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.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$798,508
Indirect Cost
City
State
Country
Zip Code
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
Grundy, Gabrielle J; Ramon-Maiques, Santiago; Dimitriadis, Emilios K et al. (2009) Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex. Mol Cell 35:217-27
Longo, Nancy S; Grundy, Gabrielle J; Lee, Jisoo et al. (2008) An activation-induced cytidine deaminase-independent mechanism of secondary VH gene rearrangement in preimmune human B cells. J Immunol 181:7825-34