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Probing the structure of RAG protein‐DNA intermediates in V(D)J recombination
Author(s) -
Schatz David,
Ciubotaru Mihai
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a44-d
Subject(s) - förster resonance energy transfer , synapsis , recombination signal sequences , dna , chemistry , cleavage (geology) , v(d)j recombination , single molecule fret , molecular beacon , fluorescence , recombination , recombination activating gene , biophysics , crystallography , physics , biology , gene , biochemistry , oligonucleotide , paleontology , quantum mechanics , fracture (geology)
A critical step in V(D)J recombination is synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1/2 proteins to generate the paired complex (PC). We have developed a fluorescence resonance energy transfer (FRET) assay to detect RSS synapsis and to investigate the configuration of the DNA molecules in the PC. FRET requires an appropriate 12/23 RSS pair, divalent metal ion, and HMGB1/2. FRET was detected with all pair wise combinations of 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC using an in gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC although an ensemble of different planar configurations cannot be ruled out. The data are most easily accommodated by models in which synapsed 12‐ and 23 RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage. We are also using FRET to study DNA bending in various complexes formed by the RAG proteins and preliminary data suggest the presence of at least two distinct bends in the 12RSS and in the 23RSS. We hypothesize that these bends are important to establish the appropriate substrate geometry needed for DNA cleavage.