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J. Biol. Chem., Vol. 283, Issue 11, 6717-6727, March 14, 2008

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V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies^*

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

V(D)J recombination is a process integral to lymphocyte development. However, this process is not always benign, since certain lymphoid malignancies exhibit recurrent chromosomal abnormalities, such as translocations and deletions, that harbor molecular signatures suggesting an origin from aberrant V(D)J recombination. Translocations involving LMO2, TAL1, Ttg-1, and Hox11, as well as a recurrent interstitial deletion at 1p32 involving SIL/SCL, are cited examples of illegitimate V(D)J recombination. Previous studies using extrachromosomal substrates reveal that cryptic recombination signal sequences (cRSSs) identified near the translocation breakpoint in these examples support V(D)J recombination with efficiencies ranging from about 30- to 20,000-fold less than bona fide V(D)J recombination signals. To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.

Received for publication, December 18, 2007

^* This work was supported by American Cancer Society Research Scholar Grant RSG-01-020-05-LIB and the Nebraska LB692 Tobacco Settlement Biomedical Research Program. (to P. C. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–5.

¹ 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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