ArticleProceedings of the National Academy of Sciences of the United States of America2012
Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 45 citations in OpenAlex.
- Article
- Dynamics in Cre-loxP site-specific recombination.Current opinion in structural biology · 2024Review
- Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.Nucleic acids research · 2022Article
- Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases.Computational and structural biotechnology journal · 2022Article
- A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase.Nucleic acids research · 2020Article
- Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination.Nucleic acids research · 2020Article
- Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics.Nucleic acids research · 2019Article
- Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination.Micromachines · 2018Review
- Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.Nucleic acids research · 2018Article
- Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion.Nucleic acids research · 2016Article
- Optimal Background Estimators in Single-Molecule FRET Microscopy.Biophysical journal · 2016Article
- RNA Polymerase Pausing during Initial Transcription.Molecular cell · 2016Article
- Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system.BMC systems biology · 2016Article
- A Tet-on and Cre-loxP Based Genetic Engineering System for Convenient Recycling of Selection Markers in Penicillium oxalicum.Frontiers in microbiology · 2016Article
- Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.Nucleic acids research · 2015Article
- Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility.Journal of computer-aided molecular design · 2015Article
- The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.Microbiology spectrum · 2014Review
- Tethered fluorophore motion: studying large DNA conformational changes by single-fluorophore imaging.Biophysical journal · 2014Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
Abstract
Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.