To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcore). The k on and k off values for the binding of the enzyme to the 36-bp substrate were 1. 5 (5) in the catalytic function was established by site-directed mutagenesis for the catalytic function of the enzyme. Two alternative mechanisms have been proposed: one is a two-metal ion mechanism (6) and the other is a carboxyl-hydroxyl relay mechanism (4, 7-9).X-ray crystallographic analyses of E. coli RNase HI (6, 10, 11) and the RNase H domain of HIV-1 1 reverse transcriptase (12) showed that these RNases H have a similar structural topology, with the exception of the presence of a handle (6) or basic protrusion region (11) in E. coli RNase HI. The importance of this region for substrate binding has been demonstrated in several studies. The RNase HI domain isolated from HIV-1 reverse transcriptase is enzymatically inactive (13,14), whereas that from murine leukemia virus reverse transcriptase, which has a part of this region, is active (15, 16). Site-directed mutagenesis indicated that the positively charged residues in this region are important for substrate binding (17). Incorporation of the basic protrusion of E. coli RNase HI at the equivalent position of the RNase H domain of HIV-1 reverse transcriptase resulted in the production of the active HIV-1 RNase H domain (18,19 However, it is not fully understood how RNase H binds to its substrate. A kinetic study using synthetic nucleosides with modifications of their 2Ј-hydroxyl groups revealed the importance of the 2Ј-hydroxyl group of the nucleoside on the 3Ј-side of the cleaved phosphodiester and that of the second nucleoside 5Ј to the cleaved phosphodiester for hydrogen bonding (20). Models for the binding of the enzyme to an RNA-DNA hybrid have been proposed based upon computer docking of the structure of E. coli RNase HI (free from its substrate) with an RNA-DNA hybrid whose structure was assumed to be an A form (6, 11), was found by NMR to be an A form (7), or that was neither A nor B (21). In these models, the RNA strand upstream of the cleavage site interacts with the enzyme. None of these models assumes that either the enzyme or the substrate alters its conformation upon binding.Recently, kinetic analyses using RNA-DNA hybrids, under conditions in which the hybrid was cleaved at a unique site (22), suggest that DNA residues complementary to the RNA residues located six or seven residues upstream of the cleavage site interact with the basic protrusion region of the enzyme. Such an interaction seems to require a conformational change in the enzyme or substrate, or in both. Determination of kinetic parameters and stoichiometry of RNase HI molecules bound to substrates of various lengths would provide additional information about the binding of enzyme to substrate.Magnesium ions may also modulate protein-nucleic acid ...

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the nV helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. cali strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins rD134HIRNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp1 34 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the nV helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its 61 position impacts catalysis.Ribonuclease H (RNase H), which degrades only the RNA strand of an RNA . DNA hybrid in the presence of divalent cations, such as Mg" or Mn", is widely found in various organisms [ 1 -31. Eschericlziu coli RNase HI, which had been designated as RNase H until a second RNase H (RNase HII) was isolated from E. coli 141, is composed of a single polypeptide chain with 155 amino acid residues [S] and has a PI value of 9.0 [6]. The similarities in the primary structures of this enzyme, reverse transcriptase RNase H 17, 81, Thermus thermophilus RNase H [91, and yeast RNase H [lo] strongly suggest that these enzymes are evolutionally related. In fact, the three-dimensional structure of E. coli RNase HI [ll-131 is similar to that of the reverse-transcriptase RNase H from human immunodeficiency virus-1 (HIV-1) [14-161. Therefore, it has been anticipated that detailed analyses of the structure/function relationships of E. Correspondence to S . Kanaya, Protein Engineering ResearchFax: +81 6 872 8210. Abbreviutions. GdnHCI, guanidine hydrochloride; HIV-1, human immunodeficiency virus-1 ; PCR, polymerase chain reaction; t,,*, temperature at which an enzyme loses 50% of its activity; P,, conformational parameter for u-helical residues in proteins ; RNase, ribonuclease; [DI 34XIRNase H, mutant ribonuclease H in which aspartic acid at position 134 is replaced by a specified amino acid.Institute,

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