title: V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies.
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.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Swanson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D060588:LIM Domain Proteins; D007938:Leukemia / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D014157:Transcription Factors / Q000378:metabolism; D045567:VDJ Recombinases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008667:Metalloproteins; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D014157:Transcription Factors; D045567:VDJ Recombinases; C065072:LMO1 protein, human; C085117:LMO2 protein, human; C071236:STIL protein, human; C067143:TAL1 protein, human; C071250:TLX1 protein, human