Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site

摘要

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.