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PubMed1987

Site-specific recombination intermediates trapped with suicide substrates.

A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.

Bacteriophage lambda↗
PubMed1990

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.

Alpharetrovirus↗
PubMed1995

Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration.

The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.

Amino Acid Sequence↗
PubMed1987

Hypothesis: somatic hypermutation by gene conversion via the error prone DNA----RNA----DNA information loop.

A mechanism to explain somatic hypermutation in immunoglobulin variable region genes is proposed employing polynucleotide information transfer through the error prone DNA----RNA----DNA loop. During transcription of the rearranged V-region, the primary transcript undergoes either inappropriate termination, cleavage, or reverse transcriptase priming allowing a V-region specific reverse-transcriptase-integrase complex to synthesize a DNA copy of the rearranged V-region and integrate it, by homologous recombination, back into the normal chromosomal site. Some consequences and predictions of the hypothesis are discussed.

Animals↗
PubMed1992

The lymphoproliferative disease virus of turkeys represents a distinct class of avian type-C retrovirus.

The lymphoproliferative disease virus of turkeys (LPDV) is the etiological agent of a rapidly developing lymphoproliferative process in turkeys. To better understand the genetic relationships of LPDV to other retroviruses we determined the nucleotide sequence of its pol gene. Comparative computer analyses of the deduced amino acid sequences of the reverse transcriptase and integrase domains within pol established that LPDV represents a distinct class of avian retroviruses that is most closely related to the avian leukemia-sarcoma viruses.

Amino Acid Sequence↗
PubMed1993

Retroviral DNA integration: lessons for transposon shuffling.

Phylogenetic comparisons of retroviral IN (integrase) protein sequences have revealed homologies that extend to the retrotransposon and bacterial transposase families and have provided evidence for at least two functional domains. The N-terminal region is characterized by a Zn-finger-like array which is conserved in retrotransposons. The central region is defined by a D,D(35)E amino acid constellation which is conserved through the retrotransposons and several bacterial IS element transposases. Mutagenesis studies and biochemical analysis of the isolated central D,D(35)E domain support our original suggestion that this region contains the catalytic center of these proteins which must all share a similar enzymatic mechanism. These, and other recent findings suggest unexpected relationships between diverse pathways of nucleic acid metabolism in eukaryotes and prokaryotes.

DNA Nucleotidyltransferases↗
PubMed1993

Genetic organization and sequence of the region encoding integrative functions from Lactobacillus gasseri temperate bacteriophage phi adh.

A 2.0-kb fragment from the Lactobacillus gasseri temperate bacteriophage phi adh contained the essential genetic determinants for site-specific integration. The nucleotide sequence of this fragment was determined. An open reading frame (intG), which adjoined the phage attachment site (attP), encoded a deduced protein related to the integrase family. The organization of this region was comparable to other phage site-specific recombination systems.

Amino Acid Sequence↗
PubMed1994

Tas, a retrotransposon from the parasitic nematode Ascaris lumbricoides.

The cloned retrotransposon Tas OE3 from the genome of the parasitic nematode Ascaris lumbricoides was completely sequenced. The element is flanked by long terminal repeats (LTR) and contains three distinct regions encoding putative proteins typical for retroid elements. The first region, ORF1, encodes a putative Gag protein including a 'Leu zipper', a nucleic acid binding motif, as well as an aspartic protease domain. The second region contains an incomplete ORF (ORF2) with sequence similarities to known retroviral reverse transcriptases (RT), ribonucleases H and integrases. A third ORF, which is located adjacent to the 3' LTR, might encode an env-like protein. Based on amino-acid sequence analysis of the RT domain, Tas falls into a new subgroup of LTR-containing retrotransposons.

Amino Acid Sequence↗
PubMed2005

Target flexibility in molecular recognition.

Induced-fit effects are well known in the binding of small molecules to proteins and other macromolecular targets. Among other targets, protein kinases are particularly flexible proteins, so that such effects should be considered in attempts at structure-based inhibitor design for kinase targets. This paper outlines some recent progress in methods for including target flexibility in computational studies of molecular recognition. A focus is the "relaxed complex method," in which ligands are docked to an ensemble of conformations of the target, and the best complexes are re-scored to provide predictions of optimal binding geometries. Early applications of this method have suggested a new approach to the development of inhibitors of HIV-1 Integrase.

Computer Simulation↗
PubMed2006

Ring substituent effects on biological activity of vinyl sulfones as inhibitors of HIV-1.

In a previous study, we prepared a small library of chicoric acid analogs that possessed both potent anti-integrase and antiviral activity. It was also shown that active compounds fell into one of two groups: those that inhibited an early stage in viral replication and those that inhibited at a later stage. In this study, a series of vinyl geminal disulfone-containing compounds possessing a range of ring substituents has been synthesized to probe the impact of structure on inhibitory mechanisms. Four active compounds were identified using HIV drug susceptibility assays. Three of the inhibitors possessing either no substituents or electron-withdrawing substituents on the aromatic rings led to high levels of cytotoxicity and antiviral activity. Intrigued by the potential implications of electronic effects on activity, we probed whether the active compounds could be nonspecifically reacting via 1,4-addition. To investigate this hypothesis, the compounds were incubated with glutathione and upon LC/MS analysis, molecular ion peaks corresponding to both mono and double addition adducts were identified. Second, we synthesized analogs lacking the ability to participate in 1,4-addition and tested them for antiviral activity and cytotoxicity, and found the compounds inactive for both activities. Taken together, the studies reported herein suggest that compounds lacking electron-donating substituents on the aromatic ring are promiscuous acceptors of biological nucleophiles, whereas compounds possessing electron-donating substituents seem to resist addition or at least be more selective and significantly less toxic.

Anti-HIV Agents↗
PubMed2004

Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds.

Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2'-deoxycytidylyl-(3'>5')-2'-deoxyuridine (dCpdU) and 2'-deoxyadenylyl-(3'-->5')-2'-deoxycytidine (dApdC)] and isomeric dinucleotides [e.g., 2'-deoxycytidylyl-(3'-->5')-1'-deoxy-2'-isouridine (dCpisodU) and 1'-deoxy-2'-isoadenylyl-(3'-->5')-2'-deoxycytidine (isodApdC)], toward 5'- and 3'-exonucleases, phosphodiesterase I (PDE I) and phosphodiesterase II (PDE II) were experimentally determined and remarkable differences emerged. The study is of importance in the discovery of nuclease-stable inhibitors of HIV integrase, but may also have ramifications in the area of anti-sense oligonucleotides of therapeutic interest.

Dinucleoside Phosphates↗
PubMed2006

Conditional and inducible gene recombineering in the mouse inner ear.

Genetically engineered mice have greatly improved our understanding of gene functions and disease mechanisms. Nevertheless, the traditional knock-out approach has limitations in the overall viability of mutants. The application of the Cre/loxP system in the inner ear can help bypass this difficulty by generation of conditional gene recombineering. However, to do so requires an expression system that allows ear-specific temporally inducible, gene abrogation of one or more of the increasingly available floxed genes. To date, three approaches have been successfully used to create murine inner ear-specific Cre lines: conventional transgenesis, BAC transgenesis, and gene knock-in. Unfortunately, timing of conditional Cre activity does not extend beyond the regulatory range of the gene controlling Cre expression. Rectification of this problem requires the generation of tamoxifen or tetracycline inducible systems in the inner ear. Examination of integrase expression at different loci will facilitate studies on the expression of exogenous transgenes. These genetic applications for the mouse genome will dramatically advance in vivo gene function studies.

Animals↗
PubMed2004

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 in E. coli K12.

The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype IncJ element, R391, revealed it to be a mosaic structure, integrated into the prfC gene in E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5' end of prfC at 99.31 min on the E. coli chromosome, and confirmed this for R391. Despite disrupting prfC, the IncJ's encode novel promoter and 5' sequences, restoring function of the disrupted prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their attL and attR sites, with subsequent recombination between the attP sites on the circular forms and the attB sites in the host genomes. The attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.

Amino Acid Motifs↗
PubMed2005

Tn7 distribution in Helicobacter pylori: a selective paradox.

The presence of class 1, 2 and 3 integrons in Helicobacter pylori, which in other species are usually related to antimicrobial resistance, were looked for in 40 epidemiologically-unrelated isolates. The presence of the class 2 integrase, which led to the discovery of Tn7, was found in 15 of 35 different clones identified by lspA-glmM polymorphism. MIC values for trimethoprim and streptomycin were not affected by the presence or absence of dfrA1 and aadA1 genes. The polyclonal spread of Tn7 showed the ability of H. pylori to harbour antimicrobial resistance mechanisms, usually located on plasmids, and also suggested that other important mechanisms of resistance may also be acquired, which would result in failure of chemotherapy.

Anti-Bacterial Agents↗
PubMed2006

DNA topology and geometry in Flp and Cre recombination.

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.

Base Sequence↗
PubMed2006

Structural and genomic properties of the hyperthermophilic archaeal virus ATV with an extracellular stage of the reproductive cycle.

A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon-shaped tail-less particles, after which they develop long tails at each pointed end, at temperatures close to that of the natural habitat, 85 degrees C. The extracellularly developed tails constitute tubes, which terminate in an anchor-like structure that is not observed in the tail-less particles. A thin filament is located within the tube, which exhibits a periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable elements as well as genes, which previously have been associated only with archaeal self-transmissable plasmids. In total, it encodes 72 predicted proteins, including 11 structural proteins with molecular masses in the range of 12 to 90 kDa. Several of the larger proteins are rich in coiled coil and/or low complexity sequence domains, which are unusual for archaea. One protein, in particular P800, resembles an intermediate filament protein in its structural properties. It is modified in the two-tailed, but not in the tail-less, virion particles and it may contribute to viral tail development. Exceptionally for a crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and this process can be interrupted by different stress factors.

Acidianus↗
PubMed2005

Pharmacokinetics and metabolism of 1,5-dicaffeoylquinic acid in rats following a single intravenous administration.

1,5-Dicaffeoylquinic acid (1,5-DCQA) is a potentially important HIV-1 integrase inhibitor widely distributed in many plants. To characterize the pharmacokinetic and metabolic properties of 1,5-DCQA in rats following single intravenous administration (160 mg/kg), the plasma concentrations of 1,5-DCQA were measured by high-performance liquid chromatography (HPLC) and the metabolites formed in urine were identified by liquid chromatography-mass spectrometry (LC-MS) in parallel to diode-array detection (DAD). The results showed that the concentrations of 1,5-DCQA in plasma declined rapidly in a biphasic manner with a mean terminal half-life (t(1/2)) of 1.40 h. The mean clearance (CL) and the apparent volume of distribution (Vd(B)) of 1,5-DCQA were 0.44l/h/kg and 0.89l/kg, respectively. A total of 15 metabolites in rat urine were identified, including four isomeric O-mono-methylated (M1-M4), six isomeric O-di-methylated (M5-M10), one isomeric O-mono-methyl-glucuronidated (M11) and four isomeric O-di-methyl-glucuronidated (M12-M15) metabolites. The O-methylation positions of three important metabolites (M1, M2 and M5) were determined (3''-, 3'-, and 3',3''-) by comparing with synthesized standards. These results suggested that the disappearance of 1,5-DCQA from plasma was rapid, and that its quick urinary excretion and extensive metabolism, including methylation and glucuronidation, were two factors causing its rapid elimination from the circulation.

Animals↗