This year we continued to study the activities of the RAG1-RAG2 protein complex, specifically focusing on the effects of modifications of these proteins. Some years ago, our group showed that the N-terminal region of RAG1 contains a RING finger domain with ubiquitin ligase (E3) activity;this fragment was able to modify a nearby lysine residue (K233) within RAG1. At that time we could not determine the effects of this alteration on the DNA cleavage activity of RAG1/2 , because it was not possible to purify full-length RAG1 (we were using an active form that was missing the N-terminal region). With our present ability to obtain active full-length RAG1, we have returned to studying the effect of ubiquitylation on the DNA cleavage activity of RAG1/2. Extending previous results, now with the ubiquitylated form of the enzyme fully purified, we find that cleavage activity is stimulated by several-fold. Efforts to assess the effect of this modification inside cells are under way. In a separate investigation (in collaboration with Dr. Jay Chung, NHLBI), we found that RAG1 is phosphorylated at a specific site by AMP-activated protein kinase, and that this modification also increases the activity of RAG1/2, as well as increasing V(D)J recombination in cells. It is becoming evident that V(D)J recombination is modulated by several metabolically significant pathways.

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Kim, Min-Sung; Lapkouski, Mikalai; Yang, Wei et al. (2015) Crystal structure of the V(D)J recombinase RAG1-RAG2. Nature 518:507-11
Singh, Samarendra K; Gellert, Martin (2015) Role of RAG1 autoubiquitination in V(D)J recombination. Proc Natl Acad Sci U S A 112:8579-83
Lapkouski, Mikalai; Chuenchor, Watchalee; Kim, Min-Sung et al. (2015) Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes. J Biol Chem 290:14618-25
Um, Jee-Hyun; Brown, Alexandra L; Singh, Samarendra K et al. (2013) Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination. Proc Natl Acad Sci U S A 110:9873-8
Gupta, Shikha; Gellert, Martin; Yang, Wei (2012) Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops. Nat Struct Mol Biol 19:72-8
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