title: Kinetic analysis of the nicking and hairpin formation steps in V(D)J recombination.
abstract: The complete cleavage phase of V(D)J recombination includes four phases: binding of the active RAG complexes to the 12- or 23-signals, nicking of the signals, synapsis of the two signals, and hairpin formation at both signals concurrently. We have done time courses for the complete cleavage phase of the V(D)J recombination reaction and quantitated the amount of active RAG enzyme. We have also formulated a kinetic model for the binding, nicking, synapsis, and hairpin formation phases. We have utilized free solution enzymatic measurements for the binding and nicking phases as we do mathematical simulations of the kinetic model. This permits iteration of rate constants for the synapsis and hairpin formation phases until the model fits the observed overall cleavage time course. This process yields a rate constant for the hairpin formation that is 0.004 min(-1), which corresponds to an average catalytic cycle time of 250 min. This value is exceedingly close to a measured value of this constant that relied on wash-out of an inhibitory cofactor. The agreement indicates that this is likely to be the rate of the hairpin step over a wide range of range of conditions and irrespective of the DNA sequence of the V, D or J coding end located adjacent to the signal. These findings indicate that, under optimal in vitro conditions, the core RAG proteins carry out nicking at a rate which is nearly 150-fold faster than hairpin formation. The physiologic implications of this and other kinetic inferences of these time courses are discussed.
authors.affiliation: Department of Pathology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Room 5428, 1441 Eastlake Avenue, MC 9176, Los Angeles, CA 90033, USA.
authors.forename: Kefei
authors.initials: K
authors.lastname: Yu
authors.forename: Alex
authors.initials: A
authors.lastname: Taghva
authors.forename: Yunmei
authors.initials: Y
authors.lastname: Ma
authors.forename: Michael R
authors.initials: MR
authors.lastname: Lieber
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology*; D015321:Gene Rearrangement / Q000235:genetics*; D024243:HMGB1 Protein / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D009687:Nuclear Proteins; D011485:Protein Binding; D011995:Recombination, Genetic / Q000235:genetics*; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA