Identification of hydrogen bonds between Escherichia coli DNA polymerase I(Klenow fragment) and the minor groove of DNA by amino acid substitution of the polymerase and atomic substitution of the DNA

DNA polymerases replicate DNA with high fidelity despite the small differen ces in energy between correct and incorrect base pairs. X-ray crystallograp hic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fideli ty. The current hypothesis is that polymerases check the geometry of the ba se pairs through hydrogen bonds and steric interactions with the minor groo ve of the DNA. The mechanisms by which these interactions are related to ca talysis and fidelity are not known. In this manuscript, we have studied the se interactions using a combination of site-specific mutagenesis of Escheri chia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA. Crystal structures have predicted hydrogen bonds from Arg668 to the t erminal base on the primer (P1) and Gln849 to its base pair partner (T1). K inetic studies, however, have implicated the minor groove of the primer ter minus but not its base pair partner as being important to catalysis and fid elity. Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A. Hydrogen bonds from the DNA we re probed with three oligodeoxynucleotides which have a guanine or 3-deazag uanine (3DG) at P1, T1, or T2. We found that the pre-steady state parameter k(pol) was decreased with R668A (40-fold) and Q849A (150-fold) or with 3DG at P1 (300-fold) or T2 (25-fold). When R668A was combined with 3DG at P1 t he decrease in rate was only 80-fold, consistent with a hydrogen bond betwe en Arg668 and P1. In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold. Similar reactions between R6 68A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.