title: Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site.
abstract: The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Tsunaka
authors.forename: M
authors.initials: M
authors.lastname: Haruki
authors.forename: M
authors.initials: M
authors.lastname: Morikawa
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002918:Chymotrypsin; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009696:Nucleic Acids / Q000378:metabolism*; D010957:Plasmids; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D009696:Nucleic Acids; D001120:Arginine; D016914:Ribonuclease H; C078856:ribonuclease HI; D002918:Chymotrypsin