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. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

J Kulkosky 1, K S Jones 1, R A Katz 1, J P Mack 1, A M Skalka 1
PMCID: PMC364405  PMID: 1314954

Abstract

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beese L. S., Steitz T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. doi: 10.1002/j.1460-2075.1991.tb07917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
  3. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  4. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buechler J. A., Taylor S. S. Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase. Biochemistry. 1988 Sep 20;27(19):7356–7361. doi: 10.1021/bi00419a027. [DOI] [PubMed] [Google Scholar]
  6. Bushman F. D., Fujiwara T., Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. doi: 10.1126/science.2171144. [DOI] [PubMed] [Google Scholar]
  7. Castle E., Nowak T., Leidner U., Wengler G., Wengler G. Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology. 1985 Sep;145(2):227–236. doi: 10.1016/0042-6822(85)90156-4. [DOI] [PubMed] [Google Scholar]
  8. Davies J. F., 2nd, Hostomska Z., Hostomsky Z., Jordan S. R., Matthews D. A. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science. 1991 Apr 5;252(5002):88–95. doi: 10.1126/science.1707186. [DOI] [PubMed] [Google Scholar]
  9. Derbyshire V., Grindley N. D., Joyce C. M. The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 1991 Jan;10(1):17–24. doi: 10.1002/j.1460-2075.1991.tb07916.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engelman A., Mizuuchi K., Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell. 1991 Dec 20;67(6):1211–1221. doi: 10.1016/0092-8674(91)90297-c. [DOI] [PubMed] [Google Scholar]
  12. Fayet O., Ramond P., Polard P., Prère M. F., Chandler M. Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol. 1990 Oct;4(10):1771–1777. doi: 10.1111/j.1365-2958.1990.tb00555.x. [DOI] [PubMed] [Google Scholar]
  13. Hein J. Unified approach to alignment and phylogenies. Methods Enzymol. 1990;183:626–645. doi: 10.1016/0076-6879(90)83041-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kanaya S., Kohara A., Miura Y., Sekiguchi A., Iwai S., Inoue H., Ohtsuka E., Ikehara M. Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis. J Biol Chem. 1990 Mar 15;265(8):4615–4621. [PubMed] [Google Scholar]
  16. Katz R. A., Merkel G., Kulkosky J., Leis J., Skalka A. M. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. doi: 10.1016/0092-8674(90)90290-u. [DOI] [PubMed] [Google Scholar]
  17. Katzman M., Katz R. A., Skalka A. M., Leis J. The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration. J Virol. 1989 Dec;63(12):5319–5327. doi: 10.1128/jvi.63.12.5319-5327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katzman M., Mack J. P., Skalka A. M., Leis J. A covalent complex between retroviral integrase and nicked substrate DNA. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4695–4699. doi: 10.1073/pnas.88.11.4695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  21. Kulkosky J., Skalka A. M. HIV DNA integration: observations and interferences. J Acquir Immune Defic Syndr. 1990;3(9):839–851. [PubMed] [Google Scholar]
  22. Mizuuchi K., Craigie R. Mechanism of bacteriophage mu transposition. Annu Rev Genet. 1986;20:385–429. doi: 10.1146/annurev.ge.20.120186.002125. [DOI] [PubMed] [Google Scholar]
  23. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  24. Rowland S. J., Dyke K. G. Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol. 1990 Jun;4(6):961–975. doi: 10.1111/j.1365-2958.1990.tb00669.x. [DOI] [PubMed] [Google Scholar]
  25. Sherman P. A., Fyfe J. A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119–5123. doi: 10.1073/pnas.87.13.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taha M. K., So M., Seifert H. S., Billyard E., Marchal C. Pilin expression in Neisseria gonorrhoeae is under both positive and negative transcriptional control. EMBO J. 1988 Dec 20;7(13):4367–4378. doi: 10.1002/j.1460-2075.1988.tb03335.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Terry R., Soltis D. A., Katzman M., Cobrinik D., Leis J., Skalka A. M. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vink C., Yeheskiely E., van der Marel G. A., van Boom J. H., Plasterk R. H. Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNA termini mediated by the viral integrase protein. Nucleic Acids Res. 1991 Dec 25;19(24):6691–6698. doi: 10.1093/nar/19.24.6691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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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