Cycle-dependent Accumulation in Vivo of Transposition-competent Complexes between Recombination Signal Ends and Full-length RAG Proiteins

Hao Jiang, Ashley E. Ross, Stephen Desiderio

Research output: Contribution to journalArticle

Abstract

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 G0/G1 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.

Original languageEnglish (US)
Pages (from-to)8478-8486
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number9
DOIs
StatePublished - Feb 27 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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