OF VIROLOGY, Nov. 1977, p. 704-708 Copyright © 1977 American Society for Microbiology
JOURNAL
Vol. 24, No. 2 Printed in U.S.A.
In Vitro Transcription of the Avian Retrovirus Genome by the a Form of the Viral RNA-Directed DNA Polymerase MARC S. COLLETT,' DUANE P. GRANDGENETT,2 AND ANTHONY J. FARAS1* Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455,1 and Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 631102
Received for publication 3 June 1977
The nature of transcription of the avian retrovirus RNA genome by the a form of the viral RNA-directed DNA polymerase has been investigated. Transcription was most efficient when Mn2" was provided as the divalent metal ion. The patterns of DNA transcription using 70S RNA, 35S RNA tRNAtrP, or 35S RNA oligo(dT)12.18 template primer complexes by the a DNA polymerase were essentially identical to those obtained using the a,8 form. The a DNA polymerase appears to be deficient in the synthesis of true duplex DNA but is able to synthesize hairpin-structured DNA initiated at the 5' terminus of the viral genome on the tRNAtrP primer molecule. The RNA-directed DNA polymerase of avian myeloblastosis virus exists in two enzymatically active forms. The major species of holoenzyme, a13, consists of an equimolar ratio of two polypeptides, a and f, with molecular weights of approximately 62,000 and 92,000, respectively (13, 15,20), whereas the minor DNA polymerase, termed a, consists of only the smaller polypeptide. The larger ,8 polypeptide appears to be the primary viral gene product, since the a polypeptide is apparently derived from ,B by proteolytic cleavage (11, 27, 29, 33, 34). In addition, both polypeptides are antigenically related and share similar amino acid sequences (11, 15, 34). A third DNA polymerase species, (,if, was recently identified in virions of Rous sarcoma virus and is thought to be a precursor to the aB species
(19).
Both avian myeloblastosis virus polymerase species exhibit all three enzymatic activities associated with purified viral reverse transcriptase: RNA-directed DNA synthesis, DNA-directed DNA synthesis, and RNase H activity (1, 15, 27). Differences in their modes of action have been reported. Whereas there is some question as to the processiveness of the af3 form of the DNA polymerase (7, 24), the a form appears to be nonprocessive in nature (12, 29). However, the RNase H activity associated with the a,8 and a enzymes appears to be processive and nonprocessive, respectively (12, 22, 23). Differences have also been noted with respect to the ability of each of the enzyme forms to bind natural and synthetic nucleic acids. The a/3 enzyme has been shown to bind more avidly than a to various template primer complexes (12, 13, 29, 32). Furthermore, it has been demonstrated
that under conditions whereby the a,8 DNA polymerase binds with high affinity to the avian retrovirus primer molecule (tryptophan tRNA) involved in synthesis of virus-specific DNA (6, 8-10, 31, 39), the a subunit exhibits little or no binding to this primer RNA species (16, 18). In view of the above differences between the a and a,3 forms of avian myelobastosis virus RNA-directed DNA polymerase, we have compared the properties of viral genome transcription in vitro by these two enzymes. Our studies of viral DNA transcription in reconstructed enzymatic reactions containing purified al DNA polymerase and genome RNA have been presented elsewhere (5). In this communication, we present data from our analysis of a RNA-directed DNA polymerase transcription in reconstructed reactions. It has been previously reported that the a DNA polymerase is unable to efficiently transcribe viral 70S RNA (17, 28). However, this deficiency could be overcome by the addition of oligo(dT) as a primer, presumably by complexing with the viral RNA 3' poly(A) tract (28). Our initial studies involved a determination of the synthetic capabilities of the a DNA polymerase as a function of the divalent metal ion present in reactions using three different template-primer complexes: native 70S RNA; 35S viral subunit RNA to which purified tRNAtrP primer had been reannealed (4, 9); and 35S RNA to which oligo(dT)12-18 had been added as the primer molecule (5). The a form of the DNA polymerase exhibited a preference for Mn2, whereas the a/B form preferred Mg2", in reconstructed enzymatic reactions using all three template primer complexes (Fig. 1). 704
NOTES
VOL. 24, 1977
Using the optimal divalent ion concentration (2 mM Mn2"), we compared the size distribution of DNA transcripts synthesized from the three template* primer complexes by the a form of the RNA-directed DNA polymerase. Radiolabeled DNA products were synthesized under several different concentrations of deoxynucleoside triphosphates (3, 5), purified, and analyzed by electrophoresis under denaturing conditions in polyacrylamide gels containing 98% formamide (5). The bulk of the DNA transcribed by the a DNA polymerase from either the 70S RNA (data not shown) or 35S RNA tRNAtrP template primer complex under either "ratelimiting" (three deoxynucleotide triphosphates [dNTP's] at 60 ,uM, the fourth at 6 AM) (data not shown) or "equimolar" (all four dNTP's at 60 AM) concentrations of dNTP's was less than 200 nucleotides in length (Fig. 2A). The three discrete species of DNA observed, a major component of 185 nucleotides in length and two minor classes of 100 and 70 nucleotides in length, represent initial DNA (iDNA) transcripts primed by the tRNAtrP molecule at the 5' ter-
705
minus of the viral genome (4, 5, 36, 37; Collett and Faras, in preparation). When 35S RNA oligo(dT)12-18template primer complexes were used in reactions containing equimolar concentrations of dNTP's, the DNA product synthesized by the a DNA polymerase exhibited a broad size distribution ranging from 220 to 2,000 nucleotides in length (Fig. 2B). These patterns of a DNA polymerase transcription from the 70S RNA, 35S RNA tRNAtrP, and 35S RNA oligo(dT)12-18template- primer complexes are identical to those observed when the afl form of the DNA polymerase was used in similar reconstructed reactions under optimal conditions of enzymatic DNA synthesis (5). Increasing the concentration of dNTP's either in endogenous reverse transcriptase reactions (3, 35) or reconstructed enzymatic reactions (5) has been shown to promote the synthesis of longer DNA transcripts. To determine if increasing the substrate concentration similarly affected a DNA polymerase transcription, DNA synthesis was conducted in the presence of 1 mM each of the dNTP's and the purified DNA product an-
A. 75 _
\\
X
o
_60 _ 0
0
45
I
30
5
[M91J
Xi
10 -3
15 M
M
2
-3] 0l
LMnJ
X 10
3 M
FIG. 1. Effect of divalent metal ion on DNA synthesis in reconstructed enzymatic reactions using the a and a,/ forms of the RNA-directed DNA polymerase. Three viral template -primer complexes were used in the various enzymatic reactions (5): native 70SRNA (0, *), 35SRNA tRNA'rP (O. *), and 35SRNA oligo(dT)12 18 (A, A). Reaction mixtures contained: 10 ug of template primer complex per ml; 0.1 M Tris-hydrochloride, pH 8.1; 0.01 MMgCI2; 1.4% (vol/vol) 2-mercaptoethanol; 6 ,uM[3H]dGTP (20 Ci/mmol); 60 uM each unlabeled dATP, dTTP, and dCTP. Both a DNA polymerase and a,/ DNA polymerase were present in saturating amounts. The a DNA polymerase was purified by chromatography on poly(U)-Sepharose (14) and exhibited a single peak on sodium dodecyl sulfate-polyacrylamide gels. The a DNA polymerase preparations contained no detectable DNA exonuclease, endonuclease, or RNase activities (14). Reactions were incubated at 37°C for 60 min with varying amounts of MgCl2 (A) or MnCl2 (B), and the perchloric acid-precipitable counts were determined. The open symbols represent synthesis by the a/I form, and the solid symbols represent synthesis by the a form of the RNA-directed DNA polymerase.
706
J. VIROL.
NOTES
synthesize true duplex DNA. afI DNA polymerase-mediated transcription of 35S RNA- oligo(dT)12-18template- primer complexes results in the production of a considerausually w 0 portion (75%) of product that exhibits the ble 0 w characteristics of double-stranded DNA (Table -J 1), whereas the DNA product of a DNA polym1.) z erase transcription contains only a small portion of duplex DNA (10%). These results are in agreement with previous data (28) demonstrating that 0 viral DNA synthesis by the a enzyme on an oligo(dT) primer is insensitive to inhibition by actinomycin D, an inhibitor of double-stranded DNA synthesis (25). However, an analysis of the products of tRNAtrP-initiated DNA synthesis in the presence of low concentations of dNTP's reveals that the a DNA polymerase is able to synthesize DNA that is hairpin in nature (Table 1). Although several of the 5'-terminally DISTANCE MIGRATED (mmi) located initial DNA transcripts (4, 5, 36) of 100 (iDNAloo) and 70 (iDNA70) nucleotides in length FIG. 2. Polyacrylamide-formamide gel electropho- (Fig. 2A) are largely single stranded (Table 1), resis of DNA synthesized in reconstructed reactions using a RNA-directed DNA polymerase. Enzymatic the 185-nucleotide-length iDNA (iDNA,85) repDNA synthesis by the a form of RNA-directed DNA resents a hairpin or foldback structure, as dempolymerase on either 35S RNA- tRNA1'r (A) or 35S onstrated by its considerable resistance to SI RNA oligo (dT) 12-18 (B) template primer complexes in nuclease hydrolysis after denaturation (Table the presence of equimolar concentrations (60 PM) of 1). In addition, iDNA185 is nearly completely dNTP's was essentially as described for a,/ DNA resistant to exonuclease I hydrolysis (90%) (data polymerase-mediated synthesis (5), except 2 mM MnCl2 was used as the divalent metal ion. The puriI
(D z -J
0
fied DNA transcripts were subjected to electrophoresis in 5% polyacrylamide-98% formamide cylindrical gels (0.6 by 10 cm) as previously described (5). Gels were calibrated with respect to nucleotide length by parallel electrophoresis of the HaeIII restriction endonuclease fragments of OX 174, simian virus 40, and polyoma virus DNAs (5).
I-mLI) z
w -J
0
(-i z 0
alyzed on polyacrylamide-formamide gels. From the electropherogram presented in Fig. 3A, the DNA product synthesized on either 35S RNA- tRNAtrP or 70S RNA (data not shown) exhibited a dramatic shift in size distribution, with transcripts ranging to 2,000 nucleotides in length. Under similar reaction conditions, DNA synthesized on 35S RNA- oligo(dT)12 18 template- primer complexes exhibited an accumulation of transcripts ranging between 1,500 and 2,500 nucleotides in length (Fig. 3B). These characteristics of a DNA polymerase transcription at high concentrations of dNTP's are again identical to those previously observed for transcription by the afl form of the enzyme (5). Analysis of the secondary structure of the DNA products resulting from a DNA polymerase transcription of the viral genome, when compared with afl DNA polymerase transcripts under optimal reaction conditions, suggests that the a form may be deficient in its ability to
0. U
DISTANCE MIGRATED (mm)
FIG. 3. Polyacrylamide-formamide gel electrophoresis of a DNA polymerase transcripts synthesized in the presence of high concentrations of dNTP's. Enzymatic synthesis by the purified a DNA polymerase was carried out as described in the legend to Fig. 2, except in the presence of all four dNTP's at 1 mM. Subsequent purification and electrophoresis in 5% polyacrylamide-98% formamide gels was as previously described (5). (A) a DNA transcripts synthesized from 35S RNA tRNAtrP. (B) a DNA transcripts synthesized from 35S RNA oligo(dT)12-18-
NOTES
VOL. 24, 1977
TABLE 1. Secondary structure of various DNA transcripts synthesized in reconstructed reactions by the afi and a forms of RNA-directed DNA polymerase Si nuclease reDNA transcript" sistant (%)b Total product, cr1 polymerase .74.5 Total product, a polymerase .9.7 . .10.4 iDNA70 . .12.8 iDNAoo 68.0 iDNA15 ... a Total DNA product was synthesized by either the ad1 or a form of DNA polymerase using viral 35S RNA oligo(dT)12 18 template primer complexes as described in the legend to Fig. 2. The iDNA transcripts of various lengths (70, 100, and 185 nucleotides) were synthesized from 35S RNA tRNAP template primer complexes, using either the ac3 or a form of the viral DNA polymerase, and were purified from denaturing 5% polyacrylamide-98% formamide gels (5). b The preparation of reaction products and treatment with Si nuclease were as previously described (2; Collett and Faras, submitted for publication). The iDNA transcripts synthesized by the af? and a DNA polymerase demonstrated the same degree of SI nuclease resistance.
not shown). A more detailed characterization of this hairpin DNA will be published elsewhere (Collett and Faras, submitted for publication). Both the a and an) forms of the viral DNA polymerase synthesize the hairpin iDNA185 to similar extents (cf. profiles in Fig. 2A and reference 5). Thus, it appears that the a DNA polymerase is deficient only in the synthesis of true duplex DNA, while maintaining its ability to form hairpin DNA initiated from the tRNAtr' primer at the 5' terminus of the viral genome. Although the a form of the avian retrovirus DNA polymerase binds poorly to various natural and synthetic nucleic acids (12, 13, 29, 32), and not at all to the viral tRNAtrP primer molecule (16, 17), it still exhibits a pattern of transcription of DNA from viral genome template primer complexes identical to that of the at) form. Transcription is most efficient if Mn2+ is used as the divalent metal ion. A similar phenomenon has been demonstrated with both mammalian and reticuloendotheliosis retrovirus reverse transcriptases (17, 21, 26, 38). It is interesting to note that DNA polymerase from both of these latter sources exhibit molecular weight estimates similar to avian myeloblastosis virus a DNA polymerase (17, 26). The apparent inability of the a DNA polymerase to synthesize true duplex DNA, while still mediating the synthesis of tRNAtrP-initiated hairpin DNA, may be a function of the location of the major site of transcription on the viral genome and/or the
707
nature of the primer for the synthesis of the second DNA strand. For example, the synthesis of hairpin iDNA185 appears to result from the use of DNA transcripts that have reached the 5' terminus of the viral genome (iDNA1oo) as both primer and template for the synthesis of a complementary strand (Collett and Faras, submitted for publication). The mechanism by which true duplex DNA is synthesized in either reconstructed or endogenous reverse transcriptase reactions is currently unknown but appears to reflect a property of the ac1 form, but not the a form, of the DNA polymerase. We thank S. Kanellos and P. Wilkie for excellent technical assistance and L. Honza for typing this manuscript. This investigation was supported by Public Health Service research grants CA 18303-02, CA 20011-01, and CA-16312-04 from the National Cancer Institute. One of us (D.P.G.) is a recipient of an American Cancer Society faculty research award.
LITERATURE CITED 1. Baltimore, D., and D. F. Smoler. 1972. Association of an endoribonuclease with the avian myeloblastosis virus DNA polymerase. J. Biol. Chem. 247:7282-7287. 2. Clayman, C. IL, M. S. Collett, and A. J. Fara. 1977. In vitro transcription of the avian oncornavirus genome by the RNA-directed DNA polymerase: effect of actinomycin D on the extent of transcription. J. Virol. 23:209-212. 3. Collett, M. S., and A. J. Faras. 1975. In vitro transcription of DNA from the 70S RNA of Rous sarcoma virus: identification and characterization of various size classes of DNA transcripts. J. Virol. 16:1220-1228. 4. Collett, M. S., and A. J. Faras. 1976. Evidence for circularization of the avian oncornavirus RNA genome during proviral DNA synthesis from studies of reverse transcription in vitro. Proc. Natl. Acad. Sci. U.S.A. 73:
1329-1332. 5. Collett, M. S., and A. J. Faras. 1977. In vitro transcrip-
6.
7.
8.
9.
10.
11.
tion of the avian oncornavirus genome by the RNAdirected DNA polymerase: analysis of DNA transcripts synthesized in reconstructed enzymatic reactions. J. Virol. 22:86-96. Dahlberg, J. E., R. C. Sawyer, A. J. Faras, W. E. Levinson, H. M. Goodman, and J. M. Bishop. 1974. Transcription of DNA from the 70S RNA of Rous sarcoma virus. I. Identification of a specific 4S RNA which serves as primer. J. Virol. 13:1126-1133. Dube, D. K., and L. A. Loeb. 1976. On the association of reverse transcriptase with polymeric templates during catalysis. Biochemistry 15:3605-3611. Faras, A. J., J. E. Dahlberg, R. C. Sawyer, F. Harada, J. M. Taylor, W. E. Levinson, J. M. Bishop, and H. M. Goodman. 1974. Transcription of DNA from the 70S RNA of Rous sarcoma virus. I. Structure of a 4S RNA primer. J. Virol. 13:1134-1142. Faras, A. J., and N. A. Dibble. 1975. RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S RNA primer in uninfected cells. Proc. Natl. Acad. Sci. U.S.A. 72:859-863. Folk, W., and A. J. Faras. 1976. Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: tryptophan tRNA as the major species of primer RNA. J. Virol. 17:1049-1051. Gibson, W., and I. M. Verma. 1974. Studies on the reverse transcriptase of RNA tumor viruses. Structural
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NOTES
relatedness of two subunits of avian RNA tumor viruses. Proc. Nat. Acad. Sci. U.S.A. 71:4991-4994. 12. Grandgenett, D., and M. Green. 1974. Different mode of action of RNase H in purified a and ad RNA-directed DNA polymerase from AMV. J. Biol. Chem. 249: 5148-5152. 13. Grandgenett, D. P. 1976. Dissociation of a,8 DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide. J. Virol. 17:950-961. 14. Grandgenett, D. P. 1976. Purification of the a subunit of avian myeloblastosis virus DNA polymerase by polyuridylic acid-Sepharose. J. Virol. 20:348-350. 15. Grandgenett, D. P., G. F. Gerard, and M. Green. 1973. A single subunit from avian myeloblastosis virus with both RNA-directed DNA polymerase and ribonuclease H activity. Proc. Natl. Acad. Sci. U.S.A. 70:230-234. 16. Grandgenett, D. P., A. C. Vora, and A. J. Faras. 1976. Different states of avian myeloblastosis virus DNA polymerase and their binding capacity to primer tRNA". Virology 75:26-32. 17. Green, M., and G. Gerard. 1974. RNA-directed DNA polymerase: properties and functions in oncogenic RNA viruses and cells, p. 187412. In W. E. Cohn (ed.), Progress in nucleic acid research and molecular biology, vol. 14. Academic Press Inc., New York. 18. Haseltine, W. A., and D. Baltimore. 1976. In vitro replication of RNA tumor viruses, p. 175-213. In D. Baltimore, A. H. Huang, and D. F. Fox (ed.), ICNUCLA Symposium on Animal Virology. Academic Press Inc., New York. 19. Hizi, A., and W. Joklik. 1977. RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the a, 82 and a6 forms of the enzyme. J. Biol. Chem. 252:2281-2289. 20. Kacian, D. L, K. F. Watson, A. Burny, and S. Spiegelman. 1971. Purification of the DNA polymerase of avian myeloblastosis virus. Biochim. Biophys. Acta 246:365-383. 21. Kang, C-Y. 1975. Characterization of endogenous RNAdirected DNA polymerase activity of reticuloendotheliosis viruses. J. Virol. 16:880-886. 22. Keller, W., and R. Crough. 1972. Degradation of DNARNA hybrids by ribonuclease H and DNA polymerases of cellular and viral origin. Proc. Natl. Acad. Sci. U.S.A. 69:3360-3364. 23. Leis, J. P., I. Berkower, and J. Hurwitz. 1973. Mechanism of action of RNase H isolated from AMV and E. coli. Proc. Nad. Acad. Sci. U.S.A. 70:466-470. 24. Leis, J. P. 1976. RNA-dependent DNA polymerase activity of RNA tumor viruses. VI. Processive mode of action of the avian myeloblastosis virus polymerase. J. Virol. 19:932-939. 25. McDonnell, G. P., A. C. Garapin, W. E. Levinson, N. Quintrell, L. Fanshier, and J. M. Bishop. 1970. DNA polymerase associated with Rous sarcoma virus: delineation of two reactions with actinomycin. Nature (London) 228:433-435.
J. VIROL. 26. Mitzutani, S., and H. M. Temin. 1975. Purification and properties of spleen necrosis virus DNA polymerase. J. Virol. 16:797-806. 27. Moelling, K. 1974. Reverse transcriptase and RNase H: present in a murine virus and in both subunits of an avian virus. Cold Spring Harbor Symp. Quant. Biol. 39:969-973. 28. Moelling, K. 1976. Comparison between an avian and a murine viral reverse transcriptase-RNase H complex, p. 121-124. In J. Clemmesen and D. S. Yohn (ed.), Comparative leukemia research 1975, Bibl. Haemat. no. 43. eds. S. Karger, Basel. 29. Moelling, K. 1976. Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. J. Virol. 18:418-425. 30. Moelling, K., B. P. Bolognesi, H. Bauer, W.Buesen, H. W. Plassmann, and P. Hausen. 1971. Association of viral reverse transcriptase with an enzyme degrading RNA moiety of RNA-DNA hybrids. Nature (London) New Biol. 234:240-243. 31. Panet, A., W. A. Haseltine, D. Baltimore, G. Peters, F. Harada, and J. E. Dahlberg. 1975. Specific binding to tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase). Proc. Nae. Acad. Sci. U.S.A. 72:2535-2539. 32. Panet, A., L. M. Verma, and D. Baltimore. 1974. Role of the subunits of the avian RNA tumor virus reverse transcriptase. Cold Spring Harbor Symp. Quant. Biol. 39:919-923. 33. Papas, T. S., D. J. Marciani, K. Samuel, and J. G. Chirikjian. 1976. Mechanism of release of active a subunit from dimeric a/I avian myeloblastosis virus DNA polymerase. J. Virol. 18:904-910. 34. Rho, H. M., D. P. Grandgenett, and M. Green. 1975. Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase. J. Biol. Chem. 250:5278-5280. 35. Rothenberg, E., and D. Baltimore. 1976. Synthesis of long, representative DNA copies of the murine RNA tumor virus genome. J. Virol. 17:168-174. 36. Staskus, K. A., M. S. Collett, and A. J. Faras. 1976. Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: structural and functional localization of the major species of primer RNA on the oncornavirus genome. Virology 71:162-168. 37. Taylor, J. M., and R. Illmensee. 1975. Site on the RNA of an avian sarcoma at which primer is bound. J. Virol. 16:553-558. 38. Waite, M., and P. Allen. 1975. RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions. J. Virol. 16:872-879. 39. Waters, L C., B. C. Mullin, T. Ho, and W. K. Yang. 1975. Ability of tryptophan tRNA to hybridize with 35S RNA of avian myeloblastosis virus and to prime reverse transcription in vitro. Proc. Nad. Acad. Sci. U.S.A. 72:2155-2159.
JOURNAL
Vol. 24, No. 2 Printed in U.S.A.
In Vitro Transcription of the Avian Retrovirus Genome by the a Form of the Viral RNA-Directed DNA Polymerase MARC S. COLLETT,' DUANE P. GRANDGENETT,2 AND ANTHONY J. FARAS1* Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455,1 and Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 631102
Received for publication 3 June 1977
The nature of transcription of the avian retrovirus RNA genome by the a form of the viral RNA-directed DNA polymerase has been investigated. Transcription was most efficient when Mn2" was provided as the divalent metal ion. The patterns of DNA transcription using 70S RNA, 35S RNA tRNAtrP, or 35S RNA oligo(dT)12.18 template primer complexes by the a DNA polymerase were essentially identical to those obtained using the a,8 form. The a DNA polymerase appears to be deficient in the synthesis of true duplex DNA but is able to synthesize hairpin-structured DNA initiated at the 5' terminus of the viral genome on the tRNAtrP primer molecule. The RNA-directed DNA polymerase of avian myeloblastosis virus exists in two enzymatically active forms. The major species of holoenzyme, a13, consists of an equimolar ratio of two polypeptides, a and f, with molecular weights of approximately 62,000 and 92,000, respectively (13, 15,20), whereas the minor DNA polymerase, termed a, consists of only the smaller polypeptide. The larger ,8 polypeptide appears to be the primary viral gene product, since the a polypeptide is apparently derived from ,B by proteolytic cleavage (11, 27, 29, 33, 34). In addition, both polypeptides are antigenically related and share similar amino acid sequences (11, 15, 34). A third DNA polymerase species, (,if, was recently identified in virions of Rous sarcoma virus and is thought to be a precursor to the aB species
(19).
Both avian myeloblastosis virus polymerase species exhibit all three enzymatic activities associated with purified viral reverse transcriptase: RNA-directed DNA synthesis, DNA-directed DNA synthesis, and RNase H activity (1, 15, 27). Differences in their modes of action have been reported. Whereas there is some question as to the processiveness of the af3 form of the DNA polymerase (7, 24), the a form appears to be nonprocessive in nature (12, 29). However, the RNase H activity associated with the a,8 and a enzymes appears to be processive and nonprocessive, respectively (12, 22, 23). Differences have also been noted with respect to the ability of each of the enzyme forms to bind natural and synthetic nucleic acids. The a/3 enzyme has been shown to bind more avidly than a to various template primer complexes (12, 13, 29, 32). Furthermore, it has been demonstrated
that under conditions whereby the a,8 DNA polymerase binds with high affinity to the avian retrovirus primer molecule (tryptophan tRNA) involved in synthesis of virus-specific DNA (6, 8-10, 31, 39), the a subunit exhibits little or no binding to this primer RNA species (16, 18). In view of the above differences between the a and a,3 forms of avian myelobastosis virus RNA-directed DNA polymerase, we have compared the properties of viral genome transcription in vitro by these two enzymes. Our studies of viral DNA transcription in reconstructed enzymatic reactions containing purified al DNA polymerase and genome RNA have been presented elsewhere (5). In this communication, we present data from our analysis of a RNA-directed DNA polymerase transcription in reconstructed reactions. It has been previously reported that the a DNA polymerase is unable to efficiently transcribe viral 70S RNA (17, 28). However, this deficiency could be overcome by the addition of oligo(dT) as a primer, presumably by complexing with the viral RNA 3' poly(A) tract (28). Our initial studies involved a determination of the synthetic capabilities of the a DNA polymerase as a function of the divalent metal ion present in reactions using three different template-primer complexes: native 70S RNA; 35S viral subunit RNA to which purified tRNAtrP primer had been reannealed (4, 9); and 35S RNA to which oligo(dT)12-18 had been added as the primer molecule (5). The a form of the DNA polymerase exhibited a preference for Mn2, whereas the a/B form preferred Mg2", in reconstructed enzymatic reactions using all three template primer complexes (Fig. 1). 704
NOTES
VOL. 24, 1977
Using the optimal divalent ion concentration (2 mM Mn2"), we compared the size distribution of DNA transcripts synthesized from the three template* primer complexes by the a form of the RNA-directed DNA polymerase. Radiolabeled DNA products were synthesized under several different concentrations of deoxynucleoside triphosphates (3, 5), purified, and analyzed by electrophoresis under denaturing conditions in polyacrylamide gels containing 98% formamide (5). The bulk of the DNA transcribed by the a DNA polymerase from either the 70S RNA (data not shown) or 35S RNA tRNAtrP template primer complex under either "ratelimiting" (three deoxynucleotide triphosphates [dNTP's] at 60 ,uM, the fourth at 6 AM) (data not shown) or "equimolar" (all four dNTP's at 60 AM) concentrations of dNTP's was less than 200 nucleotides in length (Fig. 2A). The three discrete species of DNA observed, a major component of 185 nucleotides in length and two minor classes of 100 and 70 nucleotides in length, represent initial DNA (iDNA) transcripts primed by the tRNAtrP molecule at the 5' ter-
705
minus of the viral genome (4, 5, 36, 37; Collett and Faras, in preparation). When 35S RNA oligo(dT)12-18template primer complexes were used in reactions containing equimolar concentrations of dNTP's, the DNA product synthesized by the a DNA polymerase exhibited a broad size distribution ranging from 220 to 2,000 nucleotides in length (Fig. 2B). These patterns of a DNA polymerase transcription from the 70S RNA, 35S RNA tRNAtrP, and 35S RNA oligo(dT)12-18template- primer complexes are identical to those observed when the afl form of the DNA polymerase was used in similar reconstructed reactions under optimal conditions of enzymatic DNA synthesis (5). Increasing the concentration of dNTP's either in endogenous reverse transcriptase reactions (3, 35) or reconstructed enzymatic reactions (5) has been shown to promote the synthesis of longer DNA transcripts. To determine if increasing the substrate concentration similarly affected a DNA polymerase transcription, DNA synthesis was conducted in the presence of 1 mM each of the dNTP's and the purified DNA product an-
A. 75 _
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o
_60 _ 0
0
45
I
30
5
[M91J
Xi
10 -3
15 M
M
2
-3] 0l
LMnJ
X 10
3 M
FIG. 1. Effect of divalent metal ion on DNA synthesis in reconstructed enzymatic reactions using the a and a,/ forms of the RNA-directed DNA polymerase. Three viral template -primer complexes were used in the various enzymatic reactions (5): native 70SRNA (0, *), 35SRNA tRNA'rP (O. *), and 35SRNA oligo(dT)12 18 (A, A). Reaction mixtures contained: 10 ug of template primer complex per ml; 0.1 M Tris-hydrochloride, pH 8.1; 0.01 MMgCI2; 1.4% (vol/vol) 2-mercaptoethanol; 6 ,uM[3H]dGTP (20 Ci/mmol); 60 uM each unlabeled dATP, dTTP, and dCTP. Both a DNA polymerase and a,/ DNA polymerase were present in saturating amounts. The a DNA polymerase was purified by chromatography on poly(U)-Sepharose (14) and exhibited a single peak on sodium dodecyl sulfate-polyacrylamide gels. The a DNA polymerase preparations contained no detectable DNA exonuclease, endonuclease, or RNase activities (14). Reactions were incubated at 37°C for 60 min with varying amounts of MgCl2 (A) or MnCl2 (B), and the perchloric acid-precipitable counts were determined. The open symbols represent synthesis by the a/I form, and the solid symbols represent synthesis by the a form of the RNA-directed DNA polymerase.
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J. VIROL.
NOTES
synthesize true duplex DNA. afI DNA polymerase-mediated transcription of 35S RNA- oligo(dT)12-18template- primer complexes results in the production of a considerausually w 0 portion (75%) of product that exhibits the ble 0 w characteristics of double-stranded DNA (Table -J 1), whereas the DNA product of a DNA polym1.) z erase transcription contains only a small portion of duplex DNA (10%). These results are in agreement with previous data (28) demonstrating that 0 viral DNA synthesis by the a enzyme on an oligo(dT) primer is insensitive to inhibition by actinomycin D, an inhibitor of double-stranded DNA synthesis (25). However, an analysis of the products of tRNAtrP-initiated DNA synthesis in the presence of low concentations of dNTP's reveals that the a DNA polymerase is able to synthesize DNA that is hairpin in nature (Table 1). Although several of the 5'-terminally DISTANCE MIGRATED (mmi) located initial DNA transcripts (4, 5, 36) of 100 (iDNAloo) and 70 (iDNA70) nucleotides in length FIG. 2. Polyacrylamide-formamide gel electropho- (Fig. 2A) are largely single stranded (Table 1), resis of DNA synthesized in reconstructed reactions using a RNA-directed DNA polymerase. Enzymatic the 185-nucleotide-length iDNA (iDNA,85) repDNA synthesis by the a form of RNA-directed DNA resents a hairpin or foldback structure, as dempolymerase on either 35S RNA- tRNA1'r (A) or 35S onstrated by its considerable resistance to SI RNA oligo (dT) 12-18 (B) template primer complexes in nuclease hydrolysis after denaturation (Table the presence of equimolar concentrations (60 PM) of 1). In addition, iDNA185 is nearly completely dNTP's was essentially as described for a,/ DNA resistant to exonuclease I hydrolysis (90%) (data polymerase-mediated synthesis (5), except 2 mM MnCl2 was used as the divalent metal ion. The puriI
(D z -J
0
fied DNA transcripts were subjected to electrophoresis in 5% polyacrylamide-98% formamide cylindrical gels (0.6 by 10 cm) as previously described (5). Gels were calibrated with respect to nucleotide length by parallel electrophoresis of the HaeIII restriction endonuclease fragments of OX 174, simian virus 40, and polyoma virus DNAs (5).
I-mLI) z
w -J
0
(-i z 0
alyzed on polyacrylamide-formamide gels. From the electropherogram presented in Fig. 3A, the DNA product synthesized on either 35S RNA- tRNAtrP or 70S RNA (data not shown) exhibited a dramatic shift in size distribution, with transcripts ranging to 2,000 nucleotides in length. Under similar reaction conditions, DNA synthesized on 35S RNA- oligo(dT)12 18 template- primer complexes exhibited an accumulation of transcripts ranging between 1,500 and 2,500 nucleotides in length (Fig. 3B). These characteristics of a DNA polymerase transcription at high concentrations of dNTP's are again identical to those previously observed for transcription by the afl form of the enzyme (5). Analysis of the secondary structure of the DNA products resulting from a DNA polymerase transcription of the viral genome, when compared with afl DNA polymerase transcripts under optimal reaction conditions, suggests that the a form may be deficient in its ability to
0. U
DISTANCE MIGRATED (mm)
FIG. 3. Polyacrylamide-formamide gel electrophoresis of a DNA polymerase transcripts synthesized in the presence of high concentrations of dNTP's. Enzymatic synthesis by the purified a DNA polymerase was carried out as described in the legend to Fig. 2, except in the presence of all four dNTP's at 1 mM. Subsequent purification and electrophoresis in 5% polyacrylamide-98% formamide gels was as previously described (5). (A) a DNA transcripts synthesized from 35S RNA tRNAtrP. (B) a DNA transcripts synthesized from 35S RNA oligo(dT)12-18-
NOTES
VOL. 24, 1977
TABLE 1. Secondary structure of various DNA transcripts synthesized in reconstructed reactions by the afi and a forms of RNA-directed DNA polymerase Si nuclease reDNA transcript" sistant (%)b Total product, cr1 polymerase .74.5 Total product, a polymerase .9.7 . .10.4 iDNA70 . .12.8 iDNAoo 68.0 iDNA15 ... a Total DNA product was synthesized by either the ad1 or a form of DNA polymerase using viral 35S RNA oligo(dT)12 18 template primer complexes as described in the legend to Fig. 2. The iDNA transcripts of various lengths (70, 100, and 185 nucleotides) were synthesized from 35S RNA tRNAP template primer complexes, using either the ac3 or a form of the viral DNA polymerase, and were purified from denaturing 5% polyacrylamide-98% formamide gels (5). b The preparation of reaction products and treatment with Si nuclease were as previously described (2; Collett and Faras, submitted for publication). The iDNA transcripts synthesized by the af? and a DNA polymerase demonstrated the same degree of SI nuclease resistance.
not shown). A more detailed characterization of this hairpin DNA will be published elsewhere (Collett and Faras, submitted for publication). Both the a and an) forms of the viral DNA polymerase synthesize the hairpin iDNA185 to similar extents (cf. profiles in Fig. 2A and reference 5). Thus, it appears that the a DNA polymerase is deficient only in the synthesis of true duplex DNA, while maintaining its ability to form hairpin DNA initiated from the tRNAtr' primer at the 5' terminus of the viral genome. Although the a form of the avian retrovirus DNA polymerase binds poorly to various natural and synthetic nucleic acids (12, 13, 29, 32), and not at all to the viral tRNAtrP primer molecule (16, 17), it still exhibits a pattern of transcription of DNA from viral genome template primer complexes identical to that of the at) form. Transcription is most efficient if Mn2+ is used as the divalent metal ion. A similar phenomenon has been demonstrated with both mammalian and reticuloendotheliosis retrovirus reverse transcriptases (17, 21, 26, 38). It is interesting to note that DNA polymerase from both of these latter sources exhibit molecular weight estimates similar to avian myeloblastosis virus a DNA polymerase (17, 26). The apparent inability of the a DNA polymerase to synthesize true duplex DNA, while still mediating the synthesis of tRNAtrP-initiated hairpin DNA, may be a function of the location of the major site of transcription on the viral genome and/or the
707
nature of the primer for the synthesis of the second DNA strand. For example, the synthesis of hairpin iDNA185 appears to result from the use of DNA transcripts that have reached the 5' terminus of the viral genome (iDNA1oo) as both primer and template for the synthesis of a complementary strand (Collett and Faras, submitted for publication). The mechanism by which true duplex DNA is synthesized in either reconstructed or endogenous reverse transcriptase reactions is currently unknown but appears to reflect a property of the ac1 form, but not the a form, of the DNA polymerase. We thank S. Kanellos and P. Wilkie for excellent technical assistance and L. Honza for typing this manuscript. This investigation was supported by Public Health Service research grants CA 18303-02, CA 20011-01, and CA-16312-04 from the National Cancer Institute. One of us (D.P.G.) is a recipient of an American Cancer Society faculty research award.
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