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Research in Microbiology 2002Integrases encoded in archaeal genomes can be classified into seven families on the basis of their sequences. They constitute a super-family of tyrosine DNA recombinases... (Review)
Review
Integrases encoded in archaeal genomes can be classified into seven families on the basis of their sequences. They constitute a super-family of tyrosine DNA recombinases together with a number of bacterial integrases and they are likely to be responsible for the formation of integrated elements in archaeal chromosomes. An integrated element is defined as possessing an integrase, a block of foreign genes, and a direct repeat at the two ends with one repeat unit overlapping a tRNA gene. There are two types of archaeal integrated elements, the SSV viral type, including those that carry the partitioned integrase gene, intN and intC, and the pNOB8 type, including those with a tRNA gene overlapping the attL site 5' prior to an integrase gene. Both known and unknown genes are present in these integrated elements and their encoded proteins may have facilitated the adaptation of archaea during evolution.
Topics: Chromosomes, Archaeal; Gene Transfer, Horizontal; Genes, Archaeal; Genome, Archaeal; Integrases; Phylogeny; RNA, Transfer
PubMed: 12234006
DOI: 10.1016/s0923-2508(02)01331-1 -
Viruses Aug 2019Foamy viruses (FV) are retroviruses belonging to the subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines,... (Review)
Review
Foamy viruses (FV) are retroviruses belonging to the subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.
Topics: Animals; Anti-Retroviral Agents; Catalytic Domain; DNA, Viral; HIV Infections; HIV-1; Humans; Integrase Inhibitors; Integrases; Models, Molecular; Nucleoproteins; Retroviridae; Spumavirus; Terminal Repeat Sequences; Virus Integration
PubMed: 31443391
DOI: 10.3390/v11090770 -
Nucleic Acids Research Sep 2016Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being...
Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the 'reverse' reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase-DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.
Topics: Attachment Sites, Microbiological; Biological Assay; Biological Factors; Computer Simulation; DNA; Enzyme Stability; Integrases; Kinetics; Models, Biological; Plasmids; Recombination, Genetic; Thermodynamics; Viral Proteins
PubMed: 27387286
DOI: 10.1093/nar/gkw616 -
Human Gene Therapy Oct 2010The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination...
The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ΦC31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response. Western blot and immunofluorescence analyses were performed to investigate the duration of ΦC31 integrase expression in mouse liver. Integrase was expressed within 2 to 3 hr after hydrodynamic injection of a plasmid expressing ΦC31 integrase. Expression peaked between 8 and 16 hr and fell to background levels by 24-48 hr postinjection. Analysis of the amount of integrase plasmid DNA present in the liver over time suggested that the brief period of integrase expression could largely be accounted for by rapid loss of the bulk of the plasmid DNA, as well as by silencing of plasmid expression. PCR analysis of integration indicated that ΦC31 integrase carried out genomic integration of a codelivered attB-containing plasmid by 3 hr after plasmid injection. Integrase was expressed for longer times and at higher levels in transfected cultured cells compared with liver. Inhibitor studies suggested that the enzyme had a short half-life and was degraded by the 26S proteasome. The short duration of integrase expression in liver and rapid integration reaction appear to be features favorable for use in gene therapy.
Topics: Animals; Attachment Sites, Microbiological; Blotting, Southern; Blotting, Western; Cell Line; Fluorescent Antibody Technique; Gene Expression; Gene Silencing; Genetic Therapy; Genetic Vectors; HeLa Cells; Humans; Integrases; Kinetics; Liver; Mice; Mice, Inbred C57BL; Plasmids; Polymerase Chain Reaction; Recombination, Genetic; Time Factors; Transfection
PubMed: 20497035
DOI: 10.1089/hum.2010.049 -
Genes Dec 2021Diverse phylogenetic methods require a substitution model of evolution that should mimic, as accurately as possible, the real substitution process. At the protein level,...
Diverse phylogenetic methods require a substitution model of evolution that should mimic, as accurately as possible, the real substitution process. At the protein level, empirical substitution models have traditionally been based on a large number of different proteins from particular taxonomic levels. However, these models assume that all of the proteins of a taxonomic level evolve under the same substitution patterns. We believe that this assumption is highly unrealistic and should be relaxed by considering protein-specific substitution models that account for protein-specific selection processes. In order to test this hypothesis, we inferred and evaluated four new empirical substitution models for the protease and integrase of HIV and other viruses. We found that these models more accurately fit, compared with any of the currently available empirical substitution models, the evolutionary process of these proteins. We conclude that evolutionary inferences from protein sequences are more accurate if they are based on protein-specific substitution models rather than taxonomic-specific (generalist) substitution models. We also present four new empirical substitution models of protein evolution that could be useful for phylogenetic inferences of viral protease and integrase.
Topics: Algorithms; Amino Acid Substitution; Computer Simulation; Evolution, Molecular; HIV Infections; HIV Protease; HIV-1; Humans; Integrases; Models, Genetic; Models, Statistical; Phylogeny
PubMed: 35052404
DOI: 10.3390/genes13010061 -
Current Opinion in Structural Biology Feb 1999Three-dimensional structural information on the integrase family of site-specific recombinases has only recently become available, with the crystal structures of... (Review)
Review
Three-dimensional structural information on the integrase family of site-specific recombinases has only recently become available, with the crystal structures of catalytic domains, full-length proteins and protein-DNA complexes of this family reported over the past two years. These results have led to a model for the overall architecture and active site stereochemistry of the recombination pathway that addresses a number of interesting mechanistic issues.
Topics: Binding Sites; DNA; DNA Nucleotidyltransferases; Integrases; Models, Genetic; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; Recombinases; Recombination, Genetic; Stereoisomerism; Tyrosine; Viral Proteins
PubMed: 10047575
DOI: 10.1016/s0959-440x(99)80003-7 -
Journal of Virology Oct 1999Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as...
Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.
Topics: Escherichia coli; Humans; Integrases; Recombinant Proteins; Simian Immunodeficiency Virus; Substrate Specificity; Virus Integration
PubMed: 10482559
DOI: 10.1128/JVI.73.10.8104-8111.1999 -
PeerJ 2022HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material...
HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector's replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
Topics: Integrases; Genetic Vectors; Transgenes; HIV-1; Genome
PubMed: 35979475
DOI: 10.7717/peerj.13704 -
Nature Communications Nov 2022Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However,...
Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. Here, we show that a strategy of terminal differentiation improves the evolutionary stability of burdensome functions in a general manner by realizing a reproductive and metabolic division of labor. To implement this strategy, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination. With terminal differentiation, differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. We demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression and that its evolutionary stability can be improved with strategic redundancy. Further, we show this strategy can even be applied to toxic functions. Overall, this study provides an effective, generalizable approach for protecting burdensome engineered functions from evolutionary degradation.
Topics: Escherichia coli; Integrases; Promoter Regions, Genetic; Synthetic Biology; Gene Regulatory Networks
PubMed: 36357387
DOI: 10.1038/s41467-022-34361-y -
Nucleic Acids Research Dec 2019Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine...
Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.
Topics: Amino Acid Sequence; Cloning, Molecular; Escherichia coli; Exteins; Integrases; Inteins; Organisms, Genetically Modified; Protein Engineering; Protein Splicing; Recombination, Genetic; Serine; Substrate Specificity; Trans-Splicing
PubMed: 31667500
DOI: 10.1093/nar/gkz936