Intrinsic Properties of Reverse Transcriptase in ... - Semantic Scholar

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and MolecularBiology, Inc.

Vol. 264, No. 31, Issue of November 5, pp. 18808-18817,1989 Printed in U.S.A.

Intrinsic Properties of Reverse Transcriptase in Reverse Transcription* ASSOCIATED RNase H IS ESSENTIALLY REGARDED ASAN

ENDONUCLEASE (Received for publication, April 18, 1989)

Fumitaka OyamaS, Rie Kikuchi, RobertJ. Crouch#, and TsunekoUchidaq From the Mitsubishi Kasei Institute of Life Sciences, 11 Minamwoya, Machidashi, Tokyo 194, Japan

The intrinsic properties of reverse transcriptase in and Mizutani (2) in Rous sarcoma virus (RSV). This enzyme reverse transcription were studied using a synthetic, is multifunctional, having three enzymatic activitiesona partial ovalbumin mRNA with a syntheticDNA oligo- single polypeptide. They areRNA-dependent DNA-polymernucleotide annealed to the 3’-end of the RNA as a model ase, DNA-dependent DNA-polymerase, and RNase H activisubstrate (see Fig. 1). With or without concomitant ties (3). These activities are responsible for promoting many cDNA synthesis, the RNase H activity of avian myelo- processes in retrovirus replication. blastosis virus (AMV)-reverse transcriptase cleaved Based on current models of reverse transcription (3-5), the substrate at a site which would leave a hybrid of genome RNA is reverse transcribed into the first strand of between 7 and 14 base pairsbetween the 3’ termini of the RNA and DNA oligonucleotide. Variability in the complementary DNA (cDNA) by the RNA-dependent DNAexact size of the hybrid probably reflects some weak polymerase activity of reverse transcriptase using 3’-OH terbase preference for cleavage by the enzyme. These minus of tRNAas a primer. Then, the genomic RNA is short hybrids can be recognized as substrates by Esch- removed by the RNase H activity of reverse transcriptase, erichia coli RNase H and can be utilized by reverse which specifically degraded the RNA moiety in RNA-cDNA transcriptase as sites for continuation of cDNA synthe- hybrid. Synthesis of the second DNA strand is primed from sis. Substrates with 5’-triphosphorylated termini, 3‘- a unique location on the first DNA strand by DNA-dependent OH, 3’-phosphate, 3‘-end hairpin structures and 20 DNA-polymerase activity of reverse transcriptase. The rebase pair hybrids on the middle region of long RNA sulting double-stranded cDNA is inserted into the chromosome of the host cell and can express the genetic information more than 300 bases or on circular RNA were all of the retrovirus. cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essenThe processes of reverse transcription in retroviruses are tiallyregarded as an endonuclease degrading RNA quite complex and some of the details remain unclear. In moiety in RNA-DNA hybrid. The modes of action of particular, it has been thought that RNase H activity removes reverse transcriptase from murine leukemia virus and genomic RNA by 3’”5‘ exonucleolytic degradation after it Rous-associated virus 2 were thesame as that of AMV- has served as a template for the synthesis of the first strand reverse transcriptase, except that the size of the re- of DNA (6). However, recent results indicate that RNase H maining hybrid and the specificity for cleavage deactivity could remove the poly(A) tail(7)andthetRNA pended on thereversetranscriptase. We propose a primer (8) probably by endonucleolytic cleavage, and that possible model to explain themode of action of RNase RNase H activity could create the proper primer RNA by H and RNA-dependent DNA polymerase activities in selective degradation of the template RNA to assist the inireverse transcription. tiation of the second strand of cDNA (9-11). RNase H associated with reverse transcriptase exhibits what appears to be two incompatible activities, that is exonuclease and endonuclease. Reverse transcription of the retrovirus genome RNA into A key point to be able to understand thecomplex processes double-stranded DNA is an essential feature of retrovirus of reverse transcription indetail would be to characterize the replication. This process is catalyzed by a retrovirus reverse intrinsic properties of these three activities associated with transcriptase, first independently described in 1970 by Balti- reverse transcriptase and their interactions during reverse more (1) in murine leukemia virus (MuLV)’ and by Temin transcription. For this purpose, we used the purified avian myeloblastosis virus (AMV)-reverse transcriptase and a sim* The costs of publication of this article were defrayed in part by ple model substrate, a truncated-ovalbumin mRNA synthethe payment of page charges. This article must therefore be hereby sized in vitro as a template and the complementary deoxyrimarked “aduertisement” in accordance with 18 U.S.C. Section 1734 bonucleotide oligomer (20 bases long) as primer. solely to indicate this fact. We present in thisreport studieson the intrinsic properties 4 Present address: Institute for Comprehensive Medical Science, of RNase H and RNA-dependent DNA-polymerase activities Fujita-Gakuen Health University, Toyoake, Aichi, 470-11, Japan. during the synthesis of the first cDNA strand. We also propose § Present address: Laboratory of Molecular Genetics, Dept. of Health and Human Development, NIH, Bethesda, MD 20892. a model to explain the mode of action of RNase H and RNA7 To whom correspondence should be addressed. dependent DNA-polymerase activities in reverse transcripThe abbreviations used are: MuLV, murine leukemia virus; AMV, tion. avian myeloblastosis virus; RSV, Rous sarcoma virus; RAVB, Rousassociated virus2; ddNTP, 2’,3’-dideoxyribonucleotidetriphosphate; dNTP, deoxyribonucleotide triphosphate;HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid PIPES, 1,4-piperazinediethanesulfonic acid.

EXPERIMENTALPROCEDURES

Materials-SP6-RNA polymerase, pGEM2, and RQ1 DNase were purchased from Promega Biotec. HinfI, SacI, and PstI were from

18808

RNase H Activity of Reverse Transcriptase

Is a n Endonuclease

18809

Toyobo, Japan. Labeled nucleotides, [w3’P]GTP, [cx-~’P]~CTP, andwith boric acid). Full length RNAs were located by autoradiography, [Y-~*P]ATP were from Amersham, England. Escherichia coli RNase excised, and eluted by soaking in a buffer (0.1 M CH&OOK, 20 mM H, alkaline phosphatase (from E. coli A19), T4 RNA ligase, polynu- Tris-HCI, pH 7.6, 1 mM EDTA). The eluted RNA was collected by cleotide kinase, polynucleotide phosphorylase, and RAV2-reverse ethanol precipitation. Preparation of Circular RNAlso-32P-Labeled RNA,, (15 pmol) transcriptase were from Takarashuzo, Japan. AMV-reverse transcriptase was from Seikagaku-kogyo, Japan (or Midwest Bio-Products). was annealed to primer I1 (7.5 prnol) at 60 “C for 5 min and then MuLV-reverse transcriptase was from Bethesda Research Laborato- incubated at 37 “C for 30 min followed by further incubation with T4 ries. 2’,3’-Dideoxyribonucleotidetriphosphates (ddNTPs) were from RNA ligase (125 units) at 10 “C for overnight in 50 pl of a buffer solution (50 mM HEPES, pH7.5,20 mM MgCI’, 3.3 mM dithiothreitol, Pharmacia, Sweden. Four dNTPs were from Sigma. Plasmid Construction-SP6-ovalbumin cDNA was constructed 6 PM ATP and 10% Me’SO). The reaction volume was increased to from SP6 promoter and the fragment of chicken ovalbumin double- 150 pl, and the buffer concentration was changed to 170 mM Trisstranded-cDNA. The plasmid containing the entire chicken ovalbu- HCI, pH 8.0, 10 mM MgCl’, 1 mM EDTA, 20 mM potassium phosmin cDNA cloned in pCR was a generous giftfrom Prof. P. Chambon phate, pH 8.0, 2 mM dithiothreitol, and 0.3 M NaC1. This mixture was further incubated with polynucleotide phosphorylase (0.5 unit) (Laboratoire de Genetique Moleculaire des Eucaryotes, Centre National de la Recherche Scientifique, Strasburg). The DNA was di- at 37 “C for 40 min to degrade the remaining linear RNA and any noncovalent circular RNA. After incubation, the reaction mixture gested with SacI and PstI. The 360-base pair restriction fragment containing exon 1-exon 4 was purified by 6%-polyacrylamide gel was extracted with phenol and then ether. The circular RNA,,, was electrophoresis and introduced into the SacI and PstI sites of a recovered by ethanol precipitation. The separationof circular RNA1,0 pGEM2 to yield a plasmid ~ C O V , - ~ . T h e P C Owas V ~ -linearized ~ by from linear RNA,, was carried out by electrophoresis on 8% gel HinfI or PstI and transcribed by SP6 RNA-polymerase to yield exon under the denaturingcondition according to Sanger et al. (13). Labeling of 5’ Terminus of Template RNA-Dephosphorylation of 1-exon 2 (160 bases) or exon 1-exon 4 (377 bases), respectively, which 5’ terminus of the template RNA (5 pmol) was performed in 50 ~1 were designated RNA,,o or RNA377, respectively (see Fig. 1A). Synthesis of the 20-Base DNAs-The 20-base primer I, comple- mixture (100 mM EDTA, pH 8.0) by alkaline phosphatase (4 units). mentary to bases 3-22 upstream from 3’-end of the 160-base template The 5’-end of the resulting RNA was labeled by [Y-~’P]ATP and T4RNA, and primer 11, complementary to the 10 bases at 5’-end of polynucleotide kinase. Resulting labeled RNA was purified by polyRNA,, followed by the 10 bases complementary to the 3’-end, were acrylamide gel electrophoresis as described above. Labeling of 3‘ Terminus of Template RNA or Primer-Cytidine 3‘, prepared by a DNA synthesizer (Applied Biosystem, model 338) (Fig. 13). 5”bisphosphate was prepared as described by Bruce and Uhlenbeck Transcription and Purification of the RNAs-Transcription reac- (14) with [Y-~’P]ATP. Theresulting [5’-32P]pCp was ligated to the tion was carried out in the absence or presence of [cY-~’P]GTP 3’ terminus of thetemplate RNA or primer by T 4 RNA ligase essentially as described by Melton et al. (12). The synthesized RNAs according to the procedure of Bruce and Uhlenbeck (14). were applied to a 6%-polyacrylamide gel containing 7 M urea and Assay System forRNase H Activity of AMV-reverse Transcriptase TBE buffer (90 mM Tris-borate, 25 mM EDTA adjusted to pH 8.3 or E. coli-One pmol of the 32P-labeledtemplate RNA was dissolved

A

sac1

SPB

promofffr

FIG. 1. A, scheme for preparation of the templateRNAs from ovalbumin cDNA in SP6 transcription system. SP6ovalbumin DNA was constructed from SP6 promoter and thefragment of chick ovalbumin cDNA. DNA was linearized by Hinfl or PstI and transcribed by SP6 RNA-polymerase to generate RNAlGOor RNA377. The RNA was gel purified and used as a template for the cDNA synthesis. The circular RNA was prepared by circulizing RNA1, with T 4 RNA ligase in thepresence of primer I1 as described under “Experimental Procedures.” 8 : lane a, 3”terminal nucleotide sequence ofRNA,, withcomplementary DNA strandafter extension of the cDNA strand by 18 bases. The primer I ( 0) was complementary to bases 3-22 upstream of 3’-end of the RNA,,,. The arrow indicates a major cleavage site by E. coli RNase H or AMV-reverse transcriptase (see Fig. 2, lanes 2 and 3 ) .Lane b, the nucleotide sequence of the ligated region of the circular RNA,,,. The primer I1 was complementary to each 10 bases from 5’-end and 3’-end of the RNA1,. The arrow indicates a cleavage site by AMV-reverse transcriptase.

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150 160 CUUUGAUAAA CUUCCAGGAU GAAUACACGG AAUUCGAGCU 20

= primer II

18810

RNase H Activity of Reverse Transcriptase

in an annealing buffer (57 mM Tris-HCI, pH 8.3, 83 mM KCI, 8 mM M g W and annealed a t 60 "C for 5 min in the presence of 1pmol of 20 base primer and thenincubated a t 37 "Cfor 30 min. The assay for RNase H activity of AMV-, MuLV-, and RAV2-reversetranscriptases or RNase H from E. coli was carried out in the hybrid mixture (20 pl) containing 34 mM Tris-HCI, pH 8.3,50 mM KCI, 5 mM MgCI,, 4 mM dithiothreitol, and 8 units of reverse transcriptases or 20 units of RNase H purified from a recombinant strain of E. coli. In thecase of the RNase H assay coupled with cDNA extension, 110 p~ of one dideoxy-NTP and each of the other three dNTPs were added to the reaction mixture. The hybrid mixture was incubated a t 45 "C (AMVand RAV2-reverse transcriptases) or 37 "C (MuLV-reverse transcriptase and E. coli RNase H) for 20 min. After incubation, the reaction mixtures were extracted with phenol. The processed RNA was recovered by ethanol precipitation and was dissolved in a formamidedye sample buffer (80% formamide, 1 mM EDTA, 40 mM PIPES, pH 6.4, 0.1% bromphenol blue and 0.1% Xylene cyanol), and analyzed by electrophoresis on sequencing gel containing 6-or 20%-polyacrylamide and 7 M urea. Determination of the Cleavage Site of the Circular RNAIW-Primer 11 by AM V-reverse Transcriptase-The circular RNAIW hybridized to 5'-''P-labeled primer I1 was prepared and cleaved by AMV-reverse transcriptase as at42 "C described above. After incubation, the mixture was heated a t 60 "C for 5 min and then cooled on ice for 2 min. In order to degrade the primer 11, 4 units of RQ1 DNase was added to themixture, and then incubated a t 37 "C for 15 min. The reaction mixture was extracted with phenol and the resulting RNA was recovered by ethanol precipitation. The processed RNA was hybridized to the primer I as described above. The primer extension was carried out in the mixture (20 pl) containing 34 mM Tris-HCI, pH 8.3, 50 mMKC1, 5 mM MgCI,, 4 mM dithiothreitol, 110 p M each of dATP, dGTP, and dTTP,4 p~ [a-'*P]dCTP (10 pCi/100 pmol) and 8 units of AMV-reverse transcriptase. After incubation a t 42 "C for 20 min, the reaction was stopped by addition of 144 pl of 0.1 N NaOH. The mixture was heated a t 95 "C for 5 min to destroy the template RNA and thenneutralized by the addition of 10 pl of 2 N CH'COOH. The synthesized cDNA fragment was recovered by ethanol precipitation andanalyzed by electrophoresis on a sequencing gel.

Is an Endonuclease

Fig. lB, a and Fig. 3C, b ) . This result suggested that an 11base pair hybrid remained after AMV-reverse transcriptase associated RNase H treatment, whereas E. coli RNase H left only a 2-base pair hybrid. If an 11-basepair hybrid was stable, the 20-base synthetic DNA could serveas a primer for cDNA extension by RNA-dependent DNA-polymerase activity of this reverse transcriptase. After preincubation of AMV-reverse transcriptase with this model hybrid under conditions as described for lane 3, ddGTP and the other three dNTPs were added to the reaction mixture and incubated at 45 "C for an additional 20 min. Under this condition (Fig. 2, lane 4 ) C W A elongation ~NTPS+&TP Enzyme

Products Generated by RNase H Activity Associated with AMV-reuerse Transcriptase In the Absence of cDNA Synthe~is-~~P-Labeled template RNAlso is shownin Fig. 2, lane 1, migrating at position 166 in the sequencing ladder of cDNA produced frompre-mRNA for ovalbumin(411 bases). It means that theestimate of RNA size by using the DNA markers results in an experimental error of about 496, because the mobility ofRNA is slower than thatof DNA of the same size under this electrophoretic condition. To minimize this experimental error, we estimate the change of RNA size with RNase H cleavage bycomparing with a coelectrophoresed cDNA sequencingladder, assuming that theoriginal template is 160 bases long. When E. coli RNase H was added to thistemplate together with a primer I (Fig. 2, lane 2 and Fig. 3C, a ) , we detected a major processed-RNAof about 140 bases.This size indicates that E. coli RNase H cleaved the RNA moiety in this model hybrid at a site only 2 bases from the 3'-end of the synthetic DNA oligonucleotide (only 2 base pairs of hybrid would remain if stable. See Fig.1B, a). In contrast, AMV-reverse transcriptase generated a major processed-RNA9 bases longer than that of E. coli RNase H as shown in Fig. 2, lane 3 (See

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RESULTS

Model Substrate The scheme to obtain the synthetic ovalbumin mRNA is shown in Fig. lA.The 3"terminal nucleotide sequence of the RNA determined by O'Hare et al. (15), together with the complementary DNA strand is presented in Fig. lB, a. The purified 32P-templateRNA (160 or 377 bases long) and 20base DNA were annealed at 60 "Cfor 5 min, then incubated at 37 "Cfor 30 min to form RNA-DNA hybrid.

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FIG.2. Comparison of the RNA products by E. coli RNase H and AMV-reverse transcriptase. Lane 1, 32P-labeledR N A I ~ used as a template. RNA products obtained from the RNA-DNA hybrid of RNAI%with 20-base primer by addition of E. coli RNase H (lane2)or AMV-reversetranscriptase (lanes).Lane 4, RNA products obtained by preincubation under the condition described in lane 3, followed bythe addition of ddGTP and the other three dNTPs. Lanes 1-4 mix; a mixture of the RNA products obtained in lanes 1-4. M, molecular weight markers: 0X174/TaqI DNA fragments labeled using Klenow fragment polymerase and [w3'P]dCTP. Sequence ladder used as a reference marker is the nucleotide sequence of the partial, synthetic ovalbumin pre-mRNA as described previously (18).Lanes A-C mix, means the mixture of the sequencing ladders in lanes A, T, G, and C. The arrows of 160,150,140, and130 indicate positions on the sequence ladder based on the assumption that the position of RNAIW indicates nucleotide 160.

RNase H Activity of Reverse Transcriptase Is an Endonuclease

A AMV primer M

233

FIG. 3. A, effect of the first cDNA synthesis on the RNA products generated by RNase H activity associated with AMV-reverse transcriptase. Lane 1, the 160-bases long template RNA. Lane 2, the RNA product obtained by incubation with AMV-reverse transcriptase in the absence of the primer and dNTPs.Lane 3, the RNA products obtainedin the presence of AMV-reverse transcriptase and the primer with pCp of its 3'-end. Lane 4, the products obtained in the presence of the primer and four dNTPs. Lanes 5-8, the RNA products obtained in the presence of the primer and ddATP (lane 5) (1 base cDNA extension), ddCTP (lane 6 ) (3 bases), ddTTP (lane 7) (8 bases), orddCTP (lane 8) (18 bases) and other three dNTPs (see Fig. 1B). B, analysis of final products upon the completion of cDNA synthesis. The 5'-32P-labeled template RNA was used. RNAwas analyzed on 2O%-polyacrylamide, 7 M urea gel electrophoresis. Lane 1, the 5"end-labeled template RNA. Lane 2, the RNA products obtained by incubation with AMV-reverse transcriptase in the absence of the primer and four dNTPs. Lane 3, the RNA products obtained in the presence of AMV-reverse transcriptase, the primer, and four dNTPs. Lane 4, the RNA obtained in lane 3 followed byincubation with E. coli RNase H. Lanes 5 and 6, OH- indicates the partial cleavage product of the 5'end-labeled RNA by alkaline treatment. C, summary of cDNA extension and the resulting RNase H-mediated cleavage sites obtained in Figs. 2,3, A and B. Bold lines indicate the template RNA, and boxes, the primer I. Thin lines indicate the synthesized cDNA. a, the RNAleo (Fig. 2, lane 1 and Fig. 3A, lane 1 ) and cleavage site of E. coli RNase H (arrow) (Fig. 2, lane 2). b, no cDNA synthesis (Fig. 3A, lane 3). c-f show extension of the cDNA strand by one base (c, Fig. 3A, lane 5), 3 bases (d, Fig. 3A, lane 6 ) , 8 bases (e, Fig. 3A, lane 7)and 18 bases ( f , Fig. 3A, lane 8).g, completion of cDNA synthesis (Fig. 3A, lane 4 and Fig. 3B, lane 3 ) . Thick arrows indicate the major cleavage sites and thin arrows, the minor cleavage sites by RNase H activity associated with AMV-reverse transcriptase.

18811

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RNase H Activity of Reverse Transcriptase Is an Endonuclease

A

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FIG.4. The mode of action of

MuLV- and RAV2-reverse transcriptases. A, MuLV- and RAVP-reverse transcriptase-associated RNase H products. Lanes I and 6, the RNAlw template. Lanes 2-5 and 7-10, the products obtained by incubation with MuLVand RAVP-reverse transcriptases, respectively. Lanes 2 and 7,no primer and dNTPs. Lanes 3 and 8, plus primer and absence of the dNTPs. Lanes 4 and 9, the RNA products obtained after preincubation as in lanes 3 and 8 followed by addition of ddGTP and the other three dNTPs. Lanes 5 and 10, the products obtained in the presence of ddGTP and the other threedNTPs without the obpreincubation. E , finalproducts tained by both reverse transcriptases upon completion of cDNA synthesis. The RNAs were separated on 20% polyacrylamide, 7 M urea gel electrophoresis and analyzed by autoradiography. Lane 1, the 5"end-labeled template RNA. Lanes 2, 4, and 6, the RNA products obtained by AMV, MuLV- and RAVZreverse transcriptases, respectively. Lanes 3,5, and 7, same as lanes 2,4, and 6 followedby incubation with E. coli RNase H.Lane 8 was incomplete alkaline hydrolysate of 5'4abeled RNAlw.

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