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PubMed2006

V(D)J recombinase mediated inter-chromosomal HPRT alterations at cryptic recombination signal sequences in peripheral human T cells.

The V(D)J recombinase enzyme complex is responsible for the development of a diverse immune system by catalyzing intra-molecular rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes at specific recombination signal sequences (RSSs). This enzyme complex has also been implicated in mediating pathologic and non-pathologic intra- and inter-molecular genomic rearrangements at cryptic (Psi) RSSs outside the immune system loci in lymphoid cells. We describe here two V(D)J recombinase mediated genomic rearrangements resulting in alterations at the HPRT locus in human T-cells. These are inter-chromosomal insertions in which DNA fragments are inserted at breakpoints generated by V(D)J recombinase cleavage at Psi RSS sites in the HPRT locus at Xq26. In the first, a TCR signal ended segment from chromosome 14q11 is inserted at a Psi RSS in intron 1 of the HPRT locus. In the second, a DNA fragment from 9q22 is integrated between the coding ends generated by a V(D)J recombinase mediated HPRT deletion. Identification of these in vivo V(D)J mediated inter-chromosomal insertions at Psi RSSs in the HPRT gene supports the accumulating evidence that V(D)J recombinase can mediate mutagenic rearrangements in humans with potential pathologic consequences.

Base Sequence↗
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↗
PubMed1986

Partial purification and characterization of a recombinase from human cells.

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.

Adenosine Triphosphate↗
PubMed1988

Stable expression of immunoglobulin gene V(D)J recombinase activity by gene transfer into 3T3 fibroblasts.

Using retroviral recombination substrates, we investigated the developmental stage of expression of V(D)J recombinase activity and the activation of recombinase activity in nonlymphoid cells. V(D)J recombinase activity was detected only in pre-B cells, but a DNA transfer protocol successfully activated V(D)J recombination in an NIH 3T3 cell line. The V(D)J recombinase activity was transferred in a second round of transfection and was then stably expressed in fibroblasts at a level comparable to that of a recombinationally active pre-B cell. It is likely that expression of a single, lymphoid-specific gene in a fibroblast is sufficient to confer V(D)J recombinase activity on that cell.

Animals↗
PubMed1992

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.

Adult↗
PubMed1999

C-terminal interactions between the XerC and XerD site-specific recombinases.

Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed. Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold. Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.

Amino Acid Sequence↗
PubMed1998

beta Recombinase catalyzes inversion and resolution between two inversely oriented six sites on a supercoiled DNA substrate and only inversion on relaxed or linear substrates.

The beta recombinase, in the presence of a chromatin-associated protein such as Hbsu, catalyzes DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented six sites. Hbsu stabilizes the formation of the recombination complex (Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945). In this study we show that resolution by beta recombinase strictly requires supercoiled DNA, but inversion does not. On a substrate with two inversely oriented six sites, beta recombinase catalyzed both resolution and inversion if the DNA was supercoiled but only inversion if the substrate was relaxed or linear. Hbsu was critical for the formation of synaptic complexes; its concentration relative to that of the supercoiled DNA substrate determined whether resolution or inversion products were preferentially formed. The results suggest that the beta recombinase forms unproductive short-lived synaptic complexes between two juxtaposed inversely oriented six sites; the presence of 3 to 13 Hbsu dimers per supercoiled DNA molecule would stabilize a synaptic complex with a relative geometry of the six sites allowing beta recombinase preferentially to achieve resolution. Supercoiling probably helps to overcome an energetic barrier, since resolution does not occur in relaxed DNA. The presence of >30 Hbsu dimers per DNA molecule probably favors the formation of a recombination complex with a different geometry since the reaction is directed preferentially toward DNA inversion.

Catalysis↗
PubMed2005

E47 is required for V(D)J recombinase activity in common lymphoid progenitors.

Common lymphoid progenitors (CLPs) are the first bone marrow precursors in which V(D)J recombinase activity is up-regulated. Here, we show that loss of the transcription factor E47 produces a reduced CLP population that lacks V(D)J recombinase activity and D-J(H) rearrangements in vivo. Apart from a profound arrest before the pro-B cell stage, other downstream lymphoid progeny of CLPs are still intact in these mice albeit at reduced numbers. In contrast to the inhibition of recombinase activity in early B lineage precursors in E47-deficient animals, loss of either E47 or its cis-acting target Erag (enhancer of rag transcription) has little effect on recombinase activity in thymic T lineage precursors. Taken together, this work defines a role for E47 in regulating lineage progression at the CLP stage in vivo and describes the first transcription factor required for lineage-specific recombinase activity.

Animals↗
PubMed1997

Action of site-specific recombinases XerC and XerD on tethered Holliday junctions.

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.

Bacterial Proteins↗
PubMed2006

Regulation of type 1 fimbriae by unlinked FimB- and FimE-like recombinases in uropathogenic Escherichia coli strain CFT073.

Genomic DNA sequence analysis of the uropathogenic Escherichia coli strain CFT073 revealed that besides the fimB and fimE recombinase genes that control the type 1 pilus fim phase switch, there are three additional fimB- and fimE-like genes: ipuA, ipuB, and ipbA. Alignment of the predicted amino acid sequences showed that the five recombinases range in sequence similarity from 63 to 70%. An epidemiological survey indicates that ipuA and ipuB are present and linked next to the dsdCXA locus in 24 of 67 uropathogenic E. coli strains but are found in only 1 of 15 normal human fecal isolates. The ipbA sequence located next to the betABIT locus was found in 42 of 67 uropathogenic isolates and 8 of 15 of the commensal strains. We show that two of these recombinases, those encoded by ipuA and ipbA, can function at the type 1 pilus fim switch. In a CFT073 deletion mutant lacking all five recombinase genes, recombinant ipuA or ipbA provided in trans inverted the fim element from the off state to the on state. When a fim OFF CFT073 DeltafimBE mutant was used to infect the urinary tracts of mice, a switch to the fim on state was detected within 24 h in bacteria recovered from urine, the bladder, and the kidneys. A fim OFF CFT073 DeltafimBE ipuB ipbA mutant also demonstrated the ability to switch from the fim off state to the on state during mouse infection. CFT073 recombinase mutants derived from isolates in either the fim on or off state showed a reciprocal relationship for motility. Switches from a nonmotile to a motile phenotype and from a fim on to off genotype were observed in fim ON CFT073 DeltafimBE ipuAB ipbA mutants when ipuA or fimB was provided in trans. Together these results indicate that ipuA has fimB-like on-to-off and off-to-on fim switching activity and that ipbA has the ability to switch fim from the off to the on orientation.

Amino Acid Sequence↗
PubMed2005

DIRS-1 and the other tyrosine recombinase retrotransposons.

DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called "split" direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish and amphibia, while remnants of elements related to DIRS-1 occur in the human genome. The complete set of YR retrotransposons can be divided into two major groups, the DIRS elements and the Ngaro elements, the two groups forming distinct clades on phylogenetic trees based on alignments of RT/RH and recombinase sequences, and also having some structural distinctions. A third group of transposable elements, which we call Cryptons, also carry tyrosine recombinases. These elements do not encode a reverse transcriptase and so are believed to be DNA transposons not retrotransposons. They have been detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. Sequence comparisons suggest that the Crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a Crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon.

Amino Acid Sequence↗
PubMed2006

Tamoxifen-inducible NaV1.8-CreERT2 recombinase activity in nociceptive neurons of dorsal root ganglia.

To explore the function of genes expressed in adult mouse nociceptive neurons, we generated heterozygous knock-in mice expressing the tamoxifen-inducible Cre recombinase construct CreERT2 downstream of the Na(V)1.8 promoter. CreERT2 encodes a Cre recombinase (Cre) fused to a mutant estrogen ligand-binding domain (ERT2) that requires the presence of tamoxifen for activity. We have previously shown that heterozygous Na(V)1.8-Cre mice will delete loxP flanked genes specifically in nociceptive sensory neurons from embryonic day 14. We therefore used the same strategy of homologous recombination and mouse generation, substituting the Cre cassette with CreERT2. No functional Cre recombinase activity was found in CreERT2 mice crossed with reporter mice in the absence of tamoxifen. We found that, as with Na(V)1.8-Cre mice, functional Cre recombinase was present in nociceptive sensory neurons after tamoxifen induction in vivo. However, the percentage of dorsal root ganglion (DRG) neurons expressing functional Cre activity was much reduced (<10% of the number found in the Na(V)1.8-Cre mouse). We also examined Cre recombinase activity in sensory neurons in culture. After treatment with 1 muM tamoxifen for 48 h, 15% of DRG neurons showed Cre activity. Na(V)1.8-CreERT2 animals may thus be useful for single cell studies of the functional consequences of gene ablation in culture, but are unlikely to be useful for behavioral studies.

Alleles↗
PubMed2006

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase.

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.

Animals↗
PubMed2002

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting.

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.

Animals↗
PubMed1999

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.

Animals↗
PubMed1998

Similarities and differences among 105 members of the Int family of site-specific recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).

Amino Acid Sequence↗
PubMed2002

Smooth muscle expression of Cre recombinase and eGFP in transgenic mice.

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

Animals↗
PubMed2004

Transgenic mice expressing Cre-recombinase specifically in M- or S-cone photoreceptors.

PURPOSE: To establish lines of transgenic mice that express Cre-recombinase in M- or S-cone photoreceptors for generating cone photoreceptor-specific (conditional) mutants. METHODS: Five kilobases of 5' upstream sequence of the mouse red-green (M) opsin gene or 0.5 kb of the mouse blue (S) opsin gene was cloned into a Cre-expression plasmid. Transgenic mice were generated and characterized, and appropriate lines were established. The Cre-transgenic mice were crossed with ROSA26-lacZ mice (containing floxed beta-galactosidase gene) and analyzed to determine Cre-recombinase activity. RESULTS: Immunofluorescence study showed successful targeting of Cre-recombinase expression to cone photoreceptors. Double staining with anti-Cre antibody and anti-M- or anti-S-opsin antibody revealed specificity of Cre expression in M-opsin- and/or S-opsin-positive photoreceptors. Mating with ROSA26-lacZ mice demonstrated that Cre-recombinase was functionally active in M- or S-cones. CONCLUSIONS: Lines of transgenic mice that specifically express functional Cre-recombinase in M- or S-cones were established in this study. Because mutations in several widely expressed genes lead to photoreceptor degeneration, these transgenic mice should be valuable in generating conditional mutants to investigate the function of various genes specifically in cone photoreceptors.

Animals↗