A covalent complex between retroviral integrase and nicked substrate DNA.

Katzman, Michael; Mack, Joseph P. G.; Skalka, Anna Marie; Leis, Jonathan

doi:10.1073/pnas.88.11.4695

Cited by 43 publications

(39 citation statements)

References 23 publications

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“…At least seven IS elements were found in the chromosome (named ISBsph3 to ISBsph9), and one copy of ISBsph9 exists in plasmid pBsph (Table 3). ISBsph3, ISBsph4, ISBsph5, ISBsph6, ISBsph7, and ISBsph9 share a number of features with other IS3 family elements, including inverted repeats, similarity between transposases, and the DD-(35)-E-(7)-K or DD-(35)-E-(7)-R motifs, which are highly conserved patterns among ISs (19,33,35).…”

Section: Resultsmentioning

confidence: 99%

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

Fan

Han

et al. 2008

J Bacteriol

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Bacillus sphaericus strain C3-41 is an aerobic, mesophilic, spore-forming bacterium that has been used with great success in mosquito control programs worldwide. Genome sequencing revealed that the complete genome of this entomopathogenic bacterium is composed of a chromosomal replicon of 4,639,821 bp and a plasmid replicon of 177,642 bp, containing 4,786 and 186 potential protein-coding sequences, respectively. Comparison of the genome with other published sequences indicated that the B. sphaericus C3-41 chromosome is most similar to that of Bacillus sp. strain NRRL B-14905, a marine species that, like B. sphaericus, is unable to metabolize polysaccharides. The lack of key enzymes and sugar transport systems in the two bacteria appears to be the main reason for this inability, and the abundance of proteolytic enzymes and transport systems may endow these bacteria with exclusive metabolic pathways for a wide variety of organic compounds and amino acids. The genes shared between B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, including mobile genetic elements, membrane-associated proteins, and transport systems, demonstrated that these two species are a biologically and phylogenetically divergent group. Knowledge of the genome sequence of B. sphaericus C3-41 thus increases our understanding of the bacilli and may also offer prospects for future genetic improvement of this important biological control agent.

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Section: Resultsmentioning

confidence: 99%

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

Fan

Han

et al. 2008

J Bacteriol

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“…The source of retroelement sequences has been described previously (21,22). Figure 1 shows Circle joining assay.…”

Section: Methodsmentioning

confidence: 99%

“…The alignment of phylogenetically representative members of the set of retroelements and ISs for the residues near D-64 and near the D(35)E domain (D-121 to E-157) of RSV IN is shown in Fig. 1.The source of retroelement sequences has been described previously (21,22). Figure 1 shows Circle joining assay.…”

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confidence: 99%

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

Kulkosky¹,

Jones²,

Katz³

et al. 1992

Mol. Cell. Biol.

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549

510

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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. On the basis of similarities with enzymes that catalyze analogous reactions, we propose that the invariant D and E residues may participate in coordination of the metal cofactor (Mn2+ or Mg2') required for the catalytic activities of IN. We further speculate that a metal-DNA complex may be necessary to position both LTR and target DNA substrates for nucleophilic attack during the cleavage and joining reactions.Retroviral integration is dependent upon the interaction of three macromolecular components: (i) a virus-encoded protein, integrase (IN), (ii) specific sequences at the long terminal repeat (LTR) termini of viral "donor" DNA, and (iii) a host DNA "target." Results from the study of several different retroviral systems reveal a stepwise pathway for the interaction of these components during integration in a natural virus infection: (i) removal of a dinucleotide from the 3' ends of linear viral DNA (4) in the cytoplasm, which we refer to as the processing reaction, (ii) migration of the viral DNA to the cell nucleus within a higher-order protein complex (2), (iii) staggered cleavage of host DNA generating 5'-strand extensions, likely coupled to single-strand joining of the processed 3' hydroxyl ends of viral DNA to the 5' phosphate ends of the host target DNA, and (iv) repair and ligation of the gapped unjoined opposite strands. This final step produces a short duplication of host seq...

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“…The first step consists of the elimination of 2 nucleotides from each 3Ј end of the proviral DNA. In the second step, the resulting 3Ј-OH ends of the viral DNA are covalently joined to newly created 5Ј ends in the target DNA (11,18,39).…”

mentioning

confidence: 99%

The Karyophilic Properties of Human Immunodeficiency Virus Type 1 Integrase Are Not Required for Nuclear Import of Proviral DNA

Petit

Schwartz

Mammano

2000

J Virol

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The retroviral integrase (IN) protein catalyzes integration of the provirus into a chromosome of the infected cell, an essential step of the viral replication cycle (see references 1 and 4 for recent reviews on integration). IN is translated as part of the Gag-Pol precursor molecule, which is cleaved by the viral protease to allow viral particle maturation. Several in vitro studies have examined the biochemical properties of IN, and significant progress has been made in the understanding of its structure and of the mechanism of the integration reaction (1,4,15,31). IN catalyzes the two steps of the integration process. The first step consists of the elimination of 2 nucleotides from each 3Ј end of the proviral DNA. In the second step, the resulting 3Ј-OH ends of the viral DNA are covalently joined to newly created 5Ј ends in the target DNA (11,18,39).Retroviral IN proteins are composed of three functionally distinct domains (see Fig. 1A), all of which are required for a complete integration reaction. The N-terminal domain contains a zinc finger-like motif that stabilizes the folded structure of IN and enhances the catalytic activity of the enzyme (57). The core domain of retroviral IN contains the DDE motif to which the catalytic activity is attributed. This central domain is also involved in the recognition of the conserved nucleotide sequence at each end of the retroviral DNA. The carboxyterminal domain is the least conserved among retroviruses, possesses intrinsic DNA binding activity, and is required for 3Ј-end processing and strand transfer (10, 51). The functional form of IN is multimeric, as was suggested by in vitro evidence of multimerization and demonstrated by trans-complementation of different IN mutants (16,23,(34)(35)(36)50). We recently demonstrated that the multimerization of human immunodeficiency virus type 1 (HIV-1) IN takes place in infectious viral particles and is dependent on disulfide bond formation (46).Besides the well characterized role in the integration process, IN participates in different steps of the virus cycle. Alterations of IN sequence were found to affect viral particle morphogenesis, reverse transcription, and nuclear import of the preintegration complex (PIC) (17, 28, 55), a nucleoprotein complex composed of viral and probably cellular proteins that carries the viral genome from the cytoplasm to the nucleus of the newly infected cell (9,20,21,37,38,45).HIV-1 IN has karyophilic properties which were demonstrated by the nuclear accumulation of this protein both after transient expression of a Flag-or green fluorescent proteintagged IN (46,47) and after microinjection of HIV-1 IN fused to glutathione S-transferase (GST) (28). The GST-IN fusion protein was additionally shown to bind in vitro to karyopherin-␣ (28), a cellular mediator of nuclear transport which is specific for nuclear localization signal-bearing proteins. The interaction between IN and karyopherin-␣ was suggested to be functionally relevant in vivo, providing one additional mechanism for the nuclear import of HI...

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A covalent complex between retroviral integrase and nicked substrate DNA.

Cited by 43 publications

References 23 publications

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

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

The Karyophilic Properties of Human Immunodeficiency Virus Type 1 Integrase Are Not Required for Nuclear Import of Proviral DNA

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