Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases

Recent studies have identified amino acid side chains forming several hydro gen bonds in the DNA minor groove as potentially important in polymerase re plication of DNA. Few studies have probed these interactions on the DNA its elf. Using non-hydrogen-bonding nucleoside isosteres, we have now studied e ffects in both primer and template strands with several polymerases to inve stigate the general importance of these interactions. All six polymerases s how differences in the H-bonding effects in the minor groove. Two broad cla sses of activity are seen, with a first group of DNA polymerases (KF-, Tag, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleo side Q (9-methyl-1H-imidazo-[4,5-b]pyridine) but not the analogue Z (4-meth ylbenzimidazole), implicating a specific minor groove interaction at the fi rst extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stabil ity. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. T he positions of these interactions vary among the enzymes, and only a subse t of the interactions identified structurally appears to be functionally im portant. In addition, polymerases appear to be differently sensitive to sma ll changes in base pair geometry.