V(D)J recombination, the rearrangement of gene segments to assemble intact immunoglobulin and T cell receptor coding regions, is initiated by a recombinase made up of the RAG1 and RAG2 gene products. RAG1/2 introduces double-stranded breaks in the DNA adjacent to the coding segments. We have shown that the recombinase protein RAG1 undergoes ubiquitylation in cells. In vitro, the RING finger domain of RAG1 acts as a ubiquitin ligase (E3 enzyme) that mediates its own ubiquitylation at a highly conserved lysine residue. To our knowledge this is the first recombinase protein that has also been shown to possess E3 activity. RAG1 itself undergoes primarily mono- and to a lesser extent di-ubiquitylation, although some higher order species consistent with poly-ubiquitylation are present. The reaction is dependent on the ubiquitin activating enzyme and is best supported by a specific ubiquitin conjugating enzyme or E2, UbcH3/CDC34. Auto-ubiquitylation also requires a conserved basic region adjacent to the RING finger. Although the N-terminal domain of RAG1 is not absolutely required for DNA cleavage or rearrangement of extra-chromosomal substrates, mutation of conserved cysteine residues within the RING finger or deletion of the basic motif can severely curtail recombination activity. The involvement of the cell cycle control factor CDC34 suggests that ubiquitylation of RAG1 and RAG1 E3 activity may be linked to the cell cycle, and may help explain why certain RAG1/2 cleavage products are not resolved until the G1/S transition.
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