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Abstract
An essential step in the development of the vertebrate immune system is the DNA level rearrangement of the antigen receptor genes. This process, termed "V(D)J recombination," begins with DNA cleavage at the appropriate sites mediated by the two proteins RAG1 and RAG2. We report here that the two proteins cooperate to bind DNA with significantly higher specificity than either protein alone. Gel purification of the triple complex is performed in the absence of any cross-linking agents. Both proteins remain present in the complex, and UV cross-linking using iodouridine-containing probes shows that RAG1 makes close contacts in both the heptamer and nonamer motifs. The two proteins are also shown to associate with each other in the absence of any DNA. These findings refine our understanding of the protein-DNA interactions that accompany cleavage at the recombination signals.
Highlights
V(D)J recombination occurs through a cutting and pasting mechanism in which specific double strand DNA breaks are generated adjacent to each recombination signal sequences (RSSs)
It has been shown previously that the truncated forms of RAG1 and RAG2 used here are active in V(D)J recombination and capable of supporting the complete reaction in cells (14 – 16)
It should be noted that the truncated RAG1 is entirely lacking in a N-terminal zinc-binding ring-finger motif and the RAG2 lacks a C-terminal acidic region
Summary
V(D)J recombination occurs through a cutting and pasting mechanism in which specific double strand DNA breaks are generated adjacent to each RSS. Protein-DNA interactions were observed in complexes cross-linked by glutaraldehyde [13]; no binding by RAG1 alone was observed. Most important, using a series of probes substituting iodouracil at individual positions, we find that UV exposure of the mixed protein complex generates cross-links to RAG1 in the nonamer and in the heptamer, while RAG2 is not crosslinked.
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