Active site interactions impact phosphoryl transfer during replication of damaged and undamaged DNA by Escherichia coli DNA Polymerase I

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-tetrahy...