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PubMed1995

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.

Base Sequence↗

PubMed2006

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays.

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.

Adult↗

PubMed1991

Genetic studies of the ends of a locked-in kappa prophage in Serratia marcescens by transductional and vegetative crosses.

In temperate phage kappa of Serratia marcescens several special features of different phages are combined. The unessential genes lI, iny, cII and, at least to some extent, even the integrase gene int are not subject to negative control by the repressor, the product of gene cIII. A genetic map of the prophage was established using defective, heat-induced lysates of int- lysogens both in vegetative crosses with sus mutants of essential genes and in transduction of the four unessential genes to lysogenic recipients. Results from reciprocal four factor-crosses concerning the order of the four genes had to be included. The four genes are located near the right end of the prophage, whereas cIII lies near its left end. In vegetative phage all five genes lie in an interval between the essential genes T and U, comprising 10% of kappa's genetic map. The right prophage end appears to face at least two trp cistrons, among them the gene encoding anthranilate synthetase. lI encodes a product that masks the phage receptors in the cell wall. The gene product of iny interferes with the growth of infecting phage y. The natural function of cII is still unknown, but some of its mutants display a cold-sensitive phenotype, their plaques being clear at 30 degrees C and turbid at 37 degrees C. Bacteria with such prophages stop producing viable progeny when the cultures are shifted from 37 degrees C to 30 degrees C. These cold-sensitive mutants are partly dominant and partly recessive. Analysing a virulent mutant, a gene ant encoding an antirepressor was discovered, but so far there is no evidence that it is regulated by an extra repressor. The gene is located relatively near the left prophage end. Evidence is presented that the exogenotes in transduction with the defective lysates continue to exist for some time after a first recombinational event.

Anthranilate Synthase↗

PubMed2002

New anti-HIV agents and targets.

Virtually all the compounds that are currently used or are subject of advanced clinical trials for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside reverse transcriptase inhibitors (NRTIs): i.e., zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine and nucleotide reverse transcriptase inhibitors (NtRTIs) (i.e., tenofovir disoproxil fumarate); (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e., nevirapine, delavirdine, efavirenz, emivirine; and (iii) protease inhibitors (PIs): i.e., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir. In addition to the reverse transcriptase and protease reaction, various other events in the HIV replicative cycle can be considered as potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polycarboxylates, polyoxometalates, polynucleotides, and negatively charged albumins); (ii) viral entry, through blockade of the viral coreceptors CXCR4 (i.e., bicyclam (AMD3100) derivatives) and CCR5 (i.e., TAK-779 derivatives); (iii) virus-cell fusion, through binding to the viral envelope glycoprotein gp41 (T-20, T-1249); (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 4-aryl-2,4-dioxobutanoic acid derivatives; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (flavopiridol, fluoroquinolones). Also, various new NRTIs, NNRTIs, and PIs have been developed that possess, respectively: (i) improved metabolic characteristics (i.e., phosphoramidate and cyclosaligenyl pronucleotides by-passing the first phosphorylation step of the NRTIs), (ii) increased activity ["second" or "third" generation NNRTIs ( i.e., TMC-125, DPC-083)] against those HIV strains that are resistant to the "first" generation NNRTIs, or (iii), as in the case of PIs, a different, modified peptidic (i.e., azapeptidic (atazanavir)) or non-peptidic scaffold (i.e., cyclic urea (mozenavir), 4-hydroxy-2-pyrone (tipranavir)). Non-peptidic PIs may be expected to inhibit HIV mutant strains that have become resistant to peptidomimetic PIs.

Amino Acid Sequence↗

PubMed2006

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data.

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.

Amino Acid Sequence↗

PubMed1993

Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration.

An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.

Base Sequence↗

PubMed1993

Inhibitory effect of the polyanionic drug suramin on the in vitro HIV DNA integration reaction.

An obligatory step in retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-1 U5 LTR as substrate and supercoiled pSP65 DNA as target, we have investigated the effect of the polyanionic drug suramin on the catalytic activity of the IN protein. It was found that at stoichiometric suramin to protein ratios, suramin displays a strong inhibitory effect on both the processing and strand transfer reactions. This inhibitory effect is related to the decrease of IN protein binding efficiency to the LTR end DNA fragment.

Base Sequence↗

PubMed1993

Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction.

Retroviral growth requires as an obligatory step the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-I U5 LTR as substrate and supercoiled pSP65 DNA as target, we have measured the effect of various topoisomerase inhibitors on the functional activity of the IN protein. Among the various drugs tested, the antitumor drug 2N-Methyl, 9-hydroxyellipticinium (NMHE) displays a marked inhibitory effect on the IN-catalyzed U5 insertion. This effect is related to the DNA binding properties of the drug rather than to a selective effect on the IN protein or the DNA-IN protein complex.

Amsacrine↗

PubMed1999

Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active.

We analyzed the structural and functional organization of anti-HIV and anti-tumor proteins MAP30 and GAP31 by limited proteolysis with endopeptidases Lys-C and Glu-C (V8). MAP30 and GAP31 are resistant to proteolytic digestion under conditions of as much as 5% (w/w) proteases. In the presence of 10% (w/w) protease, the central regions of the proteins are still resistant to proteolysis, whereas the N- and C-termini are accessible. Peptide fragments were purified by FPLC on Superdex 75 columns, characterized by gel electrophoresis, identified by amino acid sequencing, and analyzed for anti-HIV, anti-tumor, and other biochemical activities. We report here that limited proteolysis yields biologically active fragments of both MAP30 and GAP31. These fragments are active against HIV-1 and tumor cells with EC(50)s in the sub-nanomolar ranges, 0.2-0.4 nM. At the dose levels used in the assays, little cytotoxicity to normal cells was observed. In addition, these fragments remain fully active in HIV-integrase inhibition and HIV-LTR topological inactivation, but not ribosome inactivation. These results demonstrate that the antiviral and anti-tumor activities of MAP30 and GAP31 are independent of ribosome inactivation activity. In addition, we demonstrate that portions of the N- and C-termini are not essential for antiviral and anti-tumor activities, but do appear to be required for ribosome inactivation. These results may provide novel strategies for rational design and targeted development of mimetic antiviral and anti-tumor therapeutics.

Amino Acid Sequence↗

PubMed2002

Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.

Amino Acid Substitution↗

PubMed1996

IS911-mediated transpositional recombination in vitro.

A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.

Bacterial Proteins↗

PubMed1997

Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD.

In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli. We have used nicked suicide substrates to trap reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif. Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inefficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis. The presence of a nick in the bottom strand of dif allows cooperative interactions between two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD. Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA. Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate.

Alkylation↗

PubMed1998

A K cation-induced conformational switch within a loop spanning segment of a DNA quadruplex containing G-G-G-C repeats.

We have identified a unique structural transition (in slow exchange on the NMR time scale) in the tertiary fold of the d(G-G-G-C-T4-G-G-G-C) quadruplex on proceeding from Na+ to K+ as counterion in aqueous solution. Both monovalent cation-dependent conformations exhibit certain common structural features, which include head-to-tail dimerization of two symmetry-related stem-hairpin loops, adjacent strands which are antiparallel to each other and adjacent stacked G(syn).G(anti). G(syn).G(anti) tetrads in the central core of the quadruplexes. The Na and K cation stabilized structures of the d(G-G-G-C-T4-G-G-G-C) quadruplexes differ in the conformations of the T-T-T-T loops, the relative alignment of G.C base-pairs positioned opposite each other through their major groove edges and potentially in the number of monovalent cation binding sites. We have identified potential K cation binding cavities within the symmetry-related T-T-T-G segments, suggesting the potential for two additional monovalent cation binding sites in the K cation-stabilized quadruplex relative to its Na cation-stabilized counterpart. Modeling studies suggest that the major groove edges of guanine residues in Watson-Crick G.C base-pairs could potentially be bridged by coordinated K cations in the d(G-G-G-C-T4-G-G-G-C) quadruplex in KCl solution in contrast to formation of G.C.G.C tetrads for the corresponding quadruplex in NaCl solution. Our results defining the molecular basis of a Na to K cation-dependent conformational switch in the loop spanning segment of the d(G-G-G-C-T4-G-G-G-C) quadruplex may have relevance to recent observations that specific K cation coordinated loop conformations within quadruplexes exhibit inhibitory activity against HIV integrase.

Binding Sites↗

PubMed1998

Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination.

The integrase class site specific recombinases FLP from Saccharomyces cerevisiae, and Cre from bacteriophage P1, have been extensively used to direct DNA rearrangements in heterologous organisms. Although their reaction mechanisms have been relatively well characterised, little comparative analysis of the two enzymes has been published. We present a comparative kinetic analysis of FLP and Cre, which identifies important differences. Gel mobility shift assays show that Cre has a higher affinity for its target, loxP (7. 4x10(10) M-1), than FLP for its target, FRT (8.92x10(8) M-1). We show that both recombinases bind the two halves of their target sites cooperatively, and that Cre shows approximately threefold higher cooperativity than FLP. Using a mathematical model describing the sequential binding of recombinase monomers to DNA, we have determined values for the association and dissociation rate constants for FLP and Cre.FLP and Cre also showed different characteristics in in vitro recombination assays. In particular, approximately tenfold more active FLP was required than Cre to optimally recombine a given quantity of excision substrate. FLP was able to reach maximum excision levels approaching 100%, whilst Cre-mediated excision did not exceed 75%. To investigate possible reasons for these differences a mathematical model describing the excision recombination reaction was established. Using measured DNA binding parameters for FLP and Cre in the model, and comparing simulated and experimental recombination data, the values of the remaining unknown parameters were determined. This analysis indicates that the synaptic complex is more stable for Cre than for FLP.

Allosteric Regulation↗

PubMed2000

Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1.

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.

Amino Acid Sequence↗

PubMed2000

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

Amino Acid Sequence↗

PubMed2000

Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre.

The Flp and Cre recombinases are members of the integrase family of tyrosine recombinases. Each protein consists of a 13 kDa NH(2)-terminal domain and a larger COOH-terminal domain that contains the active site of the enzyme. The COOH-terminal domain also contains the major determinants for the binding specificity of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attachment of a conserved nucleophilic tyrosine residue to the 3'-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding specificity and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH(2)-terminal domain of Flp to the COOH-terminal domain of Cre ("Fre") and the NH(2)-terminal domain of Cre to the COOH-terminal domain of Flp ("Clp"). These chimeras have novel binding specificities in that they bind strongly to hybrid sites containing elements from both the Flp and Cre DNA targets but poorly to the native target sites. In this study we have taken advantage of the unique binding specificities of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We find that the COOH-terminal domain of the recombinases determines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous findings that Cre could carry out trans cleavage. We discuss the variations in the experimental approaches in order to reconcile the different results.

Base Sequence↗

PubMed2001

Genetic organization of the she pathogenicity island in Shigella flexneri 2a.

In this study we report the complete nucleotide sequence and genetic organization of the she pathogenicity island (PAI) of Shigella flexneri 2a strain YSH6000T. The 46 603 bp she PAI is situated adjacent to the 3' terminus of the pheV tRNA gene and includes an imperfect direct repeat of the 3'-terminal 22 bp of the pheV gene at the right boundary of the PAI. The she PAI carries a bacteriophage P4-like integrase gene within the pheV -proximal boundary of the PAI, intact and truncated mobile genetic elements, plasmid-related sequences, open reading frames exhibiting high sequence similarity to those found on the locus of enterocyte effacement (LEE) PAI of enterohemorrhagic Escherichia coli (EHEC), and the SHI-2 PAI of S. flexneri and several other open reading frames of unknown function. The she PAI also encodes two autotransporter proteins, including SigA, a cytopathic protease that contributes to intestinal fluid accumulation and Pic, a protease with mucinase, and hemagglutinin activities. In addition, an open reading frame (orf) termed sap, has high sequence similarity to the gene encoding Antigen 43, a surface-located autotransporter protein of E. coli. The ShET1 enterotoxin genes, associated predominantly with S. flexneri 2a strains, are also located on the she PAI.

Bacterial Proteins↗