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J. Biochem. 116, 1322-1329 (1994)

Role of the Mg2+ Ion in the Escherichia coli HI Reaction

Yohtaro Uchiyama,*.t Shigenori Iwai,$ Yoshio Ueno,*.t Morio Ikehara,"g and Eiko Ohtsuka$" 'Faculty of Pharmaceutical Sciences , Science University of Tokyo, Shinjuku-ku, Tokyo 162; tResearch Institute for Biosciences, Science University of Tokyo, Noda, Chiba 278; tFaculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060; and Engineering Research Institute, Suita, Osaka 565

Received for publication, August 15, 1994

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Col+, Zn2+, or Cd2+, the phosphorothioate linkage with the Re-configuration was cleaved, while the Se-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg2+-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the K,15 values were almost the same. These results are evidence of an interaction between Mg 2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.

Key words: divalent metal cations, mechanism for catalysis, 2'-modified RNA, phospho rothioate, .

RNase H degrades only the RNA strand of a DNA-RNA droxyl relay mechanism (9), and the other is the two-diva hybrid duplex in the presence of a divalent metal cation, lent metal ion mechanism (5). Although the RNase H such as Mg" or Mn2t, producing 5'-phosphate and 3'-hy of HIV reverse transcriptase (RTase) contains two droxyl ends. This is present in various organisms Mn2+ ions at its (17), the crystal structure of E. ranging from viruses to (1). Viral RNase H is coli RNase HI complexed with Mg" (4, 6, 7) and metal present in the C-terminal domain of reverse transcriptase titration experiments by NMR (10) show only one Mg" ion (RTase) (2). The physiological function of the cellular bound to the active site of the E. coli enzyme. In these RNase H remains to be investigated, although this enzyme studies, it was proposed that the non-bridging oxygens of is suggested to be involved in DNA replication or repair (3). the scissile phosphodiester linkage were coordinated to the The viral RNase H is essential for reverse transcription of Mg" ion, and the role of this Mg" was to fix this linkage at the viral single-stranded RNA genome into double-strand the position for the reaction (7, 9). These mechanisms were ed DNA (2). Since the retroviral RNase H can be a target proposed on the basis of the structural studies performed in of antiviral therapy, especially against human immunodefi the absence of the substrate. Investigation of the interac ciency virus (HIV), studies on RNase H have been promot tions between the scissile RNA strand of the DNA.RNA ed recently. hybrid duplex and the Mg" ion in the ternary complex is Among these from various species, Escherichia required for the elucidation of the mechanism for the coli RNase HI [EC 3.1.26.4] has been studied most RNase H cleavage reaction. extensively, and its structure-function relationship has Our previous work (18) showed that the pro-SP oxygen of been investigated by X-ray (4-8), NMR the scissile phosphodiester linkage interacted with an spectroscopy (9-11), and site-directed mutagenesis (9, 12 amino acid residue of the RNase H protein, but not with the 15). The four acidic amino acid residues (Asp10, G1u48, Mg" ion, and the 2'-hydroxyl function on the 5'-side of the Asp70, and Asp134), which are fully conserved at the scissile linkage was involved in catalysis, but not in binding. corresponding positions in the sequences of various RNase In this study, the interaction of the Mg" ion with the H , are the ligands for the metal cation and form the oxygens of the phosphate residue or the 2'-hydroxyl func active center (16). tion at the cleavage site was investigated, to elucidate the Two alternative mechanisms have been proposed for the role of the metal ion in the catalytic reaction of E. coli RNase H cleavage reaction. One is the carboxylate-hy RNase HI. Direct evidence for the interaction between the metal ion and the 2'-hydroxyl of the substrate was obtained T whom correspondence should be addressed. by measuring the Mg2+-dependence of the rate constants Abbreviations: dUa, 2'-amino 2'-deoxyuridine; dU, 2'-deoxyuridine; for 2'-modified substrates. The number of Mg2+ ions DMT, 4,4'-dimethoxytrityl group; dU,, 2'-fluoro-2'-deoxyuridine; essential for catalysis and the affinity of the Mg2+ ion for the HIV, human immunodeficiency virus; Cm, 2'-0-methylcytidine; G., RNase H complexes with the substrates were determined 2'-O-methylguanosine; Um, 2'-O-methyluridine; RTase, reverse transcriptase. by Hill plots.

1322 J. Biochem. Mechanism for RNase H Reaction 1323

ml/min at 50°C. The retention times were 13.5 min for MATERIALSAND METHODS rGGAGAUGAC, 12.6 min for r(GGAGA)dUar(GAC), 18.5 min for r(GGAGA)dU,r(GAC), and 15.4 min for r(GGA Preparation of Oligonucleotides-Oligonucleotides (Fig. GA)dUr(GAC). The separation of the isomers of rGGAG 1) were synthesized on an Applied Biosystems 394 DNA/ AUPSGAC was not satisfactory under these conditions. RNA synthesizer by the standard phosphoramidite meth These isomers were separated by isocratic elution with 6% ods (19), starting from 1.0 pmol of the 3'-terminal nu acetonitrile in 0.1 M TEAA (pH 7.0). The retention times cleoside on the controlled-pore glass (CPG) support, using were 34.7 min for the Re-isomer, and 41.0 min for the commercially available reagents (MilliGen Biosearch for SP-isomer. The purified oligonucleotides were analyzed by RNA and Applied Biosystems for DNA). The 9-mer con anion-exchange HPLC on a DEAF 2SW column (4.6 x 250 taining 2'-0-methyl nucleosides, GmUmCmd(ATCT)CmCm mm, Tosoh). Elution was performed with a linear gradient (Xmrepresents the 2'-0-methyl nucleoside), was prepared of ammonium formate (from 0.4 to 1.0 M during 30 min) in as described in the literature (20). Modified nucleosides, 20% acetonitrile, at a flow rate of 1.0 ml/min at 50°C. The 2'-trifluoroacetylamino-2'-deoxyuridine and 2'-fluoro-2' retention times were 22.7 min for rGGAGAUGAC, 21.0 deoxyuridine, were prepared as described (21, 22) and min for r(GGAGA)dUar(GAC), 22.8 min for r(GGAGA) phosphitylated with 2-cyanoethyl N,N-diisopropylchloro dU,r(GAC), 22.6 min for r(GGAGA)dUr(GAC), 26.4 min phosphoroamidite (23) after protection of the 5'-hydroxyl for RP-rGGAGAUPSGAC, and 26.7 min for SP-rGGAGA function with a 4,4'-dimethoxytrityl (DMT) group. The UPSGAC. phosphorothioate linkage was introduced by sulfurization Labeling of the 5'-End of the 9-mer RNA--The oligo with tetraethylthiuram disulfide before capping (24). After ribonucleotides (20 pmol) were labeled using [y-32P]ATP cleavage from the CPG with a mixture of aqueous ammonia (10 pCi) and T4 polynucleotide kinase (from E. coli strain and ethanol (3 : 1, v/v) at 25'C for 2 h and removal of the A19, Takara Shuzo, 3 units) in 50 mM Tris-HC1 (pH 8.0), acyl protecting groups at 55'C for 16 h, the 5'-DMT 10 mM MgCl2, and 5 mM dithiothreitol (5 ul). After incu derivatives were isolated on a column (0.7 x 15 cm) of bation at 37'C for 30 min, the mixtures were diluted with preparative C18 125 A (Millipore). Elution was performed water to 200 p 1and passed through a NENSORB 20 column with a linear gradient of acetonitrile, from 0 to 40% (v/v) (Du Pont). in 0.1 M triethylammonium acetate (TEAA) buffer (pH Assignment of the Absolute Configuration of the Phos 7.0). The 5'-DMT group was removed with hydrochloric phorothioate-The absolute configuration at the P chiral acid at pH 2.0 (25). The fully deprotected oligonucleotides center was assigned enzymatically using ribonuclease A. were purified by reverse-phase HPLC and anion-exchange This enzyme cuts the RP-isomer of the phosphorothioate HPLC. Reverse-phase HPLC was carried out on an Iner linkage almost exclusively (26). The labeled oligonucleo tosil ODS-2 column (4.6 X 250 mm, GL Sciences). For the tides (0.1 pmol) were mixed with tRNA (0.3 A260 unit), 2'-modified 9-mer RNA, elution was performed with a then treated with ribonuclease A (5 ng, Sigma) in 10 pl of linear gradient of acetonitrile [from 5 to 9% (v/v) during 20 50 mM Tris-HCI buffer (pH 7.5) containing 0.1 M EDTA at min] in 0.1 M TEAA buffer (pH 7.0), at a flow rate of 1.0 25'C for 3, 6, 10, 20, 30, and 60 min. The products were separated by electrophoresis on a polyacrylamide gel containing 7 M urea. The radioactivity of each band was quantified with a Fujix BAS2000 Bioimage Analyzer. The isomer with a retention time of 34.7 min by reverse-phase HPLC was assigned as RP and that with a retention time of 41.0 min was assigned as SP. Cleavage Reaction of the Phosphorothioate Containing Substrate with Various Divalent Metal Ions-Each 5'-32P labeled isomer of rGGAGAUPSGAC (10 pmol), mixed with 1.2 molar equivalents of the complementary GmUmCm d(ATCT)CmCm, was treated with E. coli RNase HI (0.02 pmol, provided by Dr. S. Kanaya) at 30'C in 10 p l of 10 mM Tris-HC1 buffer (pH 8.0), 1 mM of a divalent metal ion (MgC12, CaC12, MnC12, CdC12, ZnC12, CoC12i SrC12j or BaC12), 50 mM NaCl, 1 mM 2-mercaptoethanol, and 0.01% bovine serum albumin. After 2 h, 20p l of loading solution (10 M urea and 50 mM EDTA) was added, and the products were separated on a 20% polyacrylamide sequencing gel (19 : 1, acrylamide/bisacrylamide) containing 7 M urea (0.3 mm X 40 cm) . They were identified by comparison with the degradation products of the 5'-32P-labeled 9-mer RNA treated with snake venom . Thio-Effects in the Cleavage Reaction-The labeled 11 rGGAGAUPSGAC (10 pmol) mixed with the complement ary strand was treated with RNase H at 3O`C for 10 min in the presence of 10 mM of a divalent metal ion, either MgCI2i MnC12, CdCI2, ZnC12, or CoCI2, in 10 pl of the same Fig. 1. The structures of the ribo-strands of substrates con buffer. The amounts of RNase H were as follows: 0.0002 taining a single modification at the cleavage site for RNase H.

Vol. 116, No. 6, 1994 1324 Y. Uchiyama et al. pmol for Mg", 0.002 pmol for Mn2+ and Cot+, and 0.2 pmol venom phosphodiesterase. for Zn2+ and Cd2+. The apparent rate constants k (min-') The Mgt+-Dependence of the Cleavage Reactions of the were obtained by dividing the initial rate by the amount of 2'-Modified Substrates-Labeled rGGAGAUGAC, r(GGA RNase H. The thio-effect represents the ratio of the GA)dUar(GAC), r(GGAGA)dUfr(GAC), and r(GGAGA) apparent rate constant for the phosphorothioate to that for dUr(GAC) (10 pmol), hybridized to the complementary the phosphodiester, in the presence of each divalent metal strand, were treated with RNase H in the presence of Mg2+ ion. The amounts of RNase H were 0.0002 pmol for rGGAG Cleavage Reaction of the 2'-Modified Substrates-The AUGAC, 0.002 pmol for r(GGAGA)dUar(GAC), 0.2 pmol 5'-32P-labeled 2'-modified substrates (10 pmol), mixed for r(GGAGA)dUar(GAC), and 2.0 pmol for r(GGAGA) with 1.2 molar equivalents of the complementary GmU,,C,,, dUr(GAC). The Mg2+-concentration was varied from 0 to d(ATCT)CmCm, were treated with RNase H (0.01 pmol for 10 mM, and the apparent rate constants were obtained as a the -OH, -NH2, and -F substrates and 1 pmol for the -H function of the Mg" concentration. substrates) in 10 pl of 10 mM Tris-HCl buffer (pH 8.0), 10 MM MgC12, 50 mM NaCl, 1 mM 2-mercaptoethanol, and RESULTS 0.01% bovine serum albumin. After incubation at 30'C (15 min or 2 h for the -OH, -NH2, and -F substrates, 1 h for the Cleavage Reaction of the Phosphorothioate Linkage in -H substrate), 10 ,ul of the loading solution (10 M urea and the Presence of Various Divalent Metal Ions-To investi 50 mM EDTA) was added to 5-pl aliquots of the reaction gate the interaction between the non-bridging oxygens of mixtures, and the products were separated on a 20% the scissile phosphodiester bond and the Mg2+ ion in the E. polyacrylamide sequencing gel (19: 1, acrylamide/bis coli RNase HI ternary complex, the cleavage reaction of the acrylamide) containing 7 M urea (0.3 mm x 40 cm). They substrates containing a phosphorothioate was analyzed in were identified by comparison with the degradation prod the presence of various divalent metal ions. A single ucts of the 5'-32P-labeled 9-mer RNA treated with snake phosphorothioate linkage with a defined configuration was

Fig. 2. RNase H cleavage of the phosphorodiester (PD) and the RP and the SP-phosphorothio ates in the presence of various divalent metal ions.

J. Biochem. Mechanism for RNase H Reaction 1325

Fig. 3. Cleavage of r(GGAGA) Xr(GAC) G,U,,,Cmd(ATCT)C,„C R, b y E. coli RNase HI, where X represents the modified uridine. The concentration of RNase H was (a) 1 nM and (b) 100 nM.

Fig. 4. Mgt}-dependence of the cleavage rate in the RNase H reaction. (a) The -OH substrate , (b) the -NH2 substrate, (c) the -F sub strate, (d) the -H substrate.

introduced into a 9-mer duplex containing 2'-0-methyl MgZ+, Mn2+, Cot+, Zn2}, or Cd2+ in the same way as the ribonucleosides, rGGAGAUp5GAC-GmUmCmd(ATCT) parent duplex containing a phosphodiester bond at this site . I n contrast, the S5-isomer was not cleaved at this site CmCm, where Ps and Xm represent the phosphorothioate , but linkage and the 2'-0-methylnucleoside, respectively. The at the neighboring phosphodiester bond . The cleavage site cleavage reaction of the phosphorothioate -containing sub was shifted to A5-U6 in the presence of Mg2+ or Mn2+ , and t strates was carried out in the presence of Mgt+, Mn2+, Ca 2+' o A5-U6 and G7-A8 in the presence of Co2+ or Zn2+ . The S Cd2+, Cot+, Zn2+, Sr2+, or Ba2+ (Fig. 2). The RP-isomer was P-isomer with Cd2+ was not cleaved at any position under cleaved at the phosphorothioate linkage in the presence of these conditions. In the presence of an alkaline earth metal

Vol. 116. No. 6, 1994 1326 Y. Uchiyama et al.

Fig. 5. Hill plots of the Mg2+ dependence of the cleavage rate. (a) The -OH substrate, (b) the -NH2 sub strate, (c) the -F substrate.

TABLE I. Thio-effects in the presence of various divalent metal ions.

other than MgZ+, i. e. Ca 2+' Sr2+, or Bat+, no cleavage of the TABLE II. The dissociation constants for the Mg" ion (K,,,,) and the Hill coefficients obtained from the Hill plots. parent or phosphorothioate duplex was observed. Comparison of the Rate Constants between the Phosphate and RP-Phosphorothioate Substrates-The apparent rate constants in the presence of 10 mM each divalent metal ion were compared between the phosphodiester and R,-phos phorothioate substrates. The Rp-phosphorothioate/phos phodiester ratios, which reveal the thio-effects, are listed in Table I. This thio-effect followed the order of Cd2+> Zn2+> plexes with the 2'-modified substrates were obtained by Mn2+>Co2+>Mg2+, which agreed with the preference of Hill plots (Table II). It was found that the KMgvalues were the metal ions for the sulfur atom, as determined by the almost the same. Those for the -NH2 and -F substrates relative binding of the divalent metal ions to ATP/3S (27). were even smaller than that for the -OH substrate. Cleavage Reaction of the 2'-Modified Substrates-Our Cleavage Reaction with the Substrate Containing Deox previous work suggested that the 2'-hydroxyl function on yuridine-The substrate analogue containing deoxyuridine, the 5'-side of the scissile linkage was involved in catalysis r(GGAGA)dUr(GAC).GmUmCmd(ATCT)CmCm, was treat but not binding (18). In this study, the 2'-OH was replaced ed with RNase H. At high enzyme concentrations, this by either -NH, (dUa), -F (dUf), or -H (dU), in order to substrate was cleaved at the same site. The rate constants investigate the interaction of the 2'-hydroxyl function with at various Mg" concentrations were measured (Fig. 4d), the Mg" ion. These 2'-modified substrates, r(GGAGA) and it was found that the cleavage rate for this substrate Xr(GAC) GmUmCmd(ATCT)CmCm (X=dUa, dUf, or dU), was the largest in the absence of the Mg" ion, reduced by were treated with RNase H. It was found that all of them the addition of Mg" up to 1 mM, and was constant at higher were cleaved at the expected site, between X and G (Fig. 3). Mg" concentrations. Mg' -Dependence of the Cleavage Rates for the 2'-Modi fied Substrates-The MgZ+-dependence of the cleavage rate DISCUSSION for the 2'-modified substrates was measured under multi ple-turnover conditions (Fig. 4). The cleavage rates for the Modified oligonucleotide duplexes have been used to study -OH , -NH,, and -F substrates increased, depending on the interactions and reactions of nucleic acids. Substitution of MgZ+concentration, within the range of 0 to 3 mM. The Hill the 2'-hydroxyl function was carried out to investigate plot for each substrate was linear at Mg" concentrations reactions (28-31), and the internucleotide phos lower than 3 mM, with a slope around 1.0 (Fig. 5). The phodiester linkage was changed into a phosphorothioate to dissociation constants for the Mg2+ ion (K,,g) in the com study the interactions between the EcoRI and

J. Biochem. Mechanism for RNase H Reaction 1327 its substrate (32). To apply these modifications to the RNase H reaction, the cleavage site must be restricted to a single position, since the RNA strand of a DNA-RNA hybrid duplex is cleaved at various sites by this enzyme. This restriction can be achieved using a 2'-0-methyl oligoribonucleotide containing a tetradeoxynucleotide gap as the strand complementary to the RNA (20). In the present study, these modifications were combined in order to elucidate the role of the metal ion in the RNase H reaction. Several 2'-modified nucleosides, such as 2' amino 2' deoxyuridine, 2'-fluoro-2'-deoxyuridine, and 2' deoxyuridine, and a phosphorothioate linkage were incor porated into the RNA strands (Fig. 1), which were annealed to the complementary strand containing 2' O-methylribo nucleotides. Mg" Ion Is Not Coordinated to the Non-Bridging Oxygens of the Phosphodiester Bond-Our previous work showed that the pro-Sp oxygen of phosphodiester at the cleavage site was not a ligand of the metal cation (18). In this work, the interaction between the pro-RP oxygen at Fig. 6. A mechanism for the RNase H reaction. A single Mg2' ion this site and the Mg" ion was investigated by detailed is essential for the catalysis. The Mg" ion interacts with the kinetics, using phosphorothioate substrate analogs in com 2'.hydroxyl function at the cleavage site by the formation of an bination with various divalent metal ions. The alteration of outer-sphere complex with a water molecule, and the proton of this water is abstracted by the 3'-leaving oxyanion. The general base that this phosphodiester linkage into the RP-phosphorothioate activates the attacking water is proposed to be Asp 70, by the did not affect the cleavage site (Fig. 2). The ratios of the site-directed mutagenesis (12) and X-ray crystallographic study (7). thio-effects for Mn2+ and Cd2+ to that for Mg" were 10 (3.1 for Mn/0.31 for Mg) and 52 (16 for Cd/0.31 for Mg), respectively (Table I). If the metal ion is coordinated to the metal ion responsible for the reaction could be analyzed by this plot (Fig. 5). The slope (the Hill coefficient) of 1.0 for pro-Re-oxygen of the phosphodiester, these values should be close to those determined by the relative binding of these the -OH substrate indicates that one independent Mg" ion ions to ATP,6S (200 and 1.7 x 106 for Mn and Cd, respec is involved in the catalytic reaction. Although the slopes for tively) (27), but this is not the case. These results indicate the -NH, and the -F substrates are not quite straight, it that the pro-RP oxygen of the phosphate residue at this site may show a slight conformational change of the sugar does not interact with the metal ion in the cleavage reac puckering in the RNase H-modified substrate complexes tion. Since the order of the thio-effect was identical with the with increment of the Mg" ion. This result agrees with the data obtained by X-ray crystallography (6) and NMR preference for the sulfur atom, metal ions such as Zn2+ and spectroscopy (10), although these structural studies were Cd2+ may stabilize the leaving group by coordination to the carried out in the absence of the substrate. Therefore, the sulfur atom of the phosphorothioate after hydrolysis, and catalytic mechanism of E. coli RNase HI is different from thereby help the release of the product. the two-metal-ion mechanism proposed for the RNase H The SP-phosphorothioate linkage was not cleaved in the domain of HIV RTase, and, as discussed later, this single presence of Mg", and the cleavage of this linkage was not metal ion is involved in the catalysis by forming an restored in the presence of other divalent metal ions, Mn2+, outer-sphere complex with the 2'-OH at the cleavage site. Cot+, Zn2+, and Cd2+, which can be coordinated to a sulfur The Mg2+ Ion Interacts with the 2'-OH by Forming an atom more strongly than Mg'} (Fig. 2). These results Outer-Sphere Complex-The apparent rate constants for confirmed that the pro-Se-oxygen does not interact with the the -OH, -NH2, and -F substrates increased depending on metal ion in the cleavage reaction. the Mg2+ concentration, while that for the -H substrate did No cleavage was observed in the presence of an alkaline not increase and was much smaller than those for the other earth metal other than Mg2+ under the usual conditions. In 2'-modified substrates (Fig. 4). Since, the affinity of these the X-ray crystallographic study of the complexes with substrates for the enzyme is almost the same (18), these alkaline earth metals, these metal ions were found at the results demonstrate an interaction between the Mg2+ ion same position as Mg2+ in the active site of E. coli RNase HI and the 2'-functional group, which is essential for the (6), but our observation suggests that the exchange rate of normal RNase H reaction. these metals is too large to allow the formation of a stable The affinity of the Mg2+ ion for the enzyme-substrate complex for the reaction. complex (KM,) was not affected by substituting the -NH2 or A Single Mg" Ion Is Required for Catalysis-Determi -F group for the 2'-OH (Table II). Since the 2'-fluorine atom nation of the number of divalent metal ions essential for the cannot be a ligand for the Mg" ion, these results demon catalysis is very important for the analysis of the phospho strate that the metal ion interacts by coordination to a ryl-transfer reaction of RNase H. In this study, the number water molecule which forms a hydrogen bond with the of divalent metal ions was determined from the kinetics of 2'-functional group at the cleavage site (Fig. 6). In the the cleavage reaction. The Hill plot for the RNase H complex with the -NH2 substrate, this amino group can be reaction with the -OH substrate was linear, showing that a proton donor in the hydrogen bond, in the same way as the the enzyme-substrate complex was formed throughout the OH substrate. In the case of the -F substrate, a similar range of Mg" concentrations and that the binding of the

Vol. 116, No. 6, 1994 1328 Y. Uchiyama et al. complex can be formed, since the fluorine atom can be a Morikawa, K. (1992) Structural details of ribonuclease H from proton acceptor when the water molecule becomes the Escherichia coli as refined to an atomic resolution. J. Mol. Biol. proton donor, but this water molecule is less reactive. 223,1029-1052 Consequently, almost the same Km, value was obtained for 7. Katayanagi, K., Okumura, M., and Morikawa, K. (1993) Crystal structure of Escherichia coli RNase HI in complex with Mg" at the complex with the -F substrate, although the reaction 2.8 A resolution: Proof for a single Mg" -. Proteins: rate was reduced to a great extent. Struct. Funct. Genet. 17, 337-346 In the reaction with the -H substrate, cleavage was 8. Katayanagi, K., Ishikawa, M., Okumura, M., Ariyoshi, M., observed without Mg". The rate constant was the largest in Kanaya, S., Kawano, Y., Suzuki, M., Tanaka, I., and Morikawa, the absence of Mg" and was constant at Mg" concentra K. (1993) Crystal structures of ribonuclease HI active site tions higher than 1 mM (Fig. 4d). A different reaction mutants from Escherichia coli. J. Biol. Chem. 268, 22092-22099 mechanism, independent of Mg", can be assumed for this 9. Nakamura, H., Oda, Y., Iwai, S., Inoue, H., Ohtsuka, E., Kanaya, S., Kimura, S., Katsuda, C., Katayanagi, K., Morikawa, K., substrate, in which one of the acidic amino acid residues at Miyashiro, H., and Ikehara, M. (1991) How does RNase H the active site plays a role as a general-acid-catalyst in the recognize a DNA-RNA hybrid? Proc. Natl. Acad. Sci. USA 88, absence of Mg". After forming a complex with Mg2 }, this 11535-11539 amino acid was used as a ligand to Mg", and the normal 10. Oda, Y., Nakamura, H., Kanaya, S., and Ikehara, M. (1991) RNase H reaction was observed. However, the reaction rate Binding of metal ions to E. coli RNase HI observed by 'H-''N was very slow, even slower than that for the Mg2+-indepen heteronuclear 2D NMR. J. Biomol. NMR 1, 247 255 dent reaction, due to the lack of interaction between the It. Oda, Y., Iwai, S., Ohtsuka, E., Ishikawa, M., Ikehara, M., and metal ion and the 2'-functional group. In this case, it is Nakamura, H. (1993) Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy. Nucleic Acids Res. 21, likely that this cleavage reaction without Mg" was ob 4690-4695 served because the rate constant of the cleavage reaction 12. Kanaya, S., Kohara, A., Miura, Y., Sekiguchi, A., Iwai, S., Inoue, for the -H substrate in the presence of the Mg" ion was H., Ohtsuka, E., and Ikehara, M. (1990) Identification of the very small, and the same reaction with the -OH substrate amino acid residues involved in an active site of Escherichia coli might exist in the absence of Mg", although it was much ribonuclease H by site-directed mutagenesis. J. Biol. Chem. 265, slower than the normal Mg"-dependent reaction. 4615-4621 The Role of the Metal Ion-In this study, two sources of 13. Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Role of cysteine residues in ribonuclease H from Escherichia coli. direct proof have been obtained for a model of the interac Site-directed mutagenesis and chemical modification. Biochem. tions in the RNase H ternary complex (Fig. 6). One is a J. 271, 59-66 single metal ion required for the catalytic reaction, and the 14. Kanaya, S., Katsuda-Nakai, C., and Ikehara, M. (1991) Impor other is the interaction of this metal ion with the 2'-OH at tance of the positive charge cluster in Escherichia coli ribonu the cleavage site by the formation of an outer-sphere clease HI for the effective binding of the substrate. J. Biol. Chem. complex. It is concluded that the role of the metal ion is to 266,11621-11627 15. Kanaya, S., Katayanagi, K., Morikawa, K., Inoue, H., Ohtsuka, fix this water molecule at the favorable position for hydrol E., and Ikehara, M. (1991) Effect of mutagenesis at each of five ysis. The proton of this water, whose pKa (12.4) is smaller histidine residues on enzymatic activity and stability of ribonu than that of the water unbound to Mg2+ (15.7) (33), is clease H from Escherichia coli. Eur. J. Biochem. 198, 437-440 abstracted by the 3'-leaving group after the nucleophilic 16. Doolittle, R., Feng, D.F., Johnson, M.S., and McClure, M.A. attack of the water activated by an amino acid residue (Fig. (1989) Origins and evolutionary relationships of retroviruses. Q. 6). This mechanism is supported by the results that the Rev. Biol. 64, 1-30 apparent rate constant for the -H substrate in the presence 17. Davies, J.F., Hostomska, Z., Hostomsky, Z., Jordan, S.R., and Matthews, D.A. (1991) Crystal structure of the ribonuclease H of Mg" ion was very small. In this case, the reduced rate domain of HIV-1 reverse transcriptase. Science 252, 88-95 can be attributed to the lack of a hydrogen bond in the 18. Uchiyama, Y., Miura, Y., Inoue, H., Ohtsuka, E., Ueno, Y., outer-sphere complex to fix the water molecule. This Ikehara, M., and Iwai, S. (1994) Studies of the interactions mechanism is similar to that proposed for the ribonuclease between Escherichia coli ribonuclease HI and its substrate. J. P reaction (31), although the phosphodiester is not coor Mol. 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