Cell cycle-dependent accumulation in vivo of transposition-competent complexes between recombination signal ends and full-length RAG proteins.
V(D)J recombination is initiated by a specialized transposase consisting of RAG-1 and RAG-2. Because full-length RAG proteins are insoluble under physiologic conditions, most previous analyses of RAG activity in vitro have used truncated core RAG-1 and RAG-2 fragments. These studies identified an intermediate in V(D)J recombination, the signal end complex (SEC), in which core RAG proteins remain associated with recombination signal sequences at the cleaved signal ends. From transfected cells expressing affinity-tagged RAG proteins, we have isolated in vivo assembled SECs containing full-length RAG proteins and cleaved recombination substrates. SEC formation in vivo did not require the repair proteins DNA-dependent protein kinase, Ku80, or XRCC4. In the presence of full-length RAG-2, SEC formation in vivo was cell cycle-regulated and restricted to the G(0)/G(1) phases. In contrast, complexes accumulated throughout cell cycle in cells expressing a RAG-2 CDK2 phosphorylation site mutant. Both core and full-length SECs supported transposition in vitro with similar efficiencies. Intracellular SECs, which are likely to persist in the absence of coding ends, represent potential donors whose transposition is not suppressed by the non-core regions of the RAG proteins.
Jiang
Hao
H
Department of Molecular Biology and Genetics, Program in Immunology, Institute for Cell Engineering, and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Ross
Ashley E
AE
Desiderio
Stephen
S
D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics; D002453:Cell Cycle / Q000502:physiology*; D006224:Cricetinae; D051357:Cyclin-Dependent Kinase 2; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D051379:Mice; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011995:Recombination, Genetic; D016192:Resting Phase, Cell Cycle; D014162:Transfection; D019895:Transposases / Q000378:metabolism*; D045567:VDJ Recombinases / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D058619:Maltose-Binding Proteins; D021382:Protein Sorting Signals; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D019895:Transposases; D045567:VDJ Recombinases