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 differences in energy between correct and incorrect base pairs. X-ray crystallographic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fidelity. The current hypothesis is that polymerases check the geometry of the base pairs through hydrogen bonds and steric interactions with the minor groove of the DNA. The mechanisms by which these interactions are related to catalysis and fidelity are not known. In this manuscript, we have studied these interactions using a combination of site-specific mutagenesis of Escherichia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA. Crystal structures have predicted hydrogen bonds from Arg668 to the terminal base on the primer (P1) and Gln849 to its base pair partner (T1). Kinetic studies, however, have implicated the minor groove of the primer terminus but not its base pair partner as being important to catalysis and fidelity. Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A. Hydrogen bonds from the DNA were probed with three oligodeoxynucleotides which have a guanine or 3-deazaguanine (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 the decrease in rate was only 80-fold, consistent with a hydrogen bond between Arg668 and P1. In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold. Similar reactions between R668A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.
Spratt
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American Health Foundation, 1 Dana Road, Valhalla, New York 10595, USA. tomspratt@aol.com
D019943:Amino Acid Substitution / Q000235:genetics*; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D004256:DNA Polymerase I