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J. Biol. Chem., Vol. 279, Issue 6, 4034-4044, February 6, 2004

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Full-length RAG-2, and Not Full-length RAG-1, Specifically Suppresses RAG-mediated Transposition but Not Hybrid Joint Formation or Disintegration^*

Patrick C. Swanson, Dustin Volkmer, and Lei Wang

From the Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178

RAG-1 and RAG-2 initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences flanking a pair of antigen receptor gene segments. Occasionally, the RAG proteins mediate two other alternative DNA rearrangements in vivo: the rejoining of signal and coding ends and the transposition of signal ends into unrelated DNA. In contrast, truncated, catalytically active "core" RAG proteins readily catalyze these reactions in vitro, suggesting that full-length RAG proteins directly or indirectly suppress these undesired reactions in vivo. To discriminate between direct and indirect suppression models, full-length RAG proteins were purified and characterized in vitro. From mammalian cells, full-length RAG-1 is readily purified with core RAG-2 but not full-length RAG-2 and vice versa. Despite differences in DNA binding activity, recombinase containing either core or full-length RAG-1 or RAG-2 possess comparable cleavage, rejoining, and end-processing activity, as well as similar usage preferences for canonical versus cryptic recombination signals. However, recombinase containing full-length RAG-2, but not full-length RAG-1, exhibits dramatically reduced transposition activity in vitro. These data suggest RAG-mediated transposition and rejoining are differentially regulated by the full-length RAG proteins in vivo (the former directly by RAG-2 and the latter indirectly through other factors) and argue that noncore portions of the RAG proteins have little or no direct influence over V(D)J recombinase site specificity.

View larger version (69K): [in this window] [in a new window]   FIG. 4. Full-length RAG-2 but not full-length RAG-1 suppresses transposition in vitro. A, RAG-mediated transposition of radiolabeled signal ends into a cold target plasmid covalently links the signal ends to the target DNA and linearizes the plasmid. B, wild-type or active site mutant (D600A) cMR1 coexpressed with cMR2 (WT-cMR1/cMR2 or MT-cMR1/cMR2, respectively), cMR1/FLMR2, or FLMR1/cMR2 was incubated with a radiolabeled 23-SE with HMG-1 and cold partner DNA (12-SE), under binding conditions as indicated above the gel, and then subjected to an in-gel transposition assay (see "Experimental Procedures"). An autoradiograph of the DEAE-cellulose paper to which the DNA was transferred is shown here, with the position of the signal end complex (SEC) shown at right. C, reaction products were isolated from the SECs using the autoradiograph in B and analyzed on a native linear 4–20% gradient gel. Linearized 5'-end-labeled pcDNA1 and pre-cleaved substrate (lanes 1 and 2; indicated at right) serve as markers. The percentage of recovered plasmid DNA that is linearized is quantified below the gel (%TP). Similar results were obtained with radiolabeled 12-SE substrates (data not shown).

Received for publication, October 8, 2003

Note Added in Proof—In-gel transposition assays in which MgCl₂ substitutes for CaCl₂ in the transposition buffer yield results comparable to those presented in Fig. 4C, despite an overall reduction in transposition activity.

^* This work was supported by American Cancer Society Grant RSG-01-020-01-CCE and National Institutes of Health Grant 1 R01 AI055599-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medical Microbiology and Immunology, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178. Tel.: 402-280-2716; Fax: 402-280-1875; E-mail: pswanson{at}creighton.edu.

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