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G. D. Van Duyne - One of the best experts on this subject based on the ideXlab platform.
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Geometry of the DNA Substrates in Cre-LoxP Site-Specific Recombination.
Journal of biomolecular structure & dynamics, 2000Co-Authors: F Guo, D. N. Gopaul, G. D. Van DuyneAbstract:Abstract Cre recombinase is a member of a large family of Site-specific recombination enzymes that performs a cut-and-paste operation between two specific DNA sequences. Our goal has been to understand the mechanism of this complex reaction by trapping and characterizing the three-dimensional structures of each of the reaction intermediates. This work has led to high resolution crystallographic models of (i) the initial synaptic complex, (ii) the covalent Cre- DNA intermediate, and (iii) the Holliday junction intermediate. The Cre-LoxP system appears to function by creating at the outset a protein-DNA architecture that resembles that of the Holliday junction intermediate that is eventually formed. The "arms" of the LoxP Sites are initially bent by about 75° in the synaptic complex, forming a nearly planar arrangement that is held fixed, while cleavage and strand exchange occur in the central region between the arms. The simplest view of the recombination pathway is that it contains two symmetrical halves, each of which uses this Holliday junction-like architectural framework to mediate the cleavage and ligation steps. The two halves are linked by a subtle isomerization of the Holliday intermediate that switches the roles of the recombinase subunits and allows exchange of the second pair of DNA strands. In this paper, we summarize recent structural results from our laboratory, with an emphasis on the geometry of the DNA substrates.
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Asymmetric DNA bending in the Cre-LoxP Site-specific recombination synapse
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Feng Guo, D. N. Gopaul, G. D. Van DuyneAbstract:Cre recombinase catalyzes Site-specific recombination between two 34-bp LoxP Sites in a variety of DNA substrates. At the start of the recombination pathway, the LoxP Sites are each bound by two recombinase molecules, and synapsis of the Sites is mediated by Cre–Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized LoxP Site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step at one end of the crossover region in a manner that is atypical of protein-induced DNA bends. A large negative roll (−49°) and a positive tilt (16°) open the major groove toward the center of the synapse and compress the minor groove toward the protein–DNA interface. The bend direction of the Site appears to determine which of the two DNA substrate strands will be cleaved and exchanged in the initial stages of the recombination pathway. These results provide a structural basis for the observation that exchange of DNA strands proceeds in a defined order in some tyrosine recombinase systems. The Cre-loxS synaptic complex structure supports a model in which synapsis of the LoxP Sites results in formation of a Holliday junction-like DNA architecture that is maintained through the initial cleavage and strand exchange steps in the Site-specific recombination pathway.
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Structure of the Holliday junction intermediate in Cre-LoxP Site-specific recombination.
The EMBO journal, 1998Co-Authors: D. N. Gopaul, Feng Guo, G. D. Van DuyneAbstract:We have determined the X‐ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four‐way branched structure that occurs as an intermediate in genetic recombination pathways, including Site‐specific recombination by the λ‐integrase family. Cre recombinase is an integrase family member that recombines 34 bp LoxP Sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold‐symmetric DNA intermediate that shares features with both the stacked‐X and the square conformations of the HJ that exist in the unbound state. The structures support a protein‐mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active Sites. In this model, a subtle isomerization of the Cre recombinase–HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.
Jonathon S. Coren - One of the best experts on this subject based on the ideXlab platform.
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Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant LoxP Site.
BMC research notes, 2017Co-Authors: Jonathon S. CorenAbstract:Objective Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant LoxP Sites present.
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Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant LoxP Site
BMC, 2017Co-Authors: Jonathon S. CorenAbstract:Abstract Objective Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant LoxP Sites present. Results An oligonucleotide containing a mutant LoxP 2272 Site and a XhoI restriction enzyme sequence was designed and inserted at the SfiI restriction Site located approximately 200 basepairs upstream of the lacZ gene in pBeloBAC11. Clones containing the desired insert were identified by XhoI restriction digests since an additional band was generated. This transposon-mediated deletion technology allows researchers to identify the boundaries of cis-acting elements and genes
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Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed LoxP Site
Nucleic acids research, 1997Co-Authors: Pradeep K. Chatterjee, Jonathon S. CorenAbstract:A general approach for isolating large nested deletions in P1 artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) by retrofitting with a LoxP Site-containing Tn10 mini-transposon is described. Cre-mediated recombination between the LoxP Site existing in these clones and one introduced by transposition leads to deletions and inversions of the DNA between these Sites. Large deletions are selectively recovered by transducing the retrofitted PAC or BAC clones with P1 phage. The requirement that both LoxP Sites in the cointegrate be packaged into a P1 head ensures that only large deletions are rescued. PCR analyses identified these deletions as products of legitimate recombination between LoxP Sites mediated by Cre protein. BACs produce deletions much more efficiently than PACs although the former cannot be induced to greater than unit copy in cells. Mammalian cell-responsive antibiotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis. Most importantly, the LoxP Site retrofitting and P1 transduction can be performed in the same bacterial host containing these clones directly isolated from PAC or BAC libraries. These procedures should facilitate physical and functional mapping of genes and regulatory elements in these large plasmids.
Gregory D. Van Duyne - One of the best experts on this subject based on the ideXlab platform.
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Preferential synapsis of LoxP Sites drives ordered strand exchange in Cre-LoxP Site-specific recombination.
Nature chemical biology, 2005Co-Authors: Kaushik Ghosh, Chi Kong Lau, Kushol Gupta, Gregory D. Van DuyneAbstract:The bacteriophage P1 Cre recombinase catalyzes Site-specific recombination between 34-base-pair LoxP sequences in a variety of topological contexts. This reaction is widely used to manipulate DNA molecules in applications ranging from benchtop cloning to genome modifications in transgenic animals. Despite the simple, highly symmetric nature of the Cre-LoxP system, there is strong evidence that the reaction is asymmetric; the 'bottom' strands in the recombining LoxP Sites are preferentially exchanged before the 'top' strands. Here, we address the mechanistic basis for ordered strand exchange in the Cre-LoxP recombination pathway. Using suicide substrates containing 5′-bridging phosphorothioate linkages at both cleavage Sites, fluorescence resonance energy transfer between synapsed LoxP Sites and a Cre mutant that can cleave the bridging phosphorothioate linkage but not a normal phosphodiester linkage, we showed that preferential formation of a specific synaptic complex between LoxP Sites imposes ordered strand exchange during recombination and that synapsis stimulates cleavage of LoxP Sites.
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peptide trapping of the holliday junction intermediate in cre LoxP Site specific recombination
Journal of Biological Chemistry, 2005Co-Authors: Kaushik Ghosh, Feng Guo, Chi Kong Lau, Anca M Segall, Gregory D. Van DuyneAbstract:Abstract Cre recombinase is a prototypical member of the tyrosine recombinase family of Site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate Sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the λ-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active Site geometries in the recombinase subunits.
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A Structural View of Cre- LoxP Site-Specific Recombination
Annual Review of Biophysics and Biomolecular Structure, 2002Co-Authors: Gregory D. Van DuyneAbstract:▪ Abstract Structural models of Site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process. One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products. Crystal structures of reaction intermediates in the Cre-LoxP Site-specific recombination system, together with recent biochemical studies in the field, support a “strand swapping” model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining Sites.
Margaret Clotworthy - One of the best experts on this subject based on the ideXlab platform.
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Using Single LoxP Sites to Enhance Homologous Recombination: ts Mutants in Sec1 of Dictyostelium discoideum
PloS one, 2007Co-Authors: Mark S. Bretscher, Margaret ClotworthyAbstract:BACKGROUND Dictyostelium discoideum amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes: nsfA-the gene for NEM-sensitive factor-remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: approximately 95% of selected clones were homologous recombinants. This is unusually high for Dictyostelium: homologous recombination at other loci is usually much less, usually between 0-30%, making the isolation of ts mutants much more tedious. METHODOLOGY/PRINCIPAL FINDINGS In trying to make ts mutants in sec1A, homologous recombination was found to be only approximately 25%. A new approach, involving single LoxP Sites, was investigated. LoxP Sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A Dictyostelium line containing a single LoxP Site adjacent to the 3' end of the sec1A gene was engineered. A sec1A replacement DNA also containing a single LoxP Site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to approximately 80% at this locus, presumably largely driven by intermolecular recombination between the two single LoxP Sites. CONCLUSIONS/SIGNIFICANCE A route to increase the rate of homologous recombination at a specific locus, sec1A, is described which enabled the isolation of 30 ts mutants in sec1A. One of these, sec1Ats1,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in Dictyostelium and other creatures.
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Using Single LoxP Sites to Enhance Homologous Recombination: ts Mutants in Sec1 of Dictyostelium
2007Co-Authors: S. Bretscher, Margaret ClotworthyAbstract:Background. Dictyostelium discoideum amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes: nsfA—the gene for NEM-sensitive factor—remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: ,95% of selected clones were homologous recombinants. This is unusually high for Dictyostelium: homologous recombination at other loci is usually much less, usually between 0–30%, making the isolation of ts mutants much more tedious. Methodology/Principal Findings. In trying to make ts mutants in sec1A, homologous recombination was found to be only ,25%. A new approach, involving single LoxP Sites, was investigated. LoxP Sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A Dictyostelium line containing a single LoxP Site adjacent to the 39 end of the sec1A gene was engineered. A sec1A replacement DNA also containing a single LoxP Site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to ,80% at this locus, presumably largely driven by intermolecular recombination between the two single LoxP Sites. Conclusions/Significance. A route to increase the rate of homologous recombination at a specific locus, sec1A, is described which enabled the isolation of 30 ts mutants in sec1A. One of these, sec1Ats1,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in Dictyostelium and other creatures.
Ye Qingyi - One of the best experts on this subject based on the ideXlab platform.
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reversible transfection of human melanocytes mediated by cre LoxP Site specific recombination system and sv40 large t antigen
Experimental Dermatology, 2007Co-Authors: Wang Ying, Hao Fei, Deng Jun, Yang Xichuan, Zhong Baiyu, Ye QingyiAbstract:: Objective: To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/LoxP Site-specific recombination system. Methods: The reconstructed SV40LTAg-EGFP-neo-LoxP vector was transfected into primary cultured human melanocytes with SofastTM transfection reagent and the positive cells were selected using G418. After expanding culture of these positive cell clones, the expression of SV40LTAg was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent method. After that, these positive cells were infected by virus supernatant of Cre-ERT2 retrovirus vector and Cre recombinase was induced to act by tamoxifen. On the 6th and 10th day after Cre recombinase acting, the expression of SV40LTAg was detected using the same methods as above, and cell tumorigenicity was studied using soft agar assay, athymic mouse study and karyotype analysis. On 10th day after tamoxifen treatment, cell biological characters were identified with immunofluorescent staining and transmission electron microscopy. Then these cells were transplanted into vitiligo animal model to observe their melanogenesis ability in vivo. Results: The genome DNA and total RNA were isolated from the positive cells transfected by SV40LTAg (designated as MCT) and specific 288 bp fragment was amplificated using PCR and RT-PCR methods. The results of immunofluorescence confirmed the expression of SV40LTAg in cell nucleus. On the 6th day after tamoxifen treatment in infected cells by Cre-ERT2 retrovirus vector (designated as MCT-Cre), there could be detected SV40LTAg expression, but on 10th day, there could not be detected SV40LTAg expression in cells. These results showed that the excised efficiency of Cre recombinase increased along with time prolongation, and would obtain complete recombination efficiency. The identification of MCT-Cre cell biological characters showed that these cells had normal parent-cell-like cell phenotype and no tumorigenicity in vitro. The pigmentation started in 4 weeks and formed black macula in 3 months after grafting. The pathological results showed that there had been significant melanocytes and melanin accumulation in epidermis and some hair follicle in transplanted area, which confirmed that MCT-Cre had melanogenesis function in vivo. Conclusion: Human melanocytes could be mediated by reversible transfection by SV40LTAg and Cre/LoxP Site-specific recombination system, which had stable parent-cell-like phenotypic characters and no tumorigenicity in vitro; moreover, these cells still had melanogenesis function in vivo.
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Reversible transfection of human melanocytes mediated by Cre/LoxP Site-specific recombination system and SV40 large T antigen.
Experimental dermatology, 2007Co-Authors: Wang Ying, Hao Fei, Deng Jun, Yang Xi-chuan, Zhong Bai-yu, Ye QingyiAbstract:: Objective: To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/LoxP Site-specific recombination system. Methods: The reconstructed SV40LTAg-EGFP-neo-LoxP vector was transfected into primary cultured human melanocytes with SofastTM transfection reagent and the positive cells were selected using G418. After expanding culture of these positive cell clones, the expression of SV40LTAg was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent method. After that, these positive cells were infected by virus supernatant of Cre-ERT2 retrovirus vector and Cre recombinase was induced to act by tamoxifen. On the 6th and 10th day after Cre recombinase acting, the expression of SV40LTAg was detected using the same methods as above, and cell tumorigenicity was studied using soft agar assay, athymic mouse study and karyotype analysis. On 10th day after tamoxifen treatment, cell biological characters were identified with immunofluorescent staining and transmission electron microscopy. Then these cells were transplanted into vitiligo animal model to observe their melanogenesis ability in vivo. Results: The genome DNA and total RNA were isolated from the positive cells transfected by SV40LTAg (designated as MCT) and specific 288 bp fragment was amplificated using PCR and RT-PCR methods. The results of immunofluorescence confirmed the expression of SV40LTAg in cell nucleus. On the 6th day after tamoxifen treatment in infected cells by Cre-ERT2 retrovirus vector (designated as MCT-Cre), there could be detected SV40LTAg expression, but on 10th day, there could not be detected SV40LTAg expression in cells. These results showed that the excised efficiency of Cre recombinase increased along with time prolongation, and would obtain complete recombination efficiency. The identification of MCT-Cre cell biological characters showed that these cells had normal parent-cell-like cell phenotype and no tumorigenicity in vitro. The pigmentation started in 4 weeks and formed black macula in 3 months after grafting. The pathological results showed that there had been significant melanocytes and melanin accumulation in epidermis and some hair follicle in transplanted area, which confirmed that MCT-Cre had melanogenesis function in vivo. Conclusion: Human melanocytes could be mediated by reversible transfection by SV40LTAg and Cre/LoxP Site-specific recombination system, which had stable parent-cell-like phenotypic characters and no tumorigenicity in vitro; moreover, these cells still had melanogenesis function in vivo.
