• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. May 1992; 12(5): 2331–2338.
PMCID: PMC364405

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

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)

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.8M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Beese LS, Steitz TA. 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. [PMC free article] [PubMed]
  • Bowerman B, Brown PO, Bishop JM, Varmus HE. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. [PubMed]
  • Brown PO, Bowerman B, Varmus HE, Bishop JM. 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. [PMC free article] [PubMed]
  • Brown PO, Bowerman B, Varmus HE, Bishop JM. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. [PubMed]
  • Buechler JA, Taylor SS. 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. [PubMed]
  • Bushman FD, Fujiwara T, Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. [PubMed]
  • 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. [PubMed]
  • Davies JF, 2nd, Hostomska Z, Hostomsky Z, Jordan SR, Matthews DA. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science. 1991 Apr 5;252(5002):88–95. [PubMed]
  • Derbyshire V, Grindley ND, Joyce CM. 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Fayet O, Ramond P, Polard P, Prère MF, Chandler M. Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol. 1990 Oct;4(10):1771–1777. [PubMed]
  • Hein J. Unified approach to alignment and phylogenies. Methods Enzymol. 1990;183:626–645. [PubMed]
  • Johnson MS, McClure MA, Feng DF, Gray J, Doolittle RF. 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. [PMC free article] [PubMed]
  • 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]
  • Katz RA, Merkel G, Kulkosky J, Leis J, Skalka AM. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. [PubMed]
  • Katzman M, Katz RA, Skalka AM, 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. [PMC free article] [PubMed]
  • Katzman M, Mack JP, Skalka AM, 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. [PMC free article] [PubMed]
  • Khan E, Mack JP, Katz RA, Kulkosky J, Skalka AM. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. [PMC free article] [PubMed]
  • Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. [PubMed]
  • Kulkosky J, Skalka AM. HIV DNA integration: observations and interferences. J Acquir Immune Defic Syndr. 1990;3(9):839–851. [PubMed]
  • Mizuuchi K, Craigie R. Mechanism of bacteriophage mu transposition. Annu Rev Genet. 1986;20:385–429. [PubMed]
  • Roth MJ, Schwartzberg PL, Goff SP. 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. [PubMed]
  • Rowland SJ, Dyke KG. Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol. 1990 Jun;4(6):961–975. [PubMed]
  • Sherman PA, Fyfe JA. 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. [PMC free article] [PubMed]
  • Taha MK, So M, Seifert HS, 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. [PMC free article] [PubMed]
  • Terry R, Soltis DA, Katzman M, Cobrinik D, Leis J, Skalka AM. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. [PMC free article] [PubMed]
  • Vink C, Yeheskiely E, van der Marel GA, van Boom JH, Plasterk RH. 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. [PMC free article] [PubMed]
  • Yang W, Hendrickson WA, Crouch RJ, 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. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...