Correlation between recombinase activating gene 1 ubiquitin ligase activity and V(D)J recombination.
The really interesting new gene (RING) finger ubiquitin ligase domain of the recombinase activating gene 1 (RAG1) V(D)J recombinase protein adopts a standard cross-brace architecture but co-ordinates three zinc ions as opposed to the canonical two. We demonstrated previously that disruption of the conserved zinc co-ordination sites resulted in loss of structural integrity and ubiquitin ligase (E3) activity and interfered with the ability of full-length RAG1 to support recombination. Here we present evidence that amino acids surrounding the third, non-canonical site also contribute to functional interaction with the ubiquitin conjugating (E2) enzyme CDC34, while certain residues on the RING domain's surface important for interaction between other E2-E3 pairs are less critical to the functional RAG1-CDC34 interaction in this assay. Partial reduction of ubiquitin ligase activity was significantly correlated with reduction in the ability of RAG1 to support recombination of extra-chromosomal substrates (r = 0.805, P = 0.009). While poly-ubiquitin chains could be generated, RAG1 did not promote rapid chain extension following mono-ubiquitylation of substrate, regardless of the E2 enzyme used. No single ubiquitin lysine mutant disrupted the ability of CDC34 to form ubiquitin chains on RAG1, and mass spectrometric analysis of the poly-ubiquitylated products indicated ubiquitin chain linkages through lysines 48 and 11. These data suggest that RAG1 promotes a mono-ubiquitylation reaction that is required for optimal levels of V(D)J recombination.
Simkus
Carrie
C
Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC 20057, USA.
Bhattacharyya
Anamika
A
Zhou
Ming
M
Veenstra
Timothy D
TD
Jones
Jessica M
JM
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D045567:VDJ Recombinases / Q000378:metabolism*
D018398:Homeodomain Proteins; C064658:RAG-1 protein; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D045567:VDJ Recombinases