template strand was examined by synthesizing oligodeoxynucleotides containing guanine and 3-deazaguanine and comparing the steady-state kinetics of the incorporation of all four dNTPs. The V(max)/K(m) decreased a significant amount (170-fold) only when dCTP was the co-substrate suggesting that a hydrogen bond exists only when the correct base pair is being replicated. This approach was also used to examine how the Klenow fragment interacts with the 3-position of the mutagenic base O⁶- methylguanine (O⁶mG). The V(max)/K(m) for the incorporation of dTTP opposite O⁶-methyl-3-deazaguanine (O⁶m3DG) was 1700-fold less than opposite O⁶mG. In contrast, a small 6-fold increase in V(max)/K(m) occurred for the incorporation of dCTP opposite O⁶m3DG relative to O⁶mG. This result suggests that the hydrogen bond between the Klenow fragment and O⁶mG is more important in the incorporation of dTTP opposite O⁶mG and may contribute to the mutagenicity of O⁶mG.