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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Feb 15;90(4):1276–1280. doi: 10.1073/pnas.90.4.1276

RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template.

A Telesnitsky 1, S P Goff 1
PMCID: PMC45855  PMID: 7679498

Abstract

The active sites for the polymerase and nuclease activities of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) reside in separate domains of a single polypeptide. We have studied the effects of RNase H domain mutations on DNA polymerase activity. These mutant RTs displayed decreased processivity of DNA synthesis. We also compared complexes formed between primer-templates and mutant and wild-type reverse transcriptase (RT). Although M-MuLV RT is monomeric in solution, two molecules of RT bound DNA cooperatively, suggesting that M-MuLV RT binds primer-template as a dimer. Some mutant RTs with decreased processivity failed to form the putative dimer.

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  1. Buiser R. G., DeStefano J. J., Mallaber L. M., Fay P. J., Bambara R. A. Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. J Biol Chem. 1991 Jul 15;266(20):13103–13109. [PubMed] [Google Scholar]
  2. Chao K. L., Lohman T. M. DNA-induced dimerization of the Escherichia coli Rep helicase. J Mol Biol. 1991 Oct 20;221(4):1165–1181. doi: 10.1016/0022-2836(91)90926-w. [DOI] [PubMed] [Google Scholar]
  3. Cheng N., Painter G. R., Furman P. A. Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase. Biochem Biophys Res Commun. 1991 Jan 31;174(2):785–789. doi: 10.1016/0006-291x(91)91486-v. [DOI] [PubMed] [Google Scholar]
  4. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  5. DeStefano J. J., Buiser R. G., Mallaber L. M., Myers T. W., Bambara R. A., Fay P. J. Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J Biol Chem. 1991 Apr 25;266(12):7423–7431. [PubMed] [Google Scholar]
  6. Doolittle R. F., Feng D. F., Johnson M. S., McClure M. A. Origins and evolutionary relationships of retroviruses. Q Rev Biol. 1989 Mar;64(1):1–30. doi: 10.1086/416128. [DOI] [PubMed] [Google Scholar]
  7. Dudding L. R., Nkabinde N. C., Mizrahi V. Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. Biochemistry. 1991 Oct 29;30(43):10498–10506. doi: 10.1021/bi00107a019. [DOI] [PubMed] [Google Scholar]
  8. Furfine E. S., Reardon J. E. Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. J Biol Chem. 1991 Jan 5;266(1):406–412. [PubMed] [Google Scholar]
  9. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  10. Goff S. P. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed] [Google Scholar]
  11. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hizi A., Hughes S. H., Shaharabany M. Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. Virology. 1990 Apr;175(2):575–580. doi: 10.1016/0042-6822(90)90444-v. [DOI] [PubMed] [Google Scholar]
  13. Hostomsky Z., Hostomska Z., Fu T. B., Taylor J. Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis. J Virol. 1992 May;66(5):3179–3182. doi: 10.1128/jvi.66.5.3179-3182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  15. Huber H. E., McCoy J. M., Seehra J. S., Richardson C. C. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed] [Google Scholar]
  16. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kanaya S., Katsuda-Nakai C., Ikehara M. Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. J Biol Chem. 1991 Jun 25;266(18):11621–11627. [PubMed] [Google Scholar]
  18. Katz R. A., Skalka A. M. Generation of diversity in retroviruses. Annu Rev Genet. 1990;24:409–445. doi: 10.1146/annurev.ge.24.120190.002205. [DOI] [PubMed] [Google Scholar]
  19. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  20. Kotewicz M. L., Sampson C. M., D'Alessio J. M., Gerard G. F. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res. 1988 Jan 11;16(1):265–277. doi: 10.1093/nar/16.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Le Grice S. F., Naas T., Wohlgensinger B., Schatz O. Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J. 1991 Dec;10(12):3905–3911. doi: 10.1002/j.1460-2075.1991.tb04960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levin J. G., Crouch R. J., Post K., Hu S. C., McKelvin D., Zweig M., Court D. L., Gerwin B. I. Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity. J Virol. 1988 Nov;62(11):4376–4380. doi: 10.1128/jvi.62.11.4376-4380.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luo G. X., Taylor J. Template switching by reverse transcriptase during DNA synthesis. J Virol. 1990 Sep;64(9):4321–4328. doi: 10.1128/jvi.64.9.4321-4328.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mizrahi V., Usdin M. T., Harington A., Dudding L. R. Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase. Nucleic Acids Res. 1990 Sep 25;18(18):5359–5363. doi: 10.1093/nar/18.18.5359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oyama F., Kikuchi R., Crouch R. J., Uchida T. Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease. J Biol Chem. 1989 Nov 5;264(31):18808–18817. [PubMed] [Google Scholar]
  26. Prasad V. R., Goff S. P. Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. Proc Natl Acad Sci U S A. 1989 May;86(9):3104–3108. doi: 10.1073/pnas.86.9.3104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reardon J. E., Furfine E. S., Cheng N. Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. J Biol Chem. 1991 Jul 25;266(21):14128–14134. [PubMed] [Google Scholar]
  28. Repaske R., Hartley J. W., Kavlick M. F., O'Neill R. R., Austin J. B. Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase. J Virol. 1989 Mar;63(3):1460–1464. doi: 10.1128/jvi.63.3.1460-1464.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roth M. J., Tanese N., Goff S. P. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J Biol Chem. 1985 Aug 5;260(16):9326–9335. [PubMed] [Google Scholar]
  30. Tanese N., Goff S. P. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777–1781. doi: 10.1073/pnas.85.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Telesnitsky A., Blain S. W., Goff S. P. Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. J Virol. 1992 Feb;66(2):615–622. doi: 10.1128/jvi.66.2.615-622.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Verma I. M. The reverse transcriptase. Biochim Biophys Acta. 1977 Mar 21;473(1):1–38. doi: 10.1016/0304-419x(77)90005-1. [DOI] [PubMed] [Google Scholar]
  33. Yang W., Hendrickson W. A., Crouch R. J., Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science. 1990 Sep 21;249(4975):1398–1405. doi: 10.1126/science.2169648. [DOI] [PubMed] [Google Scholar]

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