THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265. No. 28, Issue of October 5, pp. 17162-17166, 1990 Printed m U. .%A.

Site-directed Mutagenesis of Moloney Murine Leukemia Virus Reverse Transcriptase DEMONSTRATION OF LYSINE 103 IN THE NUCLEOTIDE BINDING SITE*

(Received for publication, November 27, 1989)

Amaresh Basu, Subhalakshmi Basu, and Mukund J. ModakS From the Department of Biochemistn, and Molecular Biology, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark, New Jersey07103

Lys’O3 and LYS~~’ of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal5’- phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 164% 1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys’03 and Lys42l to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys + Ala) exhibited properties that were indis- tinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA po- lymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.

Retroviral reverse transcriptases catalyze the synthesis of double-stranded DNA of encapsidated genomic RNA through a complex process (Gilboa et al., 1979). Reverse transcriptases purified from a number of retroviruses exhibit two basic activities, a DNA polymerase and an RNase activity (Verma, 1975; Gerard and Grandgenett, 1975). The DNA polymerase activity catalyzes the formation of a phosphodiester bond between the incoming dNTP and the 3’ end of a primer and can copy either RNA or DNA templates. The RNase activity only degrades RNA from an RNA. DNA hybrid duplex and is thus termed RNase H. All retroviral RTs’ are proteolytically processed from a large polyprotein precursor (Kopchick et al.,

* This research was supported in part by a grant from the New Jersey State Commission for Cancer Research. The costs of puhlica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent. Tel.: 201-456-5515. 1 The abbreviations used are: RT(s), reverse transcriptase(s);

MuLV, Moloney murine leukemia virus; PLP, pyridoxal 5’-phos- phate; Hepes, 4-(2.hydroxyethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.

1978); however, mature enzymes from different viruses differ significantly from one another in their structures. For exam- ple, MuLV-RT is a monomer of 80 kDa (Dickson et al., 1982), avian myeloblastosis virus RT is a heterodimer of 63- and 95 kDa subunits (Varmus and Swanstrom, 1984), and human immunodeficiency virus RT is a heterodimer of 66- and 51- kDa polypeptides (di Marzo Veronese et al., 1986; Lightfoote et al., 1986). Biochemical functions and properties of many reverse transcriptases have been characterized in detail; how- ever, because of the lack of sufficient quantities of the pure enzyme, progress in structural studies has been lagging. In recent years, recombinant DNA technology has made it pos- sible to produce large amounts of reverse transcriptase, in- cluding that from MuLV-RT (Kotewicz et al., 1985; Roth et al., 1985) and human immunodeficiency virus RT (Tanese et al., 1986; Hansen et al., 1987; Larder et al., 1987; Farmeri et al., 1987; Hizi et al., 1988). Thus, the availability of recombi- nant enzyme proteins has made structure function studies feasible. Our early emphasis was to investigate mechanisms of action of inhibitors that can form covalent adducts with various polymerases (Modak, 1976; Strivastava and Modak, 1980; Basu and Modak, 1987; Basu, A., et al., 1988, 1989). The characterization of the inhibitory action provided infor- mation regarding the functional role of the site(s) that reacted with the inhibitor, and the identification of amino acid resi- dues that reacted with the inhibitor, and their location, pro- vided structural definition of the involved sites. For example, pyridoxal 5’-phosphate (PLP) was found to be a substrate dNTP binding site-directed reagent (Modak, 1976; Modak and Dumaswala, 1981) for many DNA polymerases and re- verse transcriptases including MuLV-RT. Reaction of 2 spe- cific lysine residues in MuLV-RT, namely Lyslo3 and Lys4”, with PLP was found to result in the inactivation of the enzyme. The reactivity of both lysine residues was blocked in the presence of the appropriate substrate dNTPs, concomi- tant with the restoration of enzyme activity (Basu, A., et al., 1988). Thus, both Lys’03 and Lys4’l were implicated in the process of dNTP binding in MuLV-RT. In order to clarify and confirm the catalytic involvement of the individual lysine residues, we have now carried out site-directed mutagenesis of Lyslo3 and Lys4” by substituting them with uncharged amino acids, and the properties of the mutant enzymes have been compared. Results presented here suggest that only Lys’O3 is required for the catalytically active enzyme (polym- erase activity) and that this lysine is probably required in the binding/recognition of deoxynucleoside triphosphate whereas mutation at Lys@l has no effect on the catalytic activity of the enzyme.

MATERIALS AND METHODS

Restriction endonucleases and DNA-modifying enzymes were ob- tained from Bethesda Research Laboratories. Oligonucleotide primers

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Lys103 in the dNTP Binding Site of MuLV Reverse Transcriptase

for DNA sequencing and mutagenesis were obtained from Genetic Designs and purified by high pressure liquid chromatography using the Zorbax oligo column from Du Pont-New England Nuclear. Re- agents for Ml3 sequencing were purchased from New England BioLabs, and radioactive nucleotides [a-“*P]dATP (800 Ci/mmol) and [ru-32P]dTTP (800 Ci/mmol) were from Amersham Corp. Deox- ynucleotides, (rAhwo, and (dA), j000 were obtained from Pharmacia LKB Biotechnology Inc. Poly(rA) and poly(dA) primed with oligo(dT)i2-is in a 1:l molar nucleotide ratio were used as template primers for most of the experiments. Anti-rabbit IgG conjugated with alkaline phosphatase was-from Sigma. The rabbit antibody against nure MuLV-RT was a generous gift from Dr. Monica Roth of Robert Wood Johnson MedicaT School of this university. All other chemicals were of reagent grade.

Site-directed Mutagenesis

The expression plasmid pB6B15.23 (Roth et al., 1985), which contains the entire Moloney murine leukemia,virus reverse transcrip- tase gene sequence, was a kind gift from Dr. Stephen Goff of Columbia University. Mutagenesis of lysines at positions 103 and 421 was carried out according to the method of Kunkel(l985). A 1.9-kilobase KpnI-SspI fragment was isolated and subcloned in M13mp19 digested with KpnI and HincII. The recombinant M13mp19 virus (with insert) was used to infect an Escherichia coli dut--ung- strain (CJ236) to produce template DNA containing uracil residues. The synthetic oligonucleotide 5’-CGTTAAGCTACCAGGGACT-3’, which intro- duced two nucleotide changes inhe wild-type sequence, was used as the primer for the generation of the Lye?&’ to leucine mutation, whereas the oligonucleotide 5’-ACTGACAGCGGATGCAG-3’ was used to mutate Lys”i to alanine. The desired=tants were identified directly by dideoxy sequencing (Sanger et al., 1977). A l.l-kilobase KpnI-BglII fragment containing the appropriate mutations was re- cloned into the pB6B15.23 KpnI-BglII-digested vector. The mutant proteins were then expressed by the protocol described earlier (Roth et al., 1985).

Enzyme Assays

DNA Polymerase Assay-RNA-dependent DNA polymerase activ- ity of partially purified RT preparations was determined using (rA), . (dT),,-,s and dTTP as template primer and substrate, respectively. The reaction mixture, in a final volume of 100 ~1, contained 50 mM Tris-HCl, pH 8.0.2 mM dithiothreitol, 10 fig of bovine serum albumin, 1 mM MnCl,, 60 mM NaCl, 0.5 pg of (rA),.<dThz.ia, 20 WM dTTP, 0.5 &i of la-“‘PldTTP. and 2 ul of uartiallv uurified or 20 ne of nurified

. 1 I . I .

enzyme. After a 20-min incubaiion at 37 “C, reactions were termi- nated by the addition of 5% trichloroacetic acid containing 10 mM pyrophosphate. Acid-insoluble material was collected on GF/B glass fiber filters, and radioactivity was determined by scintillation spec- troscopy. For processivity studies, l-30 nM enzyme was preincubated with the appropriate template primer for 5 min in 45 ~1 of 50 mM Tris-HCI, pH 8.0, 60 mM KCl, 1 mM dithiothreitol, and 0.05% Nonidet P-40 (buffer A). Reactions were started by the addition of MgC12/MnC12 and dNTP in 5 ~1 of buffer A to give a final concentra- tion of 10 mM MgCl, or 1 mM MnC12 and 100 fiM dNTP. Reactions were stopped at regular intervals with 5 ~1 of 0.5 M EDTA. All incubations were carried out at 37 “C. Reaction products were ana- lyzed by polyacrylamide gel electrophoresis in the presence of 7 M urea as described by Huber et al. (1989).

RNase H Assay-RNase H activity in the partially purified enzyme preparations was measured by using tritiated (rA),.(dT), as sub- strate. The reaction mixture in a final volume of 100 ~1 contained 50 mM Hepes-KOH, pH 7.8, 1 mM dithiothreitol, 20 mM NaCl, 10 gg of bovine serum albumin, 1 mM MnCl*, 0.5 gg of [3H](rA),. (dT), (20,000 cpm), and 50 ng of the enzyme protein. The reactions were carried out at 37 “C for 30 min, and the acid-insoluble counts were determined as described for the DNA polymerase assays.

Purification of Reverse Transcriptase-Five hundred-milliliter cul- tures of the wild type and two mutants were grown, and enzyme induction and purification were carried out essentially as described by Roth et al. (1985). Proteins obtained after two column chromatog- raphy steps, DEAE-cellulose and phosphocellulose, were used for enzyme characterization as they were found to be 90% pure at this stage. In the case of mutant 103, which showed no polymerase activity in the crude lysate, the reverse transcriptase protein was identified by Western blot (immunoblot) analvsis of individual column frac- tions. The fractions that showed maximum reverse transcriptase protein were pooled and used for further studies.

Western Blotting-Equivalent amounts of protein after DEAE fractionation of wild-type and mutant forms of reverse transcriptase were denatured and fractionated by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. The fractionated proteins were transferred electrophoretically onto Immobilon-P membranes and reacted with polyclonal antibody raised in rabbits against pure MuLV-RT (Roth et al., 1985). Identification of the bands reacting with the MuLV-RT antibody was accomplished by reaction with an anti-rabbit antibody conjugated with alkaline phosphatase.

Template Primer Binding Assay-The ability of the mutant en- zymes to bind to template primer was examined by determining the extent of UV-mediated cross-linking of the enzyme protein with radiolabeled template primer. The protocol for cross-linking of the enzyme protein to template primer was essentially as described earlier (Basu, S., et al., 1988). The primer (dTh5, labeled at the 5’ end by polynucleotide kinase, was annealed with (dA),. The resulting tem- plate primer, (dA), . (dT),, (1 pg, 0.2 &i), was mixed with 2 pg of the enzyme and then exposed to UV irradiation for 15 min. The extent of enzyme cross-linked to template primer was determined by SDS- polyacrylamide gel electrophoresis followed by autoradiography (Basu, S., et al., 1988). We have chosen the template primer (dA),. (dT)ih for the binding studies even though it is utilized poorly by MuLV-RT for synthesis (Modak and Marcus, 1977a). This avoided the ambiguity associated with the use of (rA),. (dT)is, which can serve as template primer for polymerase as well as a substrate for RNase H activity. The observation that the catalysis of (rA), . (dT)ia-directed synthesis is inhibited by (dA),.(dT),, also implies that MuLV-RT does indeed bind to (dA),. (dT),, (Modak and Marcus, 197713). The cross-linking studies (see Fig. 1B) provide direct proof of binding of this template primer to MuLV-RT.

RESULTS AND DISCUSSION

We have shown previously that Lysio3 and Lys4s’ of MuLV- RT are specific targets of reaction by PLP, a substrate binding site-directed reagent for DNA polymerases; this finding im- plied involvement of both lysines in the process of substrate dNTP binding (Basu, A., et al., 1988). To determine the importance of the individual lysine residues, we mutated Lyslo3 to leucine and Lys4*l to alanine. The selection of leucine and alanine as substitutions for lysine was based on the fact that these are uncharged residues and have small side chains. Therefore, these substitutions are expected to have a minimal effect on the secondary structure of mutant RTs.

Enzyme proteins were purified from lysates of bacterial cultures expressing either the wild-type or mutant proteins as described under “Materials and Methods.” Fig. IA shows a Western blot analysis of bacterial extracts expressing the wild-type and mutant forms of MuLV-RT. A 71-kDa protein cross-reacting with polyclonal antibody against pure MuLV- RT was detected in all extracts, indicating that the wild-type and mutant clones were both expressing RT. Several bands representing lower molecular weight species were also seen to cross-react with the antibody in all three instances. These were probably the breakdown products of the RT protein in E. coli extracts.

Since the mutants were able to express full-length RT protein, the next step was to determine the polymerase and RNase H activities associated with the protein. Mutant 421 showed no change in polymerase activity whereas mutant 103 showed only 1% of the activity expressed by wild-type RT (Table I). Analysis of the RNase H activity indicated that both mutants had nuclease activity comparable with that of the wild-type enzyme.

Characterization of Lys’03 Mutant of MuLV-RT-The re- sults described above show clearly that mutation of lysine 103 in MuLV-RT severely affects polymerase activity. Since the polymerase reaction requires binding of both template primer and substrate dNTPs, it was essential to demonstrate whether the binding of only one or both components was affected in the mutant enzyme. In MuLV-RT, substrate dNTP binding only occurs subsequent to the binding of template primer to

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17164 Lys103 in the dNTP Binding Site of MuLV Reverse Transcriptase

Mr x lo3

FIG. 1. Immunoblotting and UV-mediated cross-linking of (dA),.[3ZP](dT),S to wild-type and mutant forms of MuLV- RT. A, wild-type and mutant forms of MuLV-RT were analyzed on 8% SDS-polyacrylamide gel and immunoblotted with anti MuLV-RT antibody. Lane I, wild type; lene 2, Lys”’ mutant; lane 3, Lys4” mutant. R, wild-type and mutant MuLV-RTs were cross-linked to (dA),. [“‘PI (dT),i by UV irradiation and analyzed by polyacrylamide gel electrophoresis as described under “Materials and Methods.” Lane I, E. coli DNA polymerase I, large fragment, taken as control; lane 2, heat-inactivated MuLV-RT; lane 3, wild type; lane 4, Lye?” mutant; lane 5, L~s’~’ mutant MuLV-RT.

TABLE I DNA polymerase and RNase H activities of wild-type and mutant

RTs DNA polymerase and RNase H activities were assayed as described

under “Materials and Methods.” The activities of the mutants were analyzed in three individual clones representing each mutant, and the sequence of each clone was confirmed by dideoxy sequencing. The amino acids underlined indicate the positions of mutation.

Position of Activity Original NW mutation Polymerase RNase H Seq”l?WX Seq”l3lCe

%

Wild-type 100 100 Lys’O.’ 1 95 LPVKKPGTN LPVKLPGTN Lye 100 98 AVLTitDAGK AVLTtiAGK -

the enzyme (Basu, A., et al., 1988). Therefore, we attempted to determine the extent of template primer binding by the mutant and the wild-type enzyme. We used both an indirect and direct approach to accomplish this goal. In the indirect approach, inactivation of RT-associated RNase H activity by inclusion of DNA template primer is used to indicate template primer binding by RT (Modak and Marcus, 1977b). In the direct approach, binding of the DNA template primer to the enzyme is measured via UV-mediated cross-linking of the enzyme to template primer. Table II summarizes the results of the RNase H experiment. The RNase H activity of all three enzymes exhibited similar sensitivity to the addition of calf thymus-activated DNA or (dA), . (dTh2-,8, indicating that DNA, which is not a substrate for RNase H, was able to bind to the enzyme via the polymerase domain and displace it from the RNase H-substrate complex (Modak and Marcus, 197713). Furthermore, the ability of the mutant enzymes to bind to template primer is also demonstrated by comparing the effi- ciency with which wild-type and mutant enzymes cross-link to labeled (dA), . (dThs, where dTls carries the label at its 5’ end (Fig. 1B). The extent of cross-linking of mutant 103 to (dA),. (dT),, was equivalent to that of the wild-type enzyme. The choice of (dA), as a template in these studies rules out the possibility that the enzyme binds to the template primer

TABLE II Effect of natural and synthetic DNA on RNase H activity of wild-

type and mutant RTS RNase H activity was assayed using 0.5 pg of ‘H-labeled (rA),.

(dT), as described under “Materials and Methods.”

DNA”

Activated DNA 1 a

kIh%T)s

Wild WP

100

II 60

RNase H activity

LYS”‘:’ Lys”’ mutant mutant

%

100 100

80 75 58 51

5 Pg 62 60 63

“Activated calf thymus DNA or (dA),.(dT)is (1:l molar ratio) at indicated concentrations was added as a challenger DNA in the reaction mixture prior to the addition of the enzyme.

via the RNase H domain. These results indicate that the Lys’O” mutant retains its ability to bind to DNA, and hence the observed loss in polymerase activity must be due to its inability to bind to substrate dNTP, as reported earlier (Basu, A., et al., 1988).

The two functional domains, i.e. the polymerase and the RNase H domains of reverse transcriptases, have been pre- dicted (Johnson et al., 1986) and have been demonstrated to reside in separate regions of the enzyme (Tanese and Goff, 1988; Kotewicz et al., 1988). The DNA polymerase domain resides in the N-terminal region whereas, the RNase H do- main is contained in the C-terminal region of the protein. Thus, our observation that the mutation of Lys”“, which lies in the N-terminal region of the enzyme, has a drastic effect on polymerase activity without affecting RNase H activity is consistent with the two-domain constitution of RT. In addi- tion, computer analysis of the amino acid sequences has revealed that Lys”” is conserved in all retroviral reverse transcriptases, but Lys4*l is not (Johnson et al., 1986; Doolittle et al., 1989). Thus, our recent mutagenesis studies and pre- vious chemical modification studies (Basu, A., et al., 1988) show clearly that Lys’“” is one of the most important residues in the polymerase active site and that it is required for the dNTP binding activity of MuLV-RT.

Characterization of Lys”‘l Mutant of MuLV-RT-Chemical modification of MuLV-RT with pyridoxal 5’-phosphate in the presence and absence of substrate dNTP had indicated clearly that Lys4*’ and Lys”” were the targets of inhibitor action (Basu, A., et al., 1988). The present mutagenesis results, however, indicate that Lys4” has no apparent role in polym- erase or RNase H reactions. In order to establish if mutation of this lysine has altered any other property of the enzyme, we carried out a detailed analysis of the template primer preferences, rates of reaction, and kinetic constants for both template and substrates. We found no significant difference between the properties of the Ly.? mutant enzyme and the wild-type enzyme (data not shown).

Previously, we had also noted the reactivity of two distinct lysine residues in the PLP-mediated inactivation of E. coli DNA polymerase I. One of the lysines in pol I was required for substrate dNTP binding (Basu and Modak, 1987) whereas the 2nd lysine was required for the processive mode of syn- thesis (Basu, S., et al., 1988). We therefore considered the possibility that lysine 421 in MuLV-RT, which does not appear to be involved in the substrate binding per se, may influence the enzyme-template primer interaction in a man- ner analogous to that found for E. coli DNA polymerase I (Basu, S., et al., 1988). Modification of LysfiZ1’ in the E. coli

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LYS10Z3 in the dNTP Binding Site of MuLV Reverse Transcriptase 17165

pol I enzyme was shown to result in the loss of the processive mode of DNA synthesis by the modified polymerase. There- fore, the mode of DNA synthesis on single-stranded template with respect to processivity and template sequence-dependent “strong stops” by the wild-type and mutant MuLV-RT were compared. Processivity on single-stranded templates was measured by the extension of labeled primers. Poly(rA) primed with 5 ‘ “P-labeled oligo(dT),-, was extended with reverse transcriptase, and products were monitored with in- creasing time (Fig. 2, panel I) and with increasing enzyme (Fig. 2, panel 10, in the presence of excess template primer. With increasing time or enzyme, the amount of products rather than product distribution changes, and both the wild- type and mutant 421 show a similar pattern of DNA synthesis. Replicative polymerases are generally processive in nature; however. in uitro MuLV-RT is not a very processive enzyme

I A,

b I

FIG. 2. Mode of DNA synthesis on single-stranded tem- plates by wild-type and mutant MuLV RT. Panel I, reverse transcriptase (5.6 nM) was preincubatedwith 80 nM (rA),. [‘“P](dT),s, and the reaction was started by the addition of TTP and Mn’+ (see “Materials and Methods”). Aliquots were withdrawn at various times and products analyzed on an 8% urea-polyacrylamide gel. Lanes 1-3 represents synthesis at 2.5, 5, and 10 min. Panel II, reactions of 1.5 nM (lane I) and 2.8 nM (lane 2) reverse transcriptase with 56 nM (rA)..(dT)rs were initiated as described above. Reactions were stopped after 10 min and products analyzed on an 8% urea-acrylamide gel. Panel III, Reactions with 20 nM reverse transcriptase and 150 nM Ml3 DNA to which a 5’ “YP-labeled 15-mer had been annealed were initiated as described under “Materials and Methods.” DNA products at different times were analyzed as above. Lanes l-3 repre- sent 10,20, and 30 min. a represents wild-type RT, b, Lys”” mutant.

(Varmus and Swanstrom, 1984). Our observation with recom- binant wild-type enzyme confirms the low processivity asso- ciated with MuLV-RT, and the mutation at Ly? does not seem to alter this characteristic (Fig. 2). The mode of DNA synthesis on primed single-stranded Ml3 DNA by wild-type and mutant 421 were also very similar (Fig. 2, panel IZI’). Furthermore, the pattern of distinct DNA sequence-depend- ent stops observed during replication of single-stranded DNA (Abbotts et al., 1988; Huber et al., 1989) also seems to be unaffected by the mutation of Lye?‘.

The results presented here indicate that Lys4”’ apparently has no catalytic role in the DNA polymerase activity of MuLV-RT. One possible explanation for the reactivity of both Lys”.’ and Lys4” to pyridoxal 5’-phosphate and their protection by the substrate dNTP may be the molecular proximity of the 2 residues in the three-dimensional structure of RT. In such a situation, the presence of substrate dNTP in the active site pocket may block Lys4” sterically and prevent its modification by PLP.

In conclusion, the mutagenesis studies reported here show clearly that lysine 103 is catalytically important and is prob- ably involved in the substrate binding process in MuLV-RT.

Acknowledgments-We thank Stephen Goff of Columbia Univer- sity College of Physicians and Surgeons for the generous gift of the plasmid used in these studies. We also thank Catherine Joyce of Yale University for helpful advice regarding the mutagenesis protocols.

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A Basu, S Basu and M J Modaktranscriptase. Demonstration of lysine 103 in the nucleotide binding site.

Site-directed mutagenesis of Moloney murine leukemia virus reverse

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