The Isolated RNase H Domain of Murine Leukemia Virus Reverse Transcriptase
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.22023
ISSN1083-351X
Autores Tópico(s)Biochemical and Molecular Research
ResumoRetroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coliRNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H. Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coliRNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H. Reverse transcriptases (RT) 1The abbreviations used are: RT, reverse transcriptase(s); AKR-MuLV: murine leukemia virus from the AKR strain of mice; HIV, human immunodeficiency virus; PPT, polypurine tract; nt, nucleotide(s); PBS, primer binding site; AEA, C-175-AkR-RNase H withE. coli handle region.1The abbreviations used are: RT, reverse transcriptase(s); AKR-MuLV: murine leukemia virus from the AKR strain of mice; HIV, human immunodeficiency virus; PPT, polypurine tract; nt, nucleotide(s); PBS, primer binding site; AEA, C-175-AkR-RNase H withE. coli handle region. are enzymes responsible for copying the retroviral genomic RNA into double-stranded DNA by a process involving both the polymerase and RNase H of the RT (1). For human immunodeficiency virus type 1 (HIV-1), RT comprises a heterodimeric protein consisting of p66 and p51 subunits (2di Marzo Veronese F. Copeland T.D. DeVico A.L. Rahman R. Oroszlan S. Gallo R.C. Sarngadharan M.G. Science. 1986; 231: 1289-1291Crossref PubMed Scopus (417) Google Scholar, 3Lightfoote M.M. Coligan J.E. Folks T.M. Fauci A.S. Martin M.A. Venkatesan S. J. Virol. 1986; 60: 771-775Crossref PubMed Google Scholar, 4Wondrak E.M. Lower J. Kurth R. J. Gen. Virol. 1986; 67: 2791-2797Crossref PubMed Scopus (29) Google Scholar). The p51 polypeptide is derived from p66 by proteolysis, removing the C-terminal (p15) RNase H domain. Both polymerase and RNase H catalytic sites are contributed by p66, with p51 acting primarily as a structural polypeptide (5Le Grice S.F. Naas T. Wohlgensinger B. Schatz O. EMBO J. 1991; 10: 3905-3911Crossref PubMed Scopus (189) Google Scholar, 6Hostomsky Z. Hostomska Z. Fu T.B. Taylor J. J. Virol. 1992; 66: 3179-3182Crossref PubMed Google Scholar). Isolated p51 protein has very poor polymerase activity and, of course, no RNase H activity. Linker-scanning mutations in HIV-1 RT (7Prasad V.R. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3104-3108Crossref PubMed Scopus (86) Google Scholar), as well as the structure determined by x-ray crystallography (8Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 9Jacobo-Molina A. Ding J. Nanni R.G. Clark A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar), show an intermingling of polymerase and RNase H regions, with one activity relying on other portions of the protein for activity (10Blain S.W. Goff S.P. J. Virol. 1995; 69: 4440-4452Crossref PubMed Google Scholar). The RT of murine leukemia virus (MuLV) differs from that of HIV-1 RT in that it is isolated as a single polypeptide chain of 76 kDa (11Herr W. J. Virol. 1984; 49: 471-478Crossref PubMed Google Scholar), which appears to form dimers when bound to nucleic acids (12Telesnitsky A. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1276-1280Crossref PubMed Scopus (87) Google Scholar,13Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar). Furthermore, MuLV RT can be divided into two separate polypeptides; the N-terminal two-thirds portion of the protein has very good polymerase activity and a protein from the C-terminal one-third is reported to be an active RNase H (14Tanese N. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1777-1781Crossref PubMed Scopus (147) Google Scholar). RNase H activity is essential for retroviral replication (15Mizrahi V. Usdin M.T. Harington A. Dudding L.R. Nucleic Acids Res. 1990; 18: 5359-5363Crossref PubMed Scopus (41) Google Scholar, 16Mizrahi V. Brooksbank R.L. Nkabinde N.C. J. Biol. Chem. 1994; 269: 19245-19249Abstract Full Text PDF PubMed Google Scholar) and is involved in several steps of replication including the following: removal of tRNA, which functions as a primer for minus-strand DNA synthesis (17Omer C.A. Faras A.J. Cell. 1982; 30: 797-805Abstract Full Text PDF PubMed Scopus (53) Google Scholar); generation of polypurine tract (PPT), the primer for second strand DNA and its subsequent removal (18Finston W.I. Champoux J.J. J. Virol. 1984; 51: 26-33Crossref PubMed Google Scholar, 19Rattray A.J. Champoux J.J. J. Virol. 1987; 61: 2843-2851Crossref PubMed Google Scholar); and degradation of the viral RNA genome (20Resnick R. Omer C.A. Faras A.J. J. Virol. 1984; 51: 813-821Crossref PubMed Google Scholar, 21Champoux J.J. Gilboa E. Baltimore D. J. Virol. 1984; 49: 686-691Crossref PubMed Google Scholar, 22Smith J.K. Cywinski A. Taylor J.M. J. Virol. 1984; 52: 314-319Crossref PubMed Google Scholar, 23Smith J.K. Cywinski A. Taylor J.M. J. Virol. 1984; 48: 200-204Crossref Google Scholar). In vivo studies demonstrate that inactivation of RNase H results in production of noninfectious virions (24Repaske R. Hartley J.W. Kavlick M.F. O'Neill R.R. Austin J.B. J. Virol. 1989; 63: 1460-1464Crossref PubMed Google Scholar, 25Schatz O. Cromme F. Naas T. Lindemann D. Mous J. Le Grice S.F.J. FESB Lett. 1989; 257: 311-314Crossref PubMed Scopus (132) Google Scholar, 26Tisdale M. Schultze T. Larder B.A. Moelling K. J. Gen. Virol. 1991; 72: 59-66Crossref PubMed Scopus (139) Google Scholar). Amino acid sequence comparison shows that HIV and MuLV retroviral RNases H share about 25% of their sequence with Escherichia coli RNase HI (27Johnson M.S. McClure M.A. Feng D.-F. Gary J. Doolittle R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7648-76527Crossref PubMed Scopus (370) Google Scholar). Much greater similarity is found when comparing the three-dimensional structures of E. coli RNase HI (28Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Science. 1990; 249: 1398-1405Crossref PubMed Scopus (451) Google Scholar, 29Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (307) Google Scholar) and the RNase H of HIV-1 reverse transcriptase in the complete RT (8Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 9Jacobo-Molina A. Ding J. Nanni R.G. Clark A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar) or in the p15 (C-terminal domain) (30Davies J.F. Hostomska Z. Hostomsky Z. Jordan S. Mathews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (524) Google Scholar). A significant exception to the structural similarity is the presence of an additional handle region in E. coli RNase HI (helices αB, αC, and αD) not found in the corresponding region of HIV-1 p15, which contains only helices αB and αD. Several studies demonstrate that αC is rich in basic amino acids and contributes significantly to binding to RNA-DNA hybrids (31Kanaya S. Katsuda-Nakai C. Ikehara M. J. Biol. Chem. 1991; 266: 11621-11627Abstract Full Text PDF PubMed Google Scholar, 32Haruki M. Noguchi E. Kanaya S. Crouch R.J. J. Biol. Chem. 1997; 272: 22015-22022Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). One construct ofE. coli RNase HI was made in which the handle region is missing and in which enzymatic activity is lost due, in part, to a decrease in binding to RNA-DNA hybrids by more than 3 orders of magnitude (32Haruki M. Noguchi E. Kanaya S. Crouch R.J. J. Biol. Chem. 1997; 272: 22015-22022Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In another case, an E. coli RNase HI protein with a different connection of the αB and αD helices retains some enzymatic activity but only in the presence of Mn2+ ions (33Keck J.L. Marqusee S. J. Biol. Chem. 1996; 271: 19883-19887Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A protein containing only the p15 domain has no RNase H activity (34Becerra S.P. Clore G.M. Gronenborn A.R. Karlstorm S.J. Stahl S.J. Wilson S.H. Wingfield P.T. FEBS Lett. 1990; 270: 76-80Crossref PubMed Scopus (42) Google Scholar, 35Schatz O. Mous J. Le Grice S.F.J. EMBO J. 1990; 9: 1171-1176Crossref PubMed Scopus (126) Google Scholar, 36Hostomsky Z. Hostomska Z. Hudson G.O. Moomaw W.W. Nodes B.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1148-1152Crossref PubMed Scopus (94) Google Scholar) but acquires activity by the following: (i) mixing with the HIV-1 polymerase domain (p51) (36Hostomsky Z. Hostomska Z. Hudson G.O. Moomaw W.W. Nodes B.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1148-1152Crossref PubMed Scopus (94) Google Scholar) or part of the connection region (37Smith J.S. Gritsman K. Roth R.M.J. J. Virol. 1994; 68: 5721-5729Crossref PubMed Google Scholar); (ii) adding a His6-tag to the RNase H domain (37Smith J.S. Gritsman K. Roth R.M.J. J. Virol. 1994; 68: 5721-5729Crossref PubMed Google Scholar, 38Smith J.S. Roth M.J. J. Virol. 1993; 67: 4037-4049Crossref PubMed Google Scholar, 39Stahl S.J. Kaufman J.D. Vikic-Topic S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar); and (iii) inserting the αC region from the E. coli RNase HI (39Stahl S.J. Kaufman J.D. Vikic-Topic S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar, 40Keck J.L. Marqusee S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2740-2744Crossref PubMed Scopus (49) Google Scholar). One somewhat surprising result obtained with the active HIV-1 RNase H proteins is their ability to cleave model tRNA primer substrates at the same site as intact RT, suggesting that at least some of the specificity elements for cleavage are present in these polymerase minus RNases H (37Smith J.S. Gritsman K. Roth R.M.J. J. Virol. 1994; 68: 5721-5729Crossref PubMed Google Scholar, 38Smith J.S. Roth M.J. J. Virol. 1993; 67: 4037-4049Crossref PubMed Google Scholar, 39Stahl S.J. Kaufman J.D. Vikic-Topic S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar, 40Keck J.L. Marqusee S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2740-2744Crossref PubMed Scopus (49) Google Scholar). The three-dimensional structure of MuLV RNase H has not yet been elucidated, but amino acid sequence alignment predicts that this RNase H has a handle region, consisting of αB, αC, and αD, similar to E. coliRNase HI (28Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Science. 1990; 249: 1398-1405Crossref PubMed Scopus (451) Google Scholar, 29Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (307) Google Scholar). Compared with E. coli RNase HI, MuLV reverse transcriptase-associated RNase H activity is very low, whether associated with the polymerase domain or not. Furthermore, the RNase H activity of MuLV RT prefers Mn2+, and MuLV RT cleaves replication intermediates essential for retroviral replication at specific sites that are different from those E. coli RNase HI cleaves (21Champoux J.J. Gilboa E. Baltimore D. J. Virol. 1984; 49: 686-691Crossref PubMed Google Scholar, 22Smith J.K. Cywinski A. Taylor J.M. J. Virol. 1984; 52: 314-319Crossref PubMed Google Scholar). Little is known about the biochemical properties of the C-terminal RNase H of MuLV RT. To further our understanding of retroviral RNases H, we have examined the C-terminal 175 amino acid portion of MuLV RT (C175-AKR-RNase H) for its activity, its binding to RNA-DNA hybrids, and its specificity of cleavage. For specificity studies, we have used a model tRNA primer site, the polypurine tract (PPT), a fragment of mRNA for ovalbumin annealed to a complementary DNA oligonucleotide, and a homopolymeric poly(rA)·poly(dT). The E. coli strain MIC1066 (rnhA-339::cat recB270(TS)) (41Cerritelli S.M. Shin D.Y. Chen H.C. Gonzales M. Crouch R.J. Biochimie ( Paris ). 1993; 75: 107-111Crossref PubMed Scopus (8) Google Scholar) was used to over-produce the C175-AKR-RNase H (AKR is the strain of mice from which the virus was isolated), AEA (C175-AKR-RNase H with the E. coli RNase HI "handle region"), in addition to E. coli RNase HI. Bacterially expressed AKR-MuLV reverse transcriptase and a polyclonal antibody against N-terminal AKR-MuLV RNase H were kind gifts from Dr. Judith Levin at NICHD, National Institutes of Health (42Post K. Guo J. Kalman E. Uchida T. Crouch R.J. Levin J.G. Biochemistry. 1993; 32: 5508-5517Crossref PubMed Scopus (34) Google Scholar). The Mega RNA transcription kit was from Ambion. Expression vectors pET15b(+) and pET21a(+), biotinylated protease thrombin, streptavidin-agarose, and His-Bind® resin were from Novagen. The Western Light™ chemiluminescent detection system was from Tropix, Inc. RNA36 oligonucleotide (5′)-biotin-GGGAUCAGUGGUUCCCAUAUCCCGGACGAGCCCCCA-(3′) was synthesized by Oligos Etc. Inc. A partially complementary DNA36(TGGGGGCTCTGCCGGGATATGGGAACCACTGATCCC) was from BioServe. The ultrapure Sequagel sequencing system was from National Diagnostics, and 6 and 8% gel mix were from Life Technologies, Inc. The sensor chip CM5, Tween P20, and amine coupling kit were from Pharmacia Biotech Inc. Streptavidin and biotin were from Pierce. The DNA segment encoding Met1 to Val155 of E. coli RNase HI and Leu496 to Leu671 of the pol gene of AKR-MuLV (11Herr W. J. Virol. 1984; 49: 471-478Crossref PubMed Google Scholar) was modified by adding restriction sites (NdeI and EspI) and cloned either into the pET15b(+) vector, which expresses a His6-tag at the N terminus of the target proteins, or into the pET21a(+) vector for expression of proteins without His6-tag. The AEA plasmid carries the DNA of AKR-MuLV encoding amino acids Leu496 to Tyr581, the E. coli RNase HI sequence for Thr69 to His114, followed by AKR-MuLV Arg629 to Leu671. Four-liter cultures of MIC1066 harboring the C175-AKR-RNase H, chimeric RNase H (AEA), or E. coli RNase HI plasmids were grown at 32 °C to anA600 value about 0.8 and then induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 32 °C. Cells were harvested and sonicated in loading buffer (5 mm imidazole, 0.1% Nonidet P-40, 0.5m NaCl, and 20 mm Tris-HCl, pH 7.9). After centrifugation at 10,000 rpm for 30 min, the supernatants were filtered through a 0.45-micron filter and loaded on a 1-ml His-Bind® resin. The column was washed with 10 ml of loading buffer and 10 ml of washing buffer (60 mm imidazole, 0.5 m NaCl, and 20 mm Tris-HCl, pH 7.9). The His6-tagged C175-AKR-RNase H, AEA, and E. coli RNase HI were eluted with 0.5 m imidazole, 0.5 m NaCl, and 20 mm Tris-HCl, pH 7.9. After dialysis against buffer A (50 mm Tris-HCl, pH 7.9, 1 mm dithiothreitol, 50 mm NaCl, 10% glycerol, 0.5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride), samples were applied to consecutively connected high performance liquid chromatography columns of TSK gels DEAE-5PW (7.5 × 75 mm) and SP-5PW (7.5 × 75 mm). The His6-tagged C175-AKR-RNase H was present in the flow-through fraction, and the His6-tagged E. coli RNase HI bound to the SP column, eluting at about 150 mm NaCl from a 50 mm to 1 m NaCl gradient, and the chimeric AEA RNase H bound to the SP column, eluting in a manner similar to the E. coli protein. The proteins of interest were pooled and concentrated by centrifugation at 5,000 rpm in a filtron device (cutoff, 10 kDa). Enzymes were stored at −70 °C in a solution containing 45% glycerol, 25 mm Tris-HCl, pH 7.9, 0.5 mm dithiothreitol, 25 mm NaCl, 0.25 mm EDTA, and 0.5 mm phenylmethylsulfonyl fluoride. Removal of His6tag was performed by treating 100 μg of His6-tagged proteins with 1 unit of biotinylated thrombin protease at 25 °C for several hours. His-Bind® resin was added to the reaction mixture and mixed for 30 min at room temperature. After spinning, the supernatant containing the protein without His6-tag was further mixed with 100 μl of streptavidin-agarose slurry for another 30 min at room temperature. After centrifugation, the supernatant was collected and dialyzed overnight at 4 °C against buffer A. RNase H activity with α-32P-labeled poly(rA)·poly(dT) as substrate was determined essentially as described (43Dirksen M.-L. Crouch R.J. J. Biol. Chem. 1981; 256: 11569-11573Abstract Full Text PDF PubMed Google Scholar) by measuring trichloroacetic acid-soluble radioactivity. In situ renaturation gel assays were performed as described (44Carl P.L. Bloom L. Crouch R.J. J. Bacteriol. 1980; 144: 28-35Crossref PubMed Google Scholar). Four different substrates were used to determine differences in products generated by the various enzymes. Details of the conditions of the assays are given in the figure legends for each substrate. Polyacrylamide gel analysis of the products was performed according to Zhan et al. (45Zhan X. Tan C.-K. Scott W.A. Mian A.M. Downey K.M. So A.G. Biochemistry. 1994; 33: 1366-1372Crossref PubMed Scopus (38) Google Scholar). The substrate for minus-strand primer removal assay was prepared according to Smith and Roth (38Smith J.S. Roth M.J. J. Virol. 1993; 67: 4037-4049Crossref PubMed Google Scholar) and Stahlet al. (39Stahl S.J. Kaufman J.D. Vikic-Topic S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar). The uniformly [α-32P]CTP-labeled polypurine substrate was synthesized according to Guo et al. (13Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar) and uniformly [α-32P]CTP-labeled ovalbumin mRNA160was prepared as described by Oyama et al. (46Oyama F. Kikuchi R. Crouch R.J. Uchida T. J. Biol. Chem. 1989; 264: 18808-18817Abstract Full Text PDF PubMed Google Scholar) and Postet al. (42Post K. Guo J. Kalman E. Uchida T. Crouch R.J. Levin J.G. Biochemistry. 1993; 32: 5508-5517Crossref PubMed Scopus (34) Google Scholar). The final products were visualized after electrophoresis on a 6 or 8% sequencing gel by autoradiography with Kodak X-Omat film or were exposed to a phosphor screen and quantified using the ImageQuant program (Molecular Dynamics). The 5′-biotinylated RNA36oligonucleotide was annealed to its complementary DNA36oligonucleotide at a ratio 1:20 in the diethyl pyrocarbonate-treated HBS buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 0.05% Tween P20, a non-ionic detergent). A sensor chip with streptavidin-modified surface was first immobilized with 20 μl of RNA-DNA hybrid and then blocked with biotin (for details see Ref. 32Haruki M. Noguchi E. Kanaya S. Crouch R.J. J. Biol. Chem. 1997; 272: 22015-22022Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 45 μl of four different concentrations of RNase H, which were dialyzed against to HBS buffer before analysis, were injected onto the RNA-DNA surface at 20 μl/min. The surface was regenerated with one injection of 10 μl of 2 m NaCl. The sensorgrams were analyzed by subtracting sensorgrams obtained from the control surface, in which a sensor chip is loaded with streptavidin followed by biotin but not with RNA-DNA. Kinetic constants were calculated using BIAcore™ evaluation software (see also Ref. 32Haruki M. Noguchi E. Kanaya S. Crouch R.J. J. Biol. Chem. 1997; 272: 22015-22022Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar for details). RNases H of E. coli, C175-AKR-RNase H, and chimeric AEA were purified from E. coli MIC1066 (rnhA-339::cat recB270(TS)) to ensure that only the overexpressed proteins contribute to the activity observed. Versions of both proteins with or without an N-terminal His6-tag were studied. Purification of the His6-tagged proteins was facilitated by affinity chromatography using nickel nitrilotriacetic acid-agarose columns. Results of analysis of the purified proteins by SDS-polyacrylamide gel electrophoresis are shown in Fig. 1 as follows: a shows a Coomassie-stained gel, and bshows a renaturation gel assay for RNase H activity. E. coliRNase H labeled with the His6-tag purified essentially as it would without the His6-tag. Expression of the C175-AKR-RNase H enzyme is relatively poor, and therefore, we purified the His6-tagged version of this protein and, in some studies, removed the His6-tag by proteolysis with thrombin. C175-AKR-RNase H is found in the flow-through of both DEAE and SP columns, in contrast to the E. coli protein, which flows through the DEAE column but binds to the SP column, eluting at around 150 mm NaCl. Some preparations of the C175-AKR-RNase H contain small amounts of a 12-kDa protein corresponding to the N-terminal portion of C175-AKR-RNase H, as judged by its binding to nickel nitrilotriacetic acid-agarose and its interaction with antisera directed against the N-terminal portion of the RNase H region. This fragment probably results from cleavage at a site previously shown to be susceptible to proteolysis in E. coli when AKR-MuLV reverse transcriptase is expressed and purified (47Levin J.G. Crouch R.J. Post K. Hu S.C. McKelvin D. Zweig M. Court D. Gerwin B. J. Virol. 1988; 62: 4376-4380Crossref PubMed Google Scholar). We could not detect any contribution of this AKR-MuLV fragment to RNase H activity or to binding to RNA-DNA hybrids. The chimeric AEA RNase H purifies in a similar manner to C175-AKR-RNase H and appears to be homogeneous (data not shown). For simplicity in the presentation in the figures, the C175-AKR-RNase H is designated as A, E. coliRNase HI as E, and the chimeric RNase H asAEA. Previous studies of MuLV RNase H have been more qualitative than quantitative but demonstrate clearly that this domain has RNase H activity (14Tanese N. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1777-1781Crossref PubMed Scopus (147) Google Scholar). One rather novel feature of MuLV reverse transcriptase is a strong preference for Mn2+ (2.5 mm) rather than Mg2+ (5 mm) as the divalent cation when assaying for either polymerization or RNase H activity. To examine activity and metal ion preference, we assayed the C175-AKR-RNase H using poly(rA)·poly(dT) as substrate. The purified soluble C175-AKR-RNase H degrades RNA, preferring Mn2+ over Mg2+ as the divalent cation (Table Iand Fig. 2). Products of C175-AKR-RNase H digestion of poly(rA)·poly(dT) were analyzed by gel electrophoresis and quantified using Molecular Dynamics PhosphorImager and ImageQuant (Table I). The specific activity of the C175-AKR-RNase H is very similar to MuLV reverse transcriptase-associated RNase H activity with a preference for Mn2+ over Mg2+ (about 10–20-fold) but differs significantly from that of E. coliRNase HI, which prefers Mg2+ over Mn2+ more than 150-fold.Table ISpecific activities using magnesium and manganeseEnzymeMg2+Mn2+Mg2+/Mn2+E. coli RNase HI7.25 × 104401180C175-AKR RNase H28.95200.06AKR MuLV RT261920.14RNase H activity was assayed in 20 μl using 5 pmol32P-labeled poly(rA)·poly(dT) at 37 °C for 10 min in the presence of either 5 mm Mg2+ or 2.5 mmMn2+. Products were analyzed on a 15% polyacrylamide gel and quantified using ImageQuant. Specific activities are nmol of product migrating at <20 nt/mg of RNase H in 10 min. Open table in a new tab RNase H activity was assayed in 20 μl using 5 pmol32P-labeled poly(rA)·poly(dT) at 37 °C for 10 min in the presence of either 5 mm Mg2+ or 2.5 mmMn2+. Products were analyzed on a 15% polyacrylamide gel and quantified using ImageQuant. Specific activities are nmol of product migrating at <20 nt/mg of RNase H in 10 min. The size distribution of products obtained with poly(rA)· poly(dT) as substrate differs markedly depending on the enzyme (Fig. 2). Since the specific activity of each enzyme is different, various amounts of RNase H were added, and the samples were removed at various times and products analyzed on polyacrylamide gels. The distribution of products in either Mg2+ or Mn2+ is very similar when AKR-MuLV RT provides the RNase H activity with the most abundant products at 10 min being 17- and 18-mer. At later times, when little or no initial substrate remains, smaller products accumulate. E. coli RNase HI products are significantly smaller than those seen with AKR-MuLV RT using Mg2+ as cation and are even smaller when Mn2+ is present in the reaction. C175-AKR-RNase H (A in Fig. 2) generates a very different set of products from RT in the presence of Mg2+ or Mn2+ with a long ladder of oligomers of increasing length at 30 min (lane 3) and products ranging from monomers to 14-mers at 150 min (lane 4). In contrast to E. coli RNase HI, C175-AKR-RNase H gives larger products in the presence of Mn2+, almost as large as those seen with AKR-MuLV RT (lanes 4). To examine cleavage of a well characterized mRNA substrate for E. coli and MuLV RNases H, we used a portion of the ovalbumin mRNA (RNA160) annealed to a 20-mer DNA primer, which starts 2 nt away from the 3′-end of the RNA (Fig. 3). Experiments performed with this and subsequent substrates used Mg2+ as the divalent cation, since it is thought to be the relevant ion in vivo. From previous studies (42Post K. Guo J. Kalman E. Uchida T. Crouch R.J. Levin J.G. Biochemistry. 1993; 32: 5508-5517Crossref PubMed Scopus (34) Google Scholar, 46Oyama F. Kikuchi R. Crouch R.J. Uchida T. J. Biol. Chem. 1989; 264: 18808-18817Abstract Full Text PDF PubMed Google Scholar), it is known that cleavage of RNA160 is due to binding of the polymerase active site at the 3′-OH of the d20-mer, placing the RNase H active site 15 base pairs from the 3′-OH giving an RNA product of 153 nucleotides (5- and 10-min time points for RT in Fig. 3). Incubation for longer times yielded additional products resulting from 3′-OH-independent cleavages (40-min RT in Fig. 3). E. coli RNase HI degrades this substrate very differently from RT-RNase H with some products arising from hydrolysis as close as three base pairs from the 3′-OH of the d20-mer. C175-AKR-RNase H gives products very similar to E. coliRNase HI; particularly notable is the absence of the 153-nt product seen with RT. The primer of minus-strand DNA of RT of MuLV is from a tRNAPro annealed to a site some 145 nt from the 5′-end of the genomic RNA of MuLV. Synthesis of the first strand of DNA is followed by initiation of plus-strand DNA synthesis at the polypurine tract (PPT). Replication of the tRNAPro-primer yields an RNA-DNA hybrid that is cleaved by RT-RNase H removing the tRNAPro. To test for C175-AKR-RNase H specificity, we used a model substrate tRNAPro, similar to those used by Smith and Roth (38Smith J.S. Roth M.J. J. Virol. 1993; 67: 4037-4049Crossref PubMed Google Scholar) and Stahl et al. (39Stahl S.J. Kaufman J.D. Vikic-Topic S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar), to analyze tRNALys3 removal by the RNase H of HIV-1. This substrate is a synthetic d69-mer with a primer binding site sequence (PBS) at its 3′-terminus to which is annealed an 18-mer RNA containing the 3′-terminal tRNAPro sequence complementary to the PBS. The Klenow fragment of E. coli DNA polymerase I is used to synthesize and label (using [α-32P]dGTP) the model substrate (Fig. 4). This defined substrate has an RNA-DNA hybrid with an RNA-DNA covalent junction similar to the natural in vivo substrate. RNase H of MuLV reverse transcriptase cleaves similar substrates to produce two products, one with a single ribonucleotide A attached to the DNA and a second which has the ribonucleotide A removed, resulting from hydrolysis at the RNA-DNA junction (48Schultz
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