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

Ming Zhang,Patrick C. Swanson

doi:10.1074/jbc.m710301200

Ming Zhang, Patrick C. Swanson

Open Access

https://doi.org/10.1074/jbc.m710301200

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Abstract

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.

Highlights

Through an alternative pathway whereby the RAG proteins introduce two nicks in close proximity on opposite DNA strands [29, 30]. To determine whether this “nick-nick” mechanism may contribute to cleavage of these cRSSs, Hox11 and SCL, the RAG proteins were incubated with a consensus 23-RSS or various cRSS substrates radiolabeled on the bottom strand (Fig. 2E)
LMO2, Ttg-1, and SIL support RAG-mediated cleavage when embedded in a plasmid substrate, but the site preference for Ttg-1 cleavage is more diverse than observed using oligonucleotide substrates
Our experimental results from in vitro cleavage assays suggest that if a putative intact oligonucleotide cRSS substrate is cleaved by the core RAG proteins to levels approaching ϳ20% of a consensus RSS, the cRSS should be expected to support detectable levels of V(D)J recombination in cell culture

Summary

EXPERIMENTAL PROCEDURES

Oligonucleotide and Plasmid Substrates—Oligonucleotides containing a consensus or cryptic RSS were synthesized and gel-purified using a commercial vendor (IDT Inc., Coralville, IA). To prepare intact or nicked duplex substrates, the top or bottom strand oligonucleotides were radiolabeled with [␥-32P]ATP using T4 polynucleotide kinase, annealed to their unlabeled complementary strand(s), and the duplex was purified on a native polyacrylamide gel as described previously [25]. Coexpressed wild-type or mutant RAG1/RAG2 proteins (100 ng) and HMGB1 (300 ng, where indicated) were incubated with substrate DNA (ϳ0.02 pmol) in a 10-␮l reaction containing sample buffer (25 mM MOPS-KOH (pH 7.0), 60 mM potassium glutamate, 100 ␮g/ml bovine serum albumin, 20% Me2SO, and 1 mM MgCl2). For competition experiments shown, binding reactions were supplemented with unlabeled intact consensus or cRSS substrate (0.1, 1, or 10 pmol, as indicated) before the addition of the RAG proteins.

CAGTCGAAA CCGCGAAGCTCTCTTGC

RESULTS

SEBs in vivo

12RSS SEB

DISCUSSION