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PubMed2001

A structural view of cre-loxp site-specific recombination.

Structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process. One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products. Crystal structures of reaction intermediates in the Cre-loxP site-specific recombination system, together with recent biochemical studies in the field, support a "strand swapping" model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining sites.

Base Sequence↗
PubMed2006

The clinical pharmacology of antiretrovirals in development.

Drugs in development for the management of HIV type 1 (HIV-1) infection include agents in existing classes and agents of novel classes. Of existing classes, new protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors are in development. Novel therapeutic approaches include the development of chemokine receptor (CCR)5 antagonists, integrase inhibitors and maturation inhibitors. CCR5 antagonists are thought to inhibit HIV-1 entry into host cells by occupying a specific site on the CCR5 receptor, preventing attachment of the HIV-1 envelope protein gp120. Integrase inhibitors are small synthetically prepared molecules that block RNA/DNA interactions and modify protein or enzyme synthesis. Data on the pharmacokinetics and pharmacodynamics of these new antiretroviral agents continue to generate interest. This review reports the known data on the pharmacokinetics of experimental antiretrovirals, and describe the main drug-drug interactions studied so far.

Animals↗
PubMed2005

Comparative analysis of sequence-specific DNA recombination systems in human embryonic stem cells.

The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage lambda integrase, phage P1 Cre recombinase, and mutant gammadelta resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent lambda integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.

Cell Line↗
PubMed2001

New developments in anti-HIV chemotherapy.

Virtually all the compounds that are currently used, or under advanced clinical trial, for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): i.e., zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), emtricitabine [(-)FTC], tenofovir (PMPA) disoproxil fumarate; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e., nevirapine, delavirdine, efavirenz, emivirine (MKC-442); and (iii) protease inhibitors (PIs): i.e., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir. In addition to the reverse transcriptase and protease step, various other events in the HIV replicative cycle are potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polyoxometalates, zintevir, negatively charged albumins, cosalane analogues); (ii) viral entry, through blockade of the viral coreceptors CXCR4 and CCR5 [bicyclams (i.e. AMD3100), polyphemusins (T22), TAK-779, MIP-1 alpha LD78 beta isoform]; (iii) virus-cell fusion, through binding to the viral glycoprotein gp41 [T-20 (DP-178), T-1249 (DP-107), siamycins, betulinic acid derivatives]; (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA) and NCp7 peptide mimics]; (v) proviral DNA integration, through integrase inhibitors such as L-chicoric acid and diketo acids (i.e. L-731,988); (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (fluoroquinolone K-12, Streptomyces product EM2487, temacrazine, CGP64222). Also, in recent years new NRTIs, NNRTIs and PIs have been developed that possess respectively improved metabolic characteristics (i.e. phosphoramidate and cyclosaligenyl pronucleotides of d4T), or increased activity against NNRTI-resistant HIV strains [second generation NNRTIs, such as capravirine and the novel quinoxaline, quinazolinone, phenylethylthiazolylthiourea (PETT) and emivirine (MKC-442) analogues], or, as in the case of PIs, a different, non-peptidic scaffold [i.e. cyclic urea (DMP 450), 4-hydroxy-2-pyrone (tipranavir)]. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating from cell-free enzymatic assays to the mode of action of these agents in intact cells. A number of compounds (i.e. zintevir and L-chicoric acid, on the one hand; and CGP64222 on the other hand) have recently been found to interact with virus-cell binding and viral entry in contrast to their proposed modes of action targeted at the integrase and transactivation process, respectively.

Amino Acid Sequence↗
PubMedDate not supplied

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis.

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Ascomycota↗
PubMed2000

Genetic characterization of gram-positive homologs of the XerCD site-specific recombinases.

Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.

Alleles↗
PubMed2000

Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle.

Polyanionic dendrimers were synthesized and evaluated for their antiviral effects. Phenyldicarboxylic acid (BRI6195) and naphthyldisulfonic acid (BRI2923) dendrimers were found to inhibit the replication of human immunodeficiency virus type 1 (HIV-1; strain III(B)) in MT-4 cells at a EC(50) of 0.1 and 0.3 microg/ml, respectively. The dendrimers were not toxic to MT-4 cells up to the highest concentrations tested (250 microg/ml). These compounds were also effective against various other HIV-1 strains, including clinical isolates, HIV-2 strains, simian immunodeficiency virus (SIV, strain MAC(251)), and HIV-1 strains that were resistant to reverse transcriptase inhibitors. HIV strains containing mutations in the envelope glycoprotein gp120 (engendering resistance to known adsorption inhibitors) displayed reduced sensitivity to the dendrimers. The compounds inhibited the binding of wild-type virus and recombinant virus (containing wild-type gp120) to MT-4 cells at concentrations comparable to those that inhibited the replication of HIV-1(III(B)) in these cells. Cellular uptake studies indicated that BRI2923, but not BRI6195, permeates into MT-4 and CEM cells. Accordingly, the naphtyldisulfonic acid dendrimer (BRI2923) proved able to inhibit later steps of the replication cycle of HIV, i.e., reverse transcriptase and integrase. NL4.3 strains resistant to BRI2923 were selected after passage of the virus in the presence of increasing concentrations of BRI2923. The virus mutants showed 15-fold reduced sensitivity to BRI2923 and cross-resistance to known adsorption inhibitors. However, these virus mutants were not cross-resistant to reverse transcriptase inhibitors or protease inhibitors. We identified several mutations in the envelope glycoprotein gp120 gene (i.e., V2, V3, and C3, V4, and C4 regions) of the BRI2923-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain, whereas no mutations were found in the reverse transcriptase or integrase genes.

Animals↗
PubMed2002

A XerD recombinase with unusual active site motifs in Streptococcus pneumoniae.

XerD belongs to the site specific recombinases of the integrase family of proteins that catalyze recombination events via a phosphotyrosine intermediate. Sequence alignments and crystal structure resolution of E. coli XerD and related enzymes demonstrated the importance of four conserved amino acids R-H-R-H that are spaced along the C-terminal domain in addition to a conserved K and the active site Y, all of which have been implicated in catalysis. The deduced amino acid sequence of the putative S. pneumoniae XerD contained three unique replacements at the conserved positions resulting in L-Q-R-L; moreover, the active site Y was the penultimate amino acid residue, and the extreme C-terminal region suggested to be involved in interaction of E. coli XerD with XerC was lacking. Severe growth defects in a loss-of-function xerD mutant are consistent with an important in vivo function of the S. pneumoniae XerD protein. Highly related xerD genes with similar unusual amino acid replacements were found in S. mitis, S. mutans and S. pyogenes but not in other Gram-positive bacteria, although the genetic environment was very similar in many species. There are at least another four genes in the S. pneumoniae KNR_7/87 genome encoding Xer related peptides, one of which was identified as the xerC homologue. The xerD and xerC genes were present in a sample of 20 S. pneumoniae strains whereas the other xer genes appear to be absent in some of the strains and are more closely related to integrases of phage and transposon origin.

Amino Acid Motifs↗
PubMed1993

Ligation of synthetic activated DNA substrates by site-specific recombinases and topoisomerase I.

The FLP protein of the 2-microns plasmid of Saccharomyces cerevisiae is a conservative site-specific recombinase that is involved in the amplification of the plasmid. This recombination reaction proceeds via the covalent attachment of the protein to the 3'-phosphoryl group at the site of the breaks through a phosphotyrosine linkage. We have recently developed an assay that measures FLP-mediated strand ligation independent of FLP-mediated cleavage and covalent attachment to the DNA. The substrate for ligation was produced by FLP-induced cleavage of the FLP recognition site followed by digestion with Pronase and was shown to contain (at least) a tyrosine residue at the 3'-PO4 terminus adjacent to the FLP cleavage sites. We have now synthesized artificial substrates that bear a tyrosine residue on the 3'-PO4 of an appropriate oligonucleotide and find that this substrate is ligated as efficiently as the previous ligation substrates that were isolated after FLP cleavage of the substrate. Analogous substrates for other members of the integrase family of recombinases (lambda integrase protein, P1-Cre protein) as well as for mammalian topoisomerase I are also active as ligation substrates with their cognate protein. This class of activated substrates should be useful in the study of breakage and reunion reactions involving DNA.

Animals↗
PubMed2000

Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection.

A large variety of natural products have been described as anti-HIV agents, and for a portion thereof the target of interaction has been identified. Cyanovirin-N, a 11-kDa protein from Cyanobacterium (blue-green alga) irreversibly inactivates HIV and also aborts cell-to-cell fusion and transmission of HIV, due to its high-affinity interaction with gp120. Various sulfated polysaccharides extracted from seaweeds (i.e., Nothogenia fastigiata, Aghardhiella tenera) inhibit the virus adsorption process. Ingenol derivatives may inhibit virus adsorption at least in part through down-regulation of CD4 molecules on the host cells. Inhibition of virus adsorption by flavanoids such as (-)epicatechin and its 3-O-gallate has been attributed to an irreversible interaction with gp120 (although these compounds are also known as reverse transcriptase inhibitors). For the triterpene glycyrrhizin (extracted from the licorice root Glycyrrhiza radix) the mode of anti-HIV action may at least in part be attributed to interference with virus-cell binding. The mannose-specific plant lectins from Galanthus, Hippeastrum, Narcissus, Epipac tis helleborine, and Listera ovata, and the N-acetylgl ucosamine-specific lectin from Urtica dioica would primarily be targeted at the virus-cell fusion process. Various other natural products seem to qualify as HIV-cell fusion inhibitors: the siamycins [siamycin I (BMY-29304), siamycin II (RP 71955, BMY 29303), and NP-06 (FR901724)] which are tricyclic 21-amino-acid peptides isolated from Streptomyces spp that differ from one another only at position 4 or 17 (valine or isoleucine in each case); the betulinic acid derivative RPR 103611, and the peptides tachyplesin and polyphemusin which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus, i.e., the 18-amino-acid peptide T22 from which T134 has been derived. Both T22 and T134 have been shown to block T-tropic X4 HIV-1 strains through a specific antagonism with the HIV corecept or CXCR4. A number of natural products have been reported to interact with the reverse transcriptase, i.e., baicalin, avarol, avarone, psychotrine, phloroglucinol derivatives, and, in particular, calanolides (from the tropical rainforest tree, Calophyllum lanigerum) and inophyllums (from the Malaysian tree, Calophyllum inophyllum). The natural marine substance illimaquinone would be targeted at the RNase H function of the reverse transcriptase. Curcumin (diferuloylmethane, from turmeric, the roots/rhizomes of Curcuma spp), dicaffeoylquinic and dicaffeoylt artaric acids, L-chicoric acid, and a number of fungal metabolites (equisetin, phomasetin, oteromycin, and integric acid) have all been proposed as HIV-1 integrase inhibitors. Yet, we have recently shown that L-c hicoric acid owes its anti-HIV activity to a specific interaction with the viral envelope gp120 rather than integrase. A number of compounds would be able to inhibit HIV-1 gene expression at the transcription level: the flavonoid chrysin (through inhibition of casein kinase II, the antibacter ial peptides melittin (from bee venom) and cecropin, and EM2487, a novel substance produced by Streptomyces. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗
PubMedDate not supplied

Novel compounds in preclinical/early clinical development for the treatment of HIV infections.

Virtually all the compounds that are currently used, or under advanced clinical trial, for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and (iii) protease inhibitors (PIs). In addition to the reverse transcriptase and protease step, various other events in the HIV replicative cycle are potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulphates, polysulphonates, polyoxometalates, zintevir, negatively charged albumins); (ii) viral entry, through blockade of the viral coreceptors CXCR4 and CCR5 [bicyclams (AMD3100), polyphemusins (T22), TAK-779]; (iii) virus-cell fusion, through binding to the viral glycoprotein gp41 [T-20 (DP-178), siamycins, betulinic acid derivatives]; (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)]; (v) proviral DNA integration, through integrase inhibitors such as L-chicoric acid; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (peptoid CGP64222, fluoroquinolone K-12, Streptomyces product EM2487). Also, in recent years new NRTIs, NNRTIs and PIs have been developed that possess, respectively, improved metabolic characteristics (i.e. phosphoramidate and cyclosaligenyl pronucleotides of d4T), or increased activity against NNRTI-resistant HIV strains, or, in the case of PIs, a different, non-peptidic scaffold. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating from cell-free enzymatic assays to the mode of action of these agents in intact cells. A number of compounds (i.e. zintevir and L-chicoric acid, on the one hand; and CGP64222 on the other hand) have recently been found to interact with virus-cell binding and viral entry in contrast to their proposed modes of action targeted at the integrase and transactivation process, respectively.

Amino Acid Sequence↗
PubMed1988

Ribosome-inhibiting proteins, retroviral reverse transcriptases, and RNase H share common structural elements.

Plant ribosome-inhibiting proteins are shown to be homologous at the domain level to RNase H from Escherichia coli and to two regions of the pol gene product of retroviral reverse transcriptases. One of these regions carries the viral integrase or int function, while the other has previously been suggested to contain the viral RNase H exo activity. Several residues conserved among the ribosome inhibitors, E. coli RNase H, and the integrase proteins are seen to occupy a prominent cleft in the tertiary structure of the ribosome inhibitor ricin, suggesting roles in binding or catalysis. It is likely that these homologous sequences represent modern derivatives of an ancient protein-folding unit capable of nucleic acid binding and modification which has been incorporated into a variety of enzyme functions.

Amino Acid Sequence↗
PubMed1997

Defining functional regions of the IS903 transposase.

The insertion sequence IS903 encodes a 307 amino acid residue protein, transposase, that is essential for transposition. It is a multi-functional DNA-binding protein that specifically recognizes the 18 bp inverted repeats at the ends of the element and also recognizes DNa non-specifically when it captures a target site. In addition, transposase performs catalytic functions when it mediates the cleavage and religation steps of transposition. We have carried out deletion and mutational analyses to define functional domains of the transposase protein. The deletion studies delineate a 99 residue region of the protein (residues 31 to 129) that specifies binding to the inverted repeat. A slightly larger maltose-binding protein-transposase fusion that includes residues 22 to 139 (Tnp 22-139) binds as efficiently and with the same specificity as the full-length transposase protein. Tnp 22-139 also induces a DNA bend similar to that of the wild-type protein, and so we conclude that all binding and bending specificity is contained within the N-terminal domain of the protein. Unlike full-length transposase, Tnp 22-139 forms additional higher-order complexes in band-shift gels suggesting that the deletion has exposed a surface(s) capable of participating in protein-protein interactions. Six highly conserved residues in the C-terminal portion of the protein were mutated to alanine. Each mutant protein was binding-proficient but defective in transposition. The phenotype of these substitutions, and their alignment with residues shown to abolish catalysis of other transposases and integrases, suggest that these are residues responsible for catalytic steps in transposition of IS903; we believe three of these residues comprise the DDE motif, conserved in transposases and integrases. Our data are consistent with IS903 transposase being composed of two domains: an N-terminal domain primarily involved in DNA binding and a C-terminal domain that is involved in catalysis.

Amino Acid Sequence↗
PubMed1997

The site-specific integration system of the temperate Streptococcus thermophilus bacteriophage phiSfi21.

The temperate bacteriophage phiSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site-specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene. A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site. A deletion within the int gene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant.

Bacteriophages↗
PubMed1997

Integration of visna virus DNA occurs and may be necessary for productive infection.

Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host-virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host-U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells.

Animals↗
PubMed1998

Coamplification and dispersion of adjacent human endogenous retroviral HERV-H and HERV-E elements; presence of spliced hybrid transcripts in normal leukocytes.

In an RT-PCR study of HERV-H spliced subgenomic transcripts, we found transcripts with HERV-H leader and protease-encoding sequences spliced to HERV-E integrase-encoding sequences in lymphocytes from healthy blood donors. In other cell types, including two T-cell leukemia cell lines, these transcripts were absent. The PCR fragments of the hybrid transcripts contained two open reading frames (ORFs). One was a hybrid HERV-H protease/HERV-E integrase ORF and the other was the HERV-E envelope surface glycoprotein ORF. Alternative splice products were also identified. The genomic DNA origin of the hybrid transcripts was shown to be a HERV-H element with a large 3'-end deletion, adjacent to a HERV-E element lacking the 5'-LTR. This hybrid structure was shown to be amplified and dispersed to six different human chromosomes. Thus, a relatively large part of full-length HERV-E elements (15-20%) is potentially under the transcriptional control of HERV-H LTRs. The HERV-H/HERV-E junction was present in multiple copies also in the chimpanzee and gorilla, but not in the orangutan or old world monkeys.

Alternative Splicing↗
PubMed1993

Integrons: novel DNA elements which capture genes by site-specific recombination.

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5' to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3' end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons. Members of the most studied class of integrons, which include the sulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5'-conserved segment. However, the length of the 3'-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3'-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.

Base Sequence↗
PubMed2001

Reverse transcription of retroviruses and LTR retrotransposons.

Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.

Amino Acid Sequence↗