Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance*

To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding ofEscherichia 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 andk off values for the binding of the enzyme to the 36-bp substrate were 1.5 × 106 m −1 s−1 and 3.2 × 10−2 s−1, respectively. Similar values were obtained with the shorter substrates. Using uncleavable 2′-O-methylated RNA-DNA substrates, values fork on and k off were 2.1 × 105 m −1s−1 and 1.3 × 10−1 s−1 in the absence of Mg2+ that were further reduced in the presence of Mg2+ to 7.4 × 103 m −1 s−1 and 2.6 × 10−2 s−1. Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp134 replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate. Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9–10 bp of RNA-DNA hybrid.

Escherichia coli ribonuclease HI (RNase HI) degrades only the RNA strand of an RNA-DNA hybrid (1) and is composed of a single polypeptide chain of 155 amino acid residues (2). It requires divalent cations such as Mg 2ϩ or Mn 2ϩ for activity (1). The involvement of the amino acid residues Asp 10 , Glu 48 , Asp 70 (3), His 124 (4), and Asp 134 (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)(8)(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 demon-strated 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). In addition, Cys 13 , Asn 16 , Asn 44 , Asn 45 (7), and Thr 43 (17) have been shown to be important for substrate binding in E. coli RNase HI. Thus, it seems likely that all amino acid residues that are involved in catalytic function and substrate binding have been identified.
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 interactions and participate in catalysis. For example, the binding of Saccharomyces cerevisiae RNase HI to double-stranded RNA is influenced by Mg 2ϩ concentration, with tight binding being detected at low concentrations and little binding in the presence of 5 mM Mg 2ϩ (23), and the DNA binding specificity of EcoRV is increased in the presence of Mg 2ϩ (24). Therefore, it is of great interest to investigate the influence of magnesium ions on binding of RNase HI to RNA-DNA substrates.
In this study, we have analyzed the interaction between E. coli RNase HI and RNA-DNA hybrids using the BIAcore system, an instrument based on surface plasmon resonance technology. This technology is useful for obtaining kinetic data on the interaction between enzyme and substrate, particularly when the enzyme is inactive. This system enabled us to distinguish between inability to bind the substrate and inability to cleave. Furthermore, utilization of a 2Ј-O-methylated substrate permitted analysis of the effect of the Mg 2ϩ ions on substrate binding.
Preparation of RNA-DNA Hybrids-The RNA oligonucleotides biotinylated on the 5Ј-amino group (Fig. 1) were obtained from Integrated DNA Technologies and Oligos, Etc. DNA oligonucleotides ( Fig. 1) were obtained from Integrated DNA Technologies. All concentrations are expressed as moles of molecules and not bp. RNA and DNA oligonucleotides (1 M) were annealed in HBS buffer by boiling for 2 min and allowed to cool to room temperature to form RNA-DNA hybrids.
Construction and Purification of Wild-type and Mutant RNase HI-E. coli RNase HI wild-type protein (28) and the mutant protein D134A, in which Asp 134 is replaced by Ala (5), were prepared as described. The enzyme with an N-terminal 6ϫHis-Tag (6ϫHis-RNase HI) was obtained as described (16,23).
A group of plasmids for overproduction of the RNase HI variants with randomized sequences, in which Ile 82 and Val 101 are connected by four random amino acid residues resulting in deletion of the basic protrusion, was constructed as follows. Because the plasmid pJAL600 has a unique BamHI site in the sequence encompassing amino acid residues 81-83, and a unique SalI site about 50-base pairs downstream of the termination codon of the rnhA gene, the rnhA gene in plasmid pJAL600 was amplified by polymerase chain reaction using BamHI site-containing 5Ј-mutagenic primer, 5Ј-CCGGATCCNSNNSNNSNNSGTCGAT-CTCTGGCAACGTCTTGATGC-3Ј and SalI site-containing 3Ј-primer 5Ј-GGGTCGACCAATTCGCAGGCGGTTGG-3Ј. In these sequences, N represents A, G, C, or T and S represents G or C, and restriction sites are underlined. Polymerase chain reaction was performed in 25 cycles with a Perkin-Elmer DNA Thermal Cycler (model PJ2000) using a Gene Amp kit (Takara Shuzo Co., Ltd.) according to the procedure recommended by the supplier. These oligodeoxyribonucleotides were synthesized by Sawady Technology Co., Ltd. After digestion of the polymerase chain reaction fragment with BamHI and SalI, the resultant 280-base pair fragment and large BamHI-SalI fragment of pJAL600 were ligated to construct pJAL600⌬BP. The mutant proteins were overproduced in E. coli HB101 harboring plasmid pJAL600 derivatives as described previously (26). The cellular production levels of these proteins were estimated by subjecting whole cell lysates to SDSpolyacrylamide gel electrophoresis on a 15% polyacrylamide gel (29), followed by staining with Coomassie Brilliant Blue (R250).
Enzymatic Activity-RNase H activity was determined either in the presence of 10 mM MgCl 2 or 1 mM MnCl 2 by measuring the radioactivity of the acid-soluble digestion product of the substrate, 3 H-labeled M13 DNA-RNA hybrid, as described previously (16), or by degradation of 32 P-labeled poly(rA)⅐poly(dT) as described (30).
Protein Concentration-Protein concentrations were determined from the UV absorption, assuming that all mutant proteins, except for ⌬BP-RNase HI, have the same absorption coefficient, A 0.1% 280 ϭ 2.0, as that of the wild-type protein (31). The absorption coefficient of ⌬BP-RNase HI was estimated as 1.65 by using ⑀ ϭ 1576 M Ϫ1 cm Ϫ1 for Tyr (ϫ 5) and 5225 M Ϫ1 cm Ϫ1 for Trp (ϫ 4) at 280 nm (32), and a molecular mass of 15,860 Da.
Immobilization of RNA-DNA Hybrids on the Sensor Surface-RNA-DNA hybrids were prepared so that the RNA strand was biotinylated at the 5Ј-end. Streptavidin was covalently linked to the dextran on the surface of research-grade CM5 sensor chips via primary amino groups using the amine coupling kit from Pharmacia. Carboxylate groups on the dextran were activated by injecting a mixture of N-hydroxysuccinimide and N-ethyl-NЈ- (3-dimethylaminopropyl)carbodiimide followed by 20 l of 25 g/ml streptavidin in 10 mM sodium acetate, pH 5.7. Ethanolamine hydrochloride, pH 8.5, was injected to block unreacted N-hydroxysuccinimide esters. Typically, the resonance unit (RU) value increase following this procedure was 500 -1000. Twenty microliters of RNA-DNA hybrid (50 -100 nM) in TBS buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM ␤-mercaptoethanol, 0.005% Tween P20, pH 8.0) were injected onto the streptavidin-modified sensor chip giving an increase of 50 -100 RU. Unoccupied streptavidin was blocked by biotin.
Determination of the Kinetic Parameters of the Binding of RNase HI to Immobilized RNA-DNA Hybrids-All the proteins and poly(rA)⅐ poly(dT) were dissolved in TBS buffer or TBS buffer containing 10 mM MgCl 2 instead of EDTA (TBSM buffer). Samples were injected at 25°C at a flow rate of 5 l/min onto the sensor chip surface on which the RNA-DNA hybrid had been immobilized, or onto a control surface on which streptavidin had been immobilized and blocked with biotin. Binding surfaces were regenerated by washing with 2 M NaCl.
Immobilization of 6ϫHis-RNase HI on the Sensor Surface-Immobilization was carried out as described (25). The CM5 sensor chip was modified with NTA. Ni 2ϩ was chelated by injecting NiCl 2 onto the NTA-modified surface, which was followed by injection of 6ϫHis-RNase HI to immobilize the protein by chelation, giving a change in RU value of 800. The 36-bp RNA-DNA hybrid was then injected onto the surface. Binding surfaces were regenerated by washing with 0.5 M EDTA.
Analysis of the Binding Data in BIAcore-Sensorgrams for the interaction of RNase HI and RNA-DNA hybrids were analyzed with BIAlogue kinetics evaluation software, as described in the standard model (34), after subtraction of the values for the interactions between the samples and streptavidin-coated control surface. The association rate constants (k on ) were calculated from the association phases of the sensorgrams at various concentrations of the analyte, using Equation 1.
k off values obtained from Equation 1 are not very reliable for low ranges (34). Therefore, k off values were calculated from the dissociation phases of the sensorgrams using Equation 2.
Association constants (K A ) were calculated using Equation 3.
Association constants (K Aeq ) can also be obtained from equilibrium levels of the analyte binding to the surface (RU eq ) using Equation 4.
FIG. 1. The sequence of RNA-DNA hybrids. RNA-DNA hybrids used for the BIAcore analysis are listed. The 5Ј-ends of the RNA strands are biotinylated.

RESULTS
Enzymes Examined Using Plasmon Resonance-Three versions of E. coli RNase HI were examined by plasmon resonance studies as follows: wild-type enzyme, a mutant (D134A) with very low RNase H activity, and a protein whose handle or basic protrusion had been removed, presumably altering its ability to bind to RNA-DNA hybrids. This latter protein is described here for the first time.
Preparation of RNase HI Variant Missing the Basic Protrusion-By studying the binding kinetics of enzymes one can distinguish whether the lower activity of the mutant form of a protein is due to poor binding to the substrate or to a defect in catalytic activity. Previously, it has been shown that the inactivity of a protein derived from the RNase H domain of HIV-1 reverse transcriptase can be overcome by inserting handle region of E. coli RNase HI into the corresponding position of the HIV-1 RNase H protein. These results support the role of the handle in binding to the RNA-DNA substrate (18,19). To further substantiate this function of the handle region, we made a deletion of amino acid residues 83-100 and replaced it with four randomly generated amino acid residues. It has previously been shown that E. coli strain MIC3001, with the rnhA-339::cat and recB270(TS) mutation, can effectively screen for genes encoding a functional RNase H molecule (35). However, no clone was obtained after deletion-substitution of the handle region of E. coli RNase HI that could support growth of MIC3001 at the restrictive temperature, suggesting that the basic protrusion of E. coli RNase HI is critical for enzyme function in vivo. We screened the clones for an RNase HI variant that can be overproduced in a soluble form. When 22 E. coli HB101 transformants bearing pJAL600 derivatives were analyzed by SDS-polyacrylamide gel electrophoresis for overproduction, only one was able to produce soluble RNase HI in amounts suitable for subsequent purification (data not shown). Plasmid DNA was isolated from this transformant, and the DNA sequence of the mutated rnhA gene was determined. The deduced amino acid sequence connecting Ile 82 and Val 101 was Arg-Thr-Asn-Ser. This mutant protein, ⌬BP-RNase HI, was recovered in a soluble form after sonication lysis, and the lysate was subjected to DE52 chromatography using a column equilibrated with 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA. The protein was eluted from the column by linearly increasing the NaCl concentration from 0 to 0.5 M. Fractions containing the protein were combined and applied to a Sephacryl-S300 (Pharmacia) (2.2 ϫ 90 cm) equilibrated with 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.1 M NaCl. Fractions containing the pure protein were combined and used for further analyses. The amount of the mutant protein ⌬BP-RNase HI purified from a 1-liter culture was about 0.64 mg.
Characterization of ⌬BP-RNase HI-No enzymatic activity was detected for the mutant protein ⌬BP-RNase HI either in the presence of the Mg 2ϩ or Mn 2ϩ ion, indicating that this mutant is completely inactive. CD spectra of the wild-type protein and of the mutant protein ⌬BP-RNase HI are shown in Fig. 2. Few spectral changes were detected between these proteins in the far ultraviolet region, in which the spectra reflect the content of the secondary structure of the protein (Fig. 2a). This result suggests that the mutant protein ⌬BP-RNase HI folds correctly. On the other hand, in the near ultraviolet region, in which the spectra reveal environments of aromatic amino acid residues, the spectra were clearly distinguishable (Fig. 2b). This change in the spectrum may be due to a local conformational change upon the deletion of the basic protrusion and/or to the loss of two tryptophan residues.
Determination of Kinetic Parameters-For the binding of RNase HI molecules to the 36-bp RNA-DNA hybrid, TBS con-taining RNase HI at concentrations ranging from 10 nM to 1.5 M were passed, at a flow rate of 5 l/min, over the surface of the sensor chip, on which the substrate had been immobilized. The slope of the ln(RU 1 /RU n ) versus RU plots in each sensorgram was linear (data not shown), indicating that dissociation of the enzyme from the substrate occurs in an apparently first-order reaction. The sensorgrams are shown in Fig. 3. When the RNase HI molecules bound to the sensor chip were dissociated by running the RNase-free buffer at a flow rate of 5 l/min, the dissociation rate constants (k off ) decreased as the initial amount of the RNase HI molecules bound to the sensor chip decreased. This change is probably due to rebinding of the protein to the RNA-DNA hybrid on the sensor chip when the hybrid is present at high densities on the surface. Increasing the flow rate to 100 l/min increased the dissociation rate constant nearly 5-fold, but the k off value was still dependent on the initial number of RNase HI molecules bound to the sensor chip (Fig. 4). Addition of the poly(rA)⅐poly(dT) competing substrate to the buffer for dissociation at 39 g/ml resulted in a 10 -20-fold increase in the dissociation rate constant (Fig. 4). The k off values were nearly constant when the initial amount of the protein bound to the sensor chip increased, indicating that the competition completely abolishes the rebinding. In the presence of this competing substrate, binding of RNase HI to the immobilized hybrid was completely inhibited (data not shown).
k on values were calculated using Equation 1. Association profiles fit satisfactorily with a mono-exponential equation for protein concentrations up to 100 nM. k s versus C plots for 10 -80 nM protein concentrations were linear (Fig. 5), indicating that, unlike the k off values, the k on values are not affected by RNase H concentrations in the binding buffer.
Similar sensorgrams were obtained when the 30-, 24-, or 18-bp RNA-DNA hybrids were used as immobilized substrates (data not shown). Kinetic parameters of the interaction be- tween RNase HI and these substrates were similar with, at most, a 2-fold variation between them (Table I).
Biphasic RNase HI Binding to RNA-DNA Hybrids-When the concentration of RNase HI in the buffer was greater than 0.1 M, RU values showed a slow increase followed by a fast increase, never reaching a plateau (Fig. 6). This suggests that two binding phases exist, a slow phase and a fast phase. The RU eq /C versus RU eq plots for the fast binding phase were obtained over the range of 25 nM to 1.5 M by using the end points of the fast binding mode as the RU eq values (Fig. 7). These plots were biphasic with two different kinetic association constants. K A values derived from linearization of the plots obtained at low RNase HI concentrations ranged from 25 to 50 nM (K Aeq1 ) and at higher RNase HI concentrations from 0.4 to 1.5 M (K Aeq2 ) (Table I). At higher RNase HI concentrations, K Aeq values were approximately one-fourth to one-seventh those calculated from data collected at lower RNase HI concentrations.
Stoichiometry of RNase HI Bound to RNA-DNA Hybrids-An increase in RU values upon binding of analyte on the surface of the chip is proportional to the mass of analyte. Therefore, using the relationship ⌬RU nucleic acids ϭ 0.8 ϫ ⌬RU protein (36), one can calculate the ratio of RNase HI and RNA-DNA hybrid from the equation R ϭ ⌬RU RNase HI /⌬RU hybrid ϫ MW hybrid /MW RNase HI ϫ 0.8; where ⌬RU hybrid is the increase in RU value upon binding of the hybrid to the streptavidin surface, ⌬RU RNase HI is the increase in RU value upon binding of RNase HI to the hybrid, and MW hybrid and MW RNase HI are the molecular weights of the hybrid and RNase HI, respectively. The RU max values used for the determination of stoichiometry were obtained from the x intercepts of the RU eq /C versus RU eq plots. The stoichiometry obtained by linear transformation of the plots at lower RNase HI concentrations (25-50 nM (n 1 )) was 1.19 Ϯ 0.07, 1.34 Ϯ 0.10, 2.17 Ϯ 0.04, and 3.18 Ϯ 0.05 for the 18-, 24-, 30-, and 36-bp RNA-DNA hybrids, respectively. The stoichiometry obtained by linear transformation of the plots at higher RNase HI concentrations of (0.4 -1.5 M (n total )) was 1.56, 1.72, 2.62, and 4.11 Ϯ 0.14 for the 18-, 24-, 30-, and 36-bp RNA-DNA hybrids, respectively. The values n total Ϫ n 1 (n 2 ), which correspond to the number of the RNase HI molecules bound to the hybrids with the smaller K A values, were 0.37, 0.38, 0.45, and 0.92 for 18-, 24-, 30-, and 36-bp RNA-DNA hybrid, respectively. The numbers of RNase HI molecules binding to the hybrids in the slow binding phase (n slow ) was estimated from the increase in RU values in the slow binding phase at 1.5 M, because, even at this concentration, the slow binding phase was far from equilibrium. The n slow values were 0.19, 0.20, 0.31, and 0.56 for 18-, 24-, 30-, and 36-bp RNA-DNA hybrid, respectively.
Binding of RNA-DNA Hybrid to Immobilized 6ϫHis-RNase HI-Kinetic measurements were also carried out using the inverse experimental system, in which RNase HI molecules are immobilized on the surface. A histidine tag fused to the N terminus of RNase HI allows the protein to be immobilized on the chip in a unique homogeneous orientation. This enables one to analyze the interactions between a single RNase HI molecule and its RNA-DNA hybrid substrate. For binding of the 36-bp RNA-DNA hybrid to RNase HI, TBS buffer (without EDTA) containing various concentrations (from 50 nM to 1.5 M) of the 36-bp RNA-DNA hybrid were injected onto the surface of the chip, on which RNase HI had been immobilized. Immediately following injection of the hybrid, RNase HI (1 M) was injected as a competitor to avoid rebinding of the hybrid to other RNase HI molecules on the surface of the sensor chip. Binding of the 36-bp hybrid to immobilized RNase HI gave sensorgrams similar to those obtained for the binding of RNase HI to the immobilized hybrid (Fig. 8a). The RU versus RU/C plot was comprised of a dominant binding phase with high affinity and an additional binding phase with low affinity (Fig.  8b), similar to those obtained with immobilized substrate. Kinetic parameters were also similar to those determined when the substrate was immobilized (Table II).
Effect of 2Ј-O-methylation of RNA on Binding of RNase HI to RNA-DNA Hybrids-When binding of RNase HI to the 36-bp 2Ј-O-methylated substrate was analyzed by BIAcore, a rapid increase of the RU value was observed, which was followed by a slow increase in RU value (Fig. 9a). Such biphasic binding is similar to that observed for the binding to unmodified sub-strate. However, the slow phase observed in binding to the modified substrate was more pronounced than that observed for the normal substrate. For the rapid binding phase, the k on value for the modified substrate was one-seventh of that for the unmodified substrate, whereas the k off value for the modified substrate was 4-fold higher than that for the unmodified substrate (Table III). These results indicate that 2Ј-O-methylation of the substrate considerably impairs interaction between enzyme and substrate. When the RU eq /C values were plotted versus RU eq over a range of 0.1-1.5 M for the rapid binding phase, a linear relationship was observed. This indicates that The dissociation rate constant (k off ) and association rate constant (k on ) were calculated using BIAlogue software (Pharmacia Biosensor). The k on values were determined from k s versus C plot for the concentrations of RNase HI from 25 to 60 nM. The association constants were calculated from the ratio k on /k off (K A ) or were determined by measuring equilibrium sensor responses and subjecting them to RU versus RU/C plot analysis (K Aeq ). K Aeq1 and K Aeq2 values were determined from the plots for lower RNase HI concentrations from 25 to 50 nM and higher concentrations from 0.4 to 1.5 M, respectively. k off is the average and S.D. of measurements from the dissociation phase from three cycles where 25, 30, and 35 nM RNase HI were injected at 5 l/min at 25°C in TBS.   the binding is monophasic (Fig. 9b). The K A value obtained from the plot concurred with the k on /k off values. The stoichiometry calculated from the RU eq value was 3.7 Ϯ 0.38. The number of RNase HI molecules bound to the 36-bp modified substrate in the slow binding phase (n slow ) was estimated from the increase in the RU value at 1.5 M to be 0.88 Ϯ 0.05.
Because the 2Ј-O-methylated substrate cannot be cleaved by RNase HI, it was possible to determine kinetic parameters for binding of RNase HI to the modified substrate (36-bp) in the presence of 10 mM MgCl 2 . Slow monophasic binding was observed in the presence of 10 mM MgCl 2 (Fig. 10). The k on value was 3.5% that obtained for the rapid binding phase in the absence of MgCl 2 (Table III). The k off value was also lower than that obtained in the absence of MgCl 2 (Table III). Binding of the enzyme to the modified substrate in the presence of 10 mM MgCl 2 was so weak that the association constant and the stoichiometry could not be determined from the RU eq /C versus RU eq plots. The number of the RNase HI molecules bound to the modified substrate in the presence of 10 mM MgCl 2 was estimated from the increase in the RU value at 1.5 M to be 2.92 Ϯ 0.11.
Effect of Asp 134 to Ala Mutation on the Binding of RNase HI to an RNA-DNA Hybrid-Kinetic parameters for binding of the mutant protein D134A to the 36-bp unmodified substrate are shown in Table IV. The k on value was similar to that obtained for the wild-type enzyme, whereas the k off value was about half that of the wild-type enzyme. Consequently, the K A value for the mutant protein D134A is slightly higher than that of the wild-type protein. This result indicates that the Asp 134 to Ala mutation does not seriously affect the binding of RNase HI to the substrate. Kinetic analysis of the enzymatic activity has shown that the Asp 134 to Glu, Gln, Ser, and Thr mutations, which greatly reduce the enzymatic activity, do not seriously affect the K m value (5). However, this finding could not completely exclude the possibility that the Asp 134 to Ala mutation almost fully inactivates the enzyme by reducing the binding affinity of the enzyme to the substrate, because no kinetic data were available for the mutant protein D134A. The current study excludes such a possibility.
Binding of ⌬BP-RNase HI to RNA-DNA Hybrid-When the binding of ⌬BP-RNase HI (9.2 M) to the 36-bp RNA-DNA hybrid was analyzed by BIAcore, a slight increase in RU value was observed, which corresponds to that observed for 2.5 nM wild-type protein (Fig. 11). From the increase of the RU level, the affinity of the ⌬BP-RNase HI for the 36-bp RNA-DNA hybrid was estimated to be 0.025% that of the wild-type protein for the same substrate. This result suggests that the basic protrusion is the major contributor to the interaction between enzyme and substrate.

DISCUSSION
Nature of Binding Properties of RNase HI to RNA-DNA Hybrid-In this study, association and dissociation of RNase HI with RNA-DNA hybrid substrates were monitored in real time using the BIAcore system. Kinetic parameters for binding of RNase HI to hybrids were constant for concentrations up to 0.1 M. When RNaseHI was injected at this concentration, we calculated that 0.9 and 2.8 RNase HI molecules were bound to the 18-and 36-bp hybrids, respectively. Similar kinetic parameters were also obtained for binding of the 36-bp hybrid to immobilized RNase HI which binds only a single substrate molecule. These results suggest that multiple RNase HI molecules bind to the substrate simultaneously and independently with little or no cooperativity. K D values obtained in this study (ϳ2 ϫ 10 Ϫ8 M) agree well with those obtained from the titration of RNA-DNA hybrids with RNase HI using data collected from changes in CD spectra upon binding to the substrate (10 Ϫ8 -10 Ϫ9 M) (37).
We observed two phases of binding (with fast and slow rates) when the concentration of RNase HI was greater than 0.1 M (Fig. 6). Of the total protein bound to the substrate at 1.5 M, ϳ10% bound at a slower "on" rate and 90% bound at a faster  Table I rate. The phase with the faster on rate consisted of two parts, 70% with high and 20% low association constants, as indicated by the biphasic RU versus RU/C plot (Fig. 5). It is likely that the binding with the higher association constant is predominant under physiological conditions under which the concentration of RNase HI is expected to be low. Therefore, it also seems likely that binding with higher affinity is productive binding, with additional, nonspecific binding. One explanation for the secondary interactions is that free RNase HI may interact with RNase HI molecules already bound to the substrate. However, multiphasic binding was also observed when RNase HI was immobilized, and the 36-bp RNA-DNA hybrid was in the mobile phase. Taken together, these results suggest an additional weaker interaction between RNase HI and the substrate that is separate from the normal binding site.
Length of the RNA-DNA Hybrid Interacts with RNase HI-The numbers of RNase HI molecules bound to the hybrids with high association constants are ϳ1.2 for the 18-bp hybrid, ϳ1.3 for the 24-bp hybrid, ϳ2.2 for the 30-bp hybrid, and ϳ3.2 for the 36-bp hybrid. Kinetic analyses, under conditions in which the RNA-DNA hybrid was cleaved at a unique site (22), or using RNA-DNA hybrids with 2Ј-O-methylnucleosides, which limits the cleavage at a single site (38), suggest that RNase HI interacts with 9 -10 bp of RNA-DNA hybrid. Using these values, one can expect the maximum number of RNase HI molecules able to bind to the 18-, 24-, 30-, and 36-bp hybrid to be 1-2, 2, 3, and 3-4, respectively. However, to completely saturate these relatively short RNA-DNA hybrids, each RNase HI molecule(s) would need to bind in such a manner as to permit the maximum number of other RNase HI molecules to bind. For example, if one RNase HI molecule were to bind to the middle of the 18-bp RNA-DNA hybrid, there would be only 4 bp at either end of the hybrid accessible to other RNase HI molecules. Thus, the observed values will always be lower than the maximum values. Therefore, our current results are consistent with a 9 -10-bp binding size (22,38).
Effect of 2Ј-O-Methylation of RNA in Hybrid on Binding of RNase HI-It has been reported that the conformations of 2Ј-O-methyl RNA-DNA hybrids are similar to those of normal RNA-DNA hybrids (39). Therefore, a 2Ј-O-methyl RNA-DNA hybrid can provide a good model with which to investigate the interaction between RNase HI and the substrate. Our data show that the association constant between RNase HI and the 2Ј-O-methylated substrate was one-thirtieth that between enzyme and the substrate. Methylation of 2Ј-OH group at the cleavage site, which greatly reduces catalytic efficiency, also slightly reduces the affinity between the enzyme and the substrate (a 5-fold difference in the K m value), possibly due to steric hindrance (9). It is reported that the 2Ј-OH group of the nucleoside adjacent to the 3Ј-side of the cleaved phosphodiester bond acts as a proton donor and acceptor and that the second nucleoside from the 5Ј-side of the cleaved phosphodiester bond acts as a proton acceptor (20). Therefore, the reduced affinity  Table I   consequent to 2Ј-O-methylation may be due to steric hindrance by the methyl groups and/or loss of the hydrogen bonds. These results support an interaction of 2Ј-OH group and RNase HI. However, the current results cannot exclude the possibility that methylation of the 2Ј-OH group of the hybrid creates a substrate that binds to the enzyme in a different manner (e.g. in the opposite orientation or with different spacing).
Effect of Magnesium Ions on the Binding of RNase HI to a 2Ј-O-Methyl RNA-DNA Hybrid-The presence of Mg 2ϩ may affect the RNase HI/RNA-DNA interaction in two ways as follows: first, Mg 2ϩ may neutralize the charge of the phosphates and decrease the ionic interaction between the basic protrusion and the RNA-DNA hybrid; and second, Mg 2ϩ binds to the acidic amino acid residues at the catalytic center of the enzyme, a process necessary for cleavage. We find that, in the presence of Mg 2ϩ , the affinity between RNase HI and 2Ј-Omethyl RNA-DNA hybrid was reduced by 83% ( Fig. 9 and Table  III). A large reduction in affinity (3-4 orders of magnitude) in the presence of Mg 2ϩ has been reported for the nonspecific interaction between EcoRV and DNA (24), a reduction that was interpreted as due to the displacement by the enzyme of Mg 2ϩ bound to DNA. Using ⌬BP-RNase HI, a protein lacking the basic protrusion, we found a drastic reduction in substrate binding (Fig. 11). Therefore, we believe the reduction in affinity in the presence of Mg 2ϩ results from the displacement of Mg 2ϩ bound to the RNA-DNA hybrid by the basic protrusion. In contrast, the specific interaction between EcoRV and DNA is not affected by Mg 2ϩ ions (24). Thus, the effect of the Mg 2ϩ ions seems to depend on its mode of interaction. Such a difference in the effect of the Mg 2ϩ ions could account for a moderate reduction in affinity of RNase HI for its substrate as compared with that of EcoRV for DNA. However, a 20% reduction in the "off" rate counteracted, in part, this reduction in affinity. In the absence of the Mg 2ϩ ion, electrostatic repulsion between the RNA-DNA hybrid and the acidic amino acid residues in the catalytic center of RNase HI (Asp 10 , Glu 48 , Asp 70 , and Asp 134 ) would be expected to decrease affinity. The catalytic Mg 2ϩ ion would eliminate the negative charge repulsion at the active site of the enzyme, resulting in less release of the enzyme from the substrate.
Enzymatic Activity and Tertiary Structure of ⌬BP-RNase HI-Keck and Marqusee (40) have recently shown that the basic protrusion of E. coli RNase HI is not essential for activity. They reported that the mutant protein, in which the amino acid residues 83-95 are replaced by six glycine residues, exhibited RNase H activity in the presence of the Mn 2ϩ ion but not in the presence of the Mg 2ϩ ion. In contrast, we showed that ⌬BP-RNase HI, in which residues 83-100 are replaced by Arg-Thr-Asn-Ser, exhibited little RNase H activity either in the presence of the Mg 2ϩ or Mn 2ϩ ion. This apparent discrepancy in the enzymatic activity of the mutant protein, which lacks the basic protrusion, may result from the difference in the size of the deleted region or in the flexibility of the linker which is substituted for the basic protrusion.
In this report, we discussed the importance of the basic protrusion for the interaction with the substrate, on an assumption that ⌬BP-RNase HI has basically the same folding topology as that of the wild-type protein. The possibility that the deletion of the basic protrusion causes a gross structural change may not be excluded, because ⌬BP-RNase HI has little enzymatic activity. However, the similarity in the far UV CD spectra between ⌬BP-RNase HI and the wild-type protein and the fact that both the RNase H domain of HIV-1 reverse tran-scriptase and the E. coli RNase HI variant with the deletion of the basic protrusion (40) fold correctly strongly suggest that ⌬BP-RNase HI also folds correctly.