Zeitschriftenartikel: „Recombinase protein“

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Price, Candice, und Isabel Darcy. „Application of a skein relation to difference topology experiments“. Journal of Knot Theory and Its Ramifications 28, Nr. 13 (November 2019): 1940016. http://dx.doi.org/10.1142/s0218216519400169.

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Difference topology is a technique used to study any protein that can stably bind to DNA. This technique is used to determine the conformation of DNA bound by protein. Motivated by difference topology experiments, we use the skein relation tangle model as a novel technique to study experiments using topoisomerase to study SMC proteins, a family of proteins that stably bind to DNA. The oriented skein relation involves an oriented knot, [Formula: see text], with a distinguished positive crossing; a knot [Formula: see text], obtained by changing the distinguished positive crossing of [Formula: see text] to a negative crossing; a knot, [Formula: see text], resulting from the non-orientation persevering resolution of the distinguished crossing; and a link [Formula: see text], the orientation preserving resolution of the distinguished crossing. We refer to [Formula: see text] as the skein quadruple. Topoisomerases are proteins that break one segment of DNA allowing a DNA segment to pass through before resealing the break. Recombinases are proteins that cut two segments of DNA and recombine them in some manner. They can act on direct repeat or inverted repeat sites, resulting in a link or knot, respectively. Thus, the skein quadruple is now viewed as [Formula: see text] circular DNA substrate, [Formula: see text] product of topoisomerase action, [Formula: see text] product of recombinase action on directed repeat sites, and [Formula: see text] product of recombinase action of inverted repeat sites.

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Briones, Gabriel, Dirk Hofreuter und Jorge E. Galán. „Cre Reporter System To Monitor the Translocation of Type III Secreted Proteins into Host Cells“. Infection and Immunity 74, Nr. 2 (Februar 2006): 1084–90. http://dx.doi.org/10.1128/iai.74.2.1084-1090.2006.

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ABSTRACT Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

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Letunic, Ivica, Supriya Khedkar und Peer Bork. „SMART: recent updates, new developments and status in 2020“. Nucleic Acids Research 49, Nr. D1 (26.10.2020): D458—D460. http://dx.doi.org/10.1093/nar/gkaa937.

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Abstract SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curated models for more than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART’s vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available.

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Marshall Stark, W., Martin R. Boocock, Femi J. Olorunniji und Sally-J. Rowland. „Intermediates in serine recombinase-mediated site-specific recombination“. Biochemical Society Transactions 39, Nr. 2 (22.03.2011): 617–22. http://dx.doi.org/10.1042/bst0390617.

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Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer–dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.

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Orth, Peter, Petra Jekow, Juan C. Alonso und Winfried Hinrichs. „Proteolytic cleavage of Gram-positive β recombinase is required for crystallization“. Protein Engineering, Design and Selection 12, Nr. 5 (Mai 1999): 371–73. http://dx.doi.org/10.1093/protein/12.5.371.

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6

Chen, J. W., B. R. Evans, S. H. Yang, H. Araki, Y. Oshima und M. Jayaram. „Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine“. Molecular and Cellular Biology 12, Nr. 9 (September 1992): 3757–65. http://dx.doi.org/10.1128/mcb.12.9.3757-3765.1992.

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The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

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Chen, J. W., B. R. Evans, S. H. Yang, H. Araki, Y. Oshima und M. Jayaram. „Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.“ Molecular and Cellular Biology 12, Nr. 9 (September 1992): 3757–65. http://dx.doi.org/10.1128/mcb.12.9.3757.

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The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

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Koornneef, Lieke, Johan A. Slotman, Esther Sleddens-Linkels, Wiggert A. van Cappellen, Marco Barchi, Attila Tóth, Joost Gribnau, Adriaan B. Houtsmuller und Willy M. Baarends. „Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure“. PLOS Genetics 18, Nr. 7 (20.07.2022): e1010046. http://dx.doi.org/10.1371/journal.pgen.1010046.

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Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.

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Meador, Kyle, Christina L. Wysoczynski, Aaron J. Norris, Jason Aoto, Michael R. Bruchas und Chandra L. Tucker. „Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent“. Nucleic Acids Research 47, Nr. 17 (09.07.2019): e97-e97. http://dx.doi.org/10.1093/nar/gkz585.

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AbstractA common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

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Dreyfus, David. „RAG-1 (Recombination Activating Gene-1) protein is closely related to herpes virus recombinases: Implications for the origins of the acquired immune system. (105.20)“. Journal of Immunology 188, Nr. 1_Supplement (01.05.2012): 105.20. http://dx.doi.org/10.4049/jimmunol.188.supp.105.20.

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Abstract The acquired immune system results from recombination of germ-line DNA V(D)J signal sequences by the RAG-1 recombinase. RAG-1 is a dde magnesium binding recombinase related to both retroviral integrases and transposases. However the closest phylogenetic relative to RAG-1 appears to be a family of recombinases encoded by the herpesviridae termed the DBP (DNA binding proteins) that catalyze strand exchange and possibly other recombination events during herpes virus replication. Both the dde magnesium binding triad and the nonamer binding regions of RAG-1 are present and conserved in the DBP. These observations suggest that initially RAG-1 arose from an ancient insertion of a herpes-like infectious agent into a deuterostome genome prior to the origins of the acquired immune system. The author proposes a model in which the primordial RAG-1 recombinase was selected through its ability to promote viral immune responses through innate immune receptors in ancient deuterostomes prior to the origins of V(D)J recombination.

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Doak, Thomas G., David J. Witherspoon, Carolyn L. Jahn und Glenn Herrick. „Selection on the Genes of Euplotes crassus Tec1 and Tec2 Transposons: Evolutionary Appearance of a Programmed Frameshift in a Tec2 Gene Encoding a Tyrosine Family Site-Specific Recombinase“. Eukaryotic Cell 2, Nr. 1 (Februar 2003): 95–102. http://dx.doi.org/10.1128/ec.2.1.95-102.2003.

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ABSTRACT The Tec1 and Tec2 transposons of the ciliate Euplotes crassus carry a gene for a tyrosine-type site-specific recombinase. The expression of the Tec2 gene apparently uses a programmed +1 frameshift. To test this hypothesis, we first examined whether this gene has evolved under purifying selection in Tec1 and Tec2. Each element carries three genes, and each has evolved under purifying selection for the function of its encoded protein, as evidenced by a dearth of nonsynonymous changes. This distortion of divergence is apparent in codons both 5′ and 3′ of the frameshift site. Thus, Tec2 transposons have diverged from each other while using a programmed +1 frameshift to produce recombinase, the function of which is under purifying selection. What might this function be? Tyrosine-type site-specific recombinases are extremely rare in eukaryotes, and Tec elements are the first known eukaryotic type II transposons to encode a site-specific recombinase. Tec elements also encode a widespread transposase. The Tec recombinase might function in transposition, resolve products of transposition (bacterial replicative transposons use recombinase or resolvase to separate joined replicons), or provide a function that benefits the ciliate host. Transposons in ciliated protozoa are removed from the macronucleus, and it has been proposed that the transposons provide this “excisase” activity.

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Lebreton, B., P. V. Prasad, M. Jayaram und P. Youderian. „Mutations that improve the binding of yeast FLP recombinase to its substrate.“ Genetics 118, Nr. 3 (01.03.1988): 393–400. http://dx.doi.org/10.1093/genetics/118.3.393.

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Abstract When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

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Borghesi, Lisa, Abbe Vallejo, Lela Kardava, Qi Yang und Jennifer Aites. „Differential regulation of V(D)J recombination in multipotent progenitors in bone marrow versus thymus (111.8)“. Journal of Immunology 188, Nr. 1_Supplement (01.05.2012): 111.8. http://dx.doi.org/10.4049/jimmunol.188.supp.111.8.

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Abstract V(D)J recombinase activity is expressed in bone marrow (BM) multipotent progenitors (MPPs) that retain the potential to differentiate into B cells in BM and T cells in thymus. However, it remains unclear whether MPPs in each organ exhibit distinct transcriptional requirements for recombinase activity, or whether thymic MPPs imprint the requirements of upstream BM MPPs from which they originate. Here we use V(D)J recombination reporter mice to examine transcriptional requirements in BM versus thymic multipotent subsets. Within BM, the frequency of recombinase+ cells increases from 5% in MPPs to 15% in common lymphoid progenitors (CLPs) and 20% in pre-pro B cells. Within thymus, 10% of early thymic progenitors (ETPs) are recombinase+. While the E47 transcription factor is expressed in both BM and thymus, E47 deficiency selectively ablates V(D)J recombination in the former. BM MPPs, CLPs and pre-pro B cells from E47 knockouts lack detectable recombinase activity. In the thymus, E47 deficiency reduces ETP numbers but V(D)J recombinase activity is maintained. Loss of the related E protein HEB does not diminish recombinase activity in ETPs while functional inhibition of E47:HEB complexes leads to a 40% reduction in TCRβ rearrangements. Thus, V(D)J recombinase activity is regulated differently in MPPs in BM and thymus. E protein transcription factors are essential for recombinase activity in multipotent BM MPPs and CLPs but are dispensable in multipotent thymic ETPs.

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O'Brien, Sean P., und Matthew P. DeLisa. „Split-Cre recombinase effectively monitors protein-protein interactions in living bacteria“. Biotechnology Journal 9, Nr. 3 (29.01.2014): 355–61. http://dx.doi.org/10.1002/biot.201300462.

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Sun, Yueru, Thomas J. McCorvie, Luke A. Yates und Xiaodong Zhang. „Structural basis of homologous recombination“. Cellular and Molecular Life Sciences 77, Nr. 1 (20.11.2019): 3–18. http://dx.doi.org/10.1007/s00018-019-03365-1.

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AbstractHomologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase.

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Stachowski, Kye, Andrew S. Norris, Devante Potter, Vicki H. Wysocki und Mark P. Foster. „Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM“. Nucleic Acids Research 50, Nr. 3 (01.02.2022): 1753–69. http://dx.doi.org/10.1093/nar/gkac032.

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Abstract Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

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Joshi, Sunil K., Kahoko Hashimoto und Pandelakis A. Koni. „Induced DNA recombination by Cre recombinase protein transduction“. genesis 33, Nr. 1 (26.04.2002): 48–54. http://dx.doi.org/10.1002/gene.10089.

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Burns, Lesley S., Stephen G. J. Smith und Charles J. Dorman. „Interaction of the FimB Integrase with thefimS Invertible DNA Element in Escherichia coliIn Vivo and In Vitro“. Journal of Bacteriology 182, Nr. 10 (15.05.2000): 2953–59. http://dx.doi.org/10.1128/jb.182.10.2953-2959.2000.

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ABSTRACT The FimB protein is a site-specific recombinase that inverts thefimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind tofimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fimswitch.

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Qian, X. H., R. B. Inman und M. M. Cox. „Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination.“ Journal of Biological Chemistry 265, Nr. 35 (Dezember 1990): 21779–88. http://dx.doi.org/10.1016/s0021-9258(18)45808-5.

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Morlino, Giovanni B., Lorenza Tizzani, Reinhard Fleer, Laura Frontali und Michele M. Bianchi. „Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis“. Applied and Environmental Microbiology 65, Nr. 11 (01.11.1999): 4808–13. http://dx.doi.org/10.1128/aem.65.11.4808-4813.1999.

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ABSTRACT Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

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Nanassy, Oliver Z., und Kelly T. Hughes. „In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase“. Genetics 149, Nr. 4 (01.08.1998): 1649–63. http://dx.doi.org/10.1093/genetics/149.4.1649.

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Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

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Yum, S. Y., S. J. Kim, J. H. Moon, W. J. Choi, J. H. Lee, B. C. Lee und G. Jang. „21 SITE-SPECIFIC RECOMBINATION USING Dre-RECOMBINASE IN PORCINE CELLS AND EMBRYOS“. Reproduction, Fertility and Development 26, Nr. 1 (2014): 125. http://dx.doi.org/10.1071/rdv26n1ab21.

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Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.

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Jones, J. M., und M. Gellert. „Autoubiquitylation of the V(D)J recombinase protein RAG1“. Proceedings of the National Academy of Sciences 100, Nr. 26 (11.12.2003): 15446–51. http://dx.doi.org/10.1073/pnas.2637012100.

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del Val, Elsa, William Nasser, Hafid Abaibou und Sylvie Reverchon. „RecA and DNA recombination: a review of molecular mechanisms“. Biochemical Society Transactions 47, Nr. 5 (18.10.2019): 1511–31. http://dx.doi.org/10.1042/bst20190558.

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Abstract Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer–monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.

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Gyohda, Atsuko, und Teruya Komano. „Purification and Characterization of the R64 Shufflon-Specific Recombinase“. Journal of Bacteriology 182, Nr. 10 (15.05.2000): 2787–92. http://dx.doi.org/10.1128/jb.182.10.2787-2792.2000.

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ABSTRACT The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

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García-Vázquez, Francisco A., Salvador Ruiz, Carmen Matás, M. José Izquierdo-Rico, Luis A. Grullón, Aitor De Ondiz, Luis Vieira, Karen Avilés-López, Alfonso Gutiérrez-Adán und Joaquín Gadea. „Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA“. REPRODUCTION 140, Nr. 2 (August 2010): 259–72. http://dx.doi.org/10.1530/rep-10-0129.

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Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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Suiwal, Shweta, Philipp Wartenberg, Ulrich Boehm, Frank Schmitz und Karin Schwarz. „A Novel Cre Recombinase Mouse Strain for Cell-Specific Deletion of Floxed Genes in Ribbon Synapse-Forming Retinal Neurons“. International Journal of Molecular Sciences 25, Nr. 3 (05.02.2024): 1916. http://dx.doi.org/10.3390/ijms25031916.

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We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons.

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Wang, Xinrui, Amelia Lauth, Tina C. Wan, John W. Lough und John A. Auchampach. „Myh6-driven Cre recombinase activates the DNA damage response and the cell cycle in the myocardium in the absence of loxP sites“. Disease Models & Mechanisms 13, Nr. 12 (08.10.2020): dmm046375. http://dx.doi.org/10.1242/dmm.046375.

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ABSTRACTRegeneration of muscle in the damaged myocardium is a major objective of cardiovascular research, for which purpose many investigators utilize mice containing transgenes encoding Cre recombinase to recombine loxP-flanked target genes. An unfortunate side effect of the Cre-loxP model is the propensity of Cre recombinase to inflict off-target DNA damage, which has been documented in various eukaryotic cell types including cardiomyocytes (CMs). In the heart, reported effects of Cre recombinase include contractile dysfunction, fibrosis, cellular infiltration and induction of the DNA damage response (DDR). During experiments on adult mice containing a widely used Myh6-merCremer transgene, the protein product of which is activated by tamoxifen, we observed large, transient, off-target effects of merCremer, some of which have not previously been reported. On Day 3 after the first of three daily tamoxifen injections, immunofluorescent microscopy of heart sections revealed that the presence of merCremer protein in myonuclei was nearly uniform, thereafter diminishing to near extinction by Day 6; during this time, cardiac function was depressed as determined by echocardiography. On Day 5, peaks of apoptosis and expression of DDR-regulatory genes were observed, highlighted by >25-fold increased expression of Brca1. Concomitantly, the expression of genes encoding cyclin-A2, cyclin-B2 and cyclin-dependent kinase 1, which regulate the G2/S cell-cycle transition, were dramatically increased (>50- to 100-fold). Importantly, immunofluorescent staining revealed that this was accompanied by peaks in Ki67, 5′-bromodeoxyuridine and phosphohistone H3 labeling in non-CMs, as well as CMs. We further document that tamoxifen-induced activation of merCremer exacerbates cardiac dysfunction following myocardial infarction. These findings, when considered in the context of previous reports, indicate that the presence of merCremer in the nucleus induces DNA damage and unscheduled cell-cycle activation. Although these effects are transient, the inclusion of appropriate controls, coupled with an awareness of the defects caused by Cre recombinase, are required to avoid misinterpreting results when using Cre-loxP models for cardiac regeneration studies.This article has an associated First Person interview with the first author of the paper.

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Inagaki, Satoko, Kazuyo Fujita, Yukiko Takashima, Kayoko Nagayama, Arifah C. Ardin, Yuki Matsumi und Michiyo Matsumoto-Nakano. „Regulation of Recombination betweengtfB/gtfCGenes inStreptococcus mutansby Recombinase A“. Scientific World Journal 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/405075.

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Streptococcus mutansproduces 3 types of glucosyltransferases (GTFs), whose cooperative action is essential for cellular adhesion. The recombinase A (RecA) protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions.S. mutansstrain MT8148 was grown in the presence of recombinant RecA (rRecA) protein, after which smooth colonies were isolated. The biological functions and sequences of thegtfBandgtfCgenes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of thegtfBandgtfCgenes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of therecAgene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions ofgtfBandgtfCgenes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.

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Xin, H. B., K. Y. Deng, M. Rishniw, G. Ji und M. I. Kotlikoff. „Smooth muscle expression of Cre recombinase and eGFP in transgenic mice“. Physiological Genomics 10, Nr. 3 (03.09.2002): 211–15. http://dx.doi.org/10.1152/physiolgenomics.00054.2002.

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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.

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Shintani, Yoshizumi, Hiroshi Yotsuyanagi, Kyoji Moriya, Hajime Fujie, Takeya Tsutsumi, Yumi Kanegae, Satoshi Kimura, Izumu Saito und Kazuhiko Koike. „Induction of apoptosis after switch-on of the hepatitis B virus X gene mediated by the Cre/loxP recombination system“. Journal of General Virology 80, Nr. 12 (01.12.1999): 3257–65. http://dx.doi.org/10.1099/0022-1317-80-12-3257.

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The HBx protein of hepatitis B virus is a multifunctional protein that is implicated in the pathogenesis of hepatocellular carcinoma by regulating gene transcription, causing cell proliferation and, as shown recently, inducing cell death. However, analysis of the effects of HBx in stable cultured cell clones has been hampered because only cell lines that adapted to the effects of HBx were selected during the establishment of cell clones. Here, we describe a system in which transcription of the X gene of hepatitis B virus is switched on by the use of the site-specific Cre recombinase. Two human liver cell lines, HLF and HepG2, were used, the former with a mutant p53 allele and the latter with wild-type p53. The stable cell clones isolated, which carried the X gene in a transcriptionally silent state, were infected with recombinant adenovirus carrying Cre recombinase. Ninety-six hours after adenovirus infection, cell clones that expressed HBx had undergone TUNEL-positive cell death with characteristics of apoptosis. Apoptosis was induced despite concomitant inactivation of the p53 protein as a result of its cytoplasmic translocation by HBx. In contrast, neither the X gene-carrying cells infected with wild-type adenovirus nor various control cells infected with Cre-expressing adenovirus exhibited apoptosis. These results indicate that the expression of HBx protein leads to liver cell apoptosis independently of the p53 pathway. The significance of HBx-induced apoptosis in natural infection is unclear, but it may contribute to the development of hepatitis and serve to spread progeny virus to neighbouring cells while evading the host immune responses.

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Dunlop, Myun Hwa, Eloïse Dray, Weixing Zhao, Joseph San Filippo, Miaw-Sheue Tsai, Stanley G. Leung, David Schild, Claudia Wiese und Patrick Sung. „Mechanistic Insights into RAD51-associated Protein 1 (RAD51AP1) Action in Homologous DNA Repair“. Journal of Biological Chemistry 287, Nr. 15 (27.02.2012): 12343–47. http://dx.doi.org/10.1074/jbc.c112.352161.

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Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

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Simpson, Destiny A., und Karla K. Rodgers. „Risky business blockade: RAG2 basic region blocks V(D)J recombinase function upon genotoxic stress in DNA damage response“. Journal of Immunology 208, Nr. 1_Supplement (01.05.2022): 107.13. http://dx.doi.org/10.4049/jimmunol.208.supp.107.13.

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Abstract The V(D)J recombinase, composed of the recombination activating gene (RAG) proteins, RAG1 and RAG2, generate functional antigen receptor genes through the repeated generation of DNA double strand breaks and DNA repair. However, this process can lead to increased genomic instability particularly in the presence of excess DNA breaks. Previously, we showed in pre-B cells that full length RAG2, a nuclear protein, will export to the cytoplasm with DNA damage in an ATM-dependent manner with eventual recovery back into the nucleus following DNA repair. We propose that re-localization of RAG2 prevents RAG-mediated DNA cleavage in the presence of excess DNA ends. Here, by using fluorescently tagged RAG proteins, we show that DNA damage-induced export of the V(D)J recombinase can be recapitulated in non-lymphoid cells. Further, we determined that mutations in the intrinsically-disordered region of RAG2 have distinct effects on this process. Specifically, a basic region mutant of RAG2 (K/R 518–524 A) alters the localization pattern of the V(D)J recombinase, and yet its pre-DNA damage localization pattern is re-established upon DNA repair. Conversely, a T490A mutant blocks relocalization of RAG2 following genotoxic stress for both the single T490A mutant and the combined T490A/basic region mutant. Together, these results demonstrate the role of the intrinsically-disordered region in RAG2 in regulating the mobility and subcellular localization of the V(D)J recombinase upon cellular conditions that may be deemed too risky for continued DNA rearrangements. Supported by NIA T32AG052363, NIH AI128137, OCAST

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Pan, G., K. Luetke und P. D. Sadowski. „Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis“. Molecular and Cellular Biology 13, Nr. 6 (Juni 1993): 3167–75. http://dx.doi.org/10.1128/mcb.13.6.3167-3175.1993.

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The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

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Pan, G., K. Luetke und P. D. Sadowski. „Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.“ Molecular and Cellular Biology 13, Nr. 6 (Juni 1993): 3167–75. http://dx.doi.org/10.1128/mcb.13.6.3167.

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The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

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Constantinescu, Andrei, und Mark S. Schlissel. „Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development“. Journal of Experimental Medicine 185, Nr. 4 (17.02.1997): 609–20. http://dx.doi.org/10.1084/jem.185.4.609.

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The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.

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Li, Huiping, Xiyou Zhou, Deborah R. Davis, Di Xu und Curt D. Sigmund. „An androgen-inducible proximal tubule-specific Cre recombinase transgenic model“. American Journal of Physiology-Renal Physiology 294, Nr. 6 (Juni 2008): F1481—F1486. http://dx.doi.org/10.1152/ajprenal.00064.2008.

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To facilitate the study of renal proximal tubules, we generated a transgenic mouse strain expressing an improved Cre recombinase (iCre) under the control of the kidney androgen-regulated protein (KAP) promoter. The transgene was expressed in the kidney of male mice but not in female mice. Treatment of female transgenic mice with androgen induced robust expression of the transgene in the kidney. We confirmed the presence of Cre recombinase activity and the cell specificity by breeding the KAP2-iCRE mice with ROSA26 reporter mice. X-Gal staining of kidney sections from male double transgenic mice showed robust staining in the epithelial cells of renal proximal tubules. β-Gal staining in female mice became evident in proximal tubules after administration of androgen. This model of inducible Cre recombinase in the renal proximal tubule should provide a novel useful tool for studying the physiological significance of genes expressed in the renal proximal tubule. This has advantages over other current models where Cre recombinase expression is constitutive, not inducible.

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Khoo, Kelvin H. P., Hayley R. Jolly und Jason A. Able. „The RAD51 gene family in bread wheat is highly conserved across eukaryotes, with RAD51A upregulated during early meiosis“. Functional Plant Biology 35, Nr. 12 (2008): 1267. http://dx.doi.org/10.1071/fp08203.

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The RADiation sensitive protein 51 (RAD51) recombinase is a eukaryotic homologue of the bacterial Recombinase A (RecA). It is required for homologous recombination of DNA during meiosis where it plays a role in processes such as homology searching and strand invasion. RAD51 is well conserved in eukaryotes with as many as four paralogues identified in vertebrates and some higher plants. Here we report the isolation and preliminary characterisation of four RAD51 gene family members in hexaploid (bread) wheat (Triticum aestivum L.). RAD51A1, RAD51A2 and RAD51D were located on chromosome group 7, and RAD51C was on chromosome group 2. Q-PCR gene expression profiling revealed that RAD51A1 was upregulated during meiosis with lower expression levels seen in mitotic tissue, and bioinformatics analysis demonstrated the evolutionary linkages of this gene family to other eukaryotic RAD51 sequences. Western blot analysis of heterologously expressed RAD51 from bread wheat has shown that it is detectable using anti-human RAD51 antibodies and that molecular modelling of the same protein revealed structural conservation when compared with yeast, human, Arabidopsis and maize RAD51A orthologues. This report has widened the knowledge base of this important protein family in plants, and highlighted the high level of structural conservation among RAD51 proteins from various species.

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Sauer, B. „Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae“. Molecular and Cellular Biology 7, Nr. 6 (Juni 1987): 2087–96. http://dx.doi.org/10.1128/mcb.7.6.2087-2096.1987.

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The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

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Sauer, B. „Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.“ Molecular and Cellular Biology 7, Nr. 6 (Juni 1987): 2087–96. http://dx.doi.org/10.1128/mcb.7.6.2087.

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The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

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Ferguson, S. E., M. A. Accavitti, D. D. Wang, C. L. Chen und C. B. Thompson. „Regulation of RAG-2 protein expression in avian thymocytes“. Molecular and Cellular Biology 14, Nr. 11 (November 1994): 7298–305. http://dx.doi.org/10.1128/mcb.14.11.7298-7305.1994.

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The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells.

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Ferguson, S. E., M. A. Accavitti, D. D. Wang, C. L. Chen und C. B. Thompson. „Regulation of RAG-2 protein expression in avian thymocytes.“ Molecular and Cellular Biology 14, Nr. 11 (November 1994): 7298–305. http://dx.doi.org/10.1128/mcb.14.11.7298.

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The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells.

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Maxwell, Megan, Jonas Bjorkman, Tam Nguyen, Peter Sharp, John Finnie, Carol Paterson, Ian Tonks, Barbara C. Paton, Graham F. Kay und Denis I. Crane. „Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype“. Molecular and Cellular Biology 23, Nr. 16 (15.08.2003): 5947–57. http://dx.doi.org/10.1128/mcb.23.16.5947-5957.2003.

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ABSTRACT Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13−/− mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.

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Lansing, Felix, Maciej Paszkowski-Rogacz, Lukas Theo Schmitt, Paul Martin Schneider, Teresa Rojo Romanos, Jan Sonntag und Frank Buchholz. „A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus“. Nucleic Acids Research 48, Nr. 1 (20.11.2019): 472–85. http://dx.doi.org/10.1093/nar/gkz1078.

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Abstract Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

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Thorslund, Tina, Fumiko Esashi und Stephen C. West. „Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1“. EMBO Journal 26, Nr. 12 (31.05.2007): 2915–22. http://dx.doi.org/10.1038/sj.emboj.7601739.

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Rajaee, Maryam, und David W. Ow. „A new location to split Cre recombinase for protein fragment complementation“. Plant Biotechnology Journal 15, Nr. 11 (20.04.2017): 1420–28. http://dx.doi.org/10.1111/pbi.12726.

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Buchholz, Frank, und A. Francis Stewart. „Alteration of Cre recombinase site specificity by substrate-linked protein evolution“. Nature Biotechnology 19, Nr. 11 (November 2001): 1047–52. http://dx.doi.org/10.1038/nbt1101-1047.

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Chen, Jing-Wen, Barbara R. Evans, Lei Zheng und Makkuni Jayaram. „Tyr60 variants of Flp recombinase generate conformationally altered protein-DNA complexes“. Journal of Molecular Biology 218, Nr. 1 (März 1991): 107–18. http://dx.doi.org/10.1016/0022-2836(91)90877-9.

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Sellers, Drew L., Jamie M. Bergen, Russell N. Johnson, Heidi Back, John M. Ravits, Philip J. Horner und Suzie H. Pun. „Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration“. Proceedings of the National Academy of Sciences 113, Nr. 9 (17.02.2016): 2514–19. http://dx.doi.org/10.1073/pnas.1515526113.

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A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

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Nagel, Claus-Henning, Katinka Döhner, Mojgan Fathollahy, Tanja Strive, Eva Maria Borst, Martin Messerle und Beate Sodeik. „Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17+)“. Journal of Virology 82, Nr. 6 (26.12.2007): 3109–24. http://dx.doi.org/10.1128/jvi.02124-07.

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ABSTRACT To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17+)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17+)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17+)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.

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