Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia...

Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O⁶-methylguanine (O⁶-mG), 8-oxo-7,8-dihydroguanine (oxoG), O²-[4-(3-pyridyl)-4-oxobutyl]thymine (O²-POB-T), and N²-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N²-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in k_pol that was proportional to k_pol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O⁶mG, which showed large decreases in k_pol, similar to that exhibited by the Watson-Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O⁶mG occurred via Watson-Crick-like structures.