In the past year, we have 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. 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 this problem. Our first observation was that full-length RAG1 is ubiquitylated with the help of a different ubiquitin transferase (E2) enzyme, UbcH5, than the RING fragment, which required Cdc34. Cdc34, in turn, is inactive on the full-length protein, revealing once again the uncertainties imposed by work with protein fragments. Our second observation was that some but not all cleavage reactions of RAG1/2 are modulated by ubiquitylation. In order to quantitate these results, it is necessary to purify the fully modified form of RAG1, and this effort is now under way. A separate effort is devoted to identifying other proteins that bind to the RAG1-RAG2 complex in cells. In an earlier year, a preliminary list of candidate proteins had been obtained by mass spectrometry and confirmed by co-immunoprecipitation studies. This group of proteins included some interesting DNA repair factors, among which we chose poly-ADP-ribose-polymerase 1 (PARP-1) for further study. In vitro, PARP-1 modifies both RAG1 and RAG2, but interestingly RAG2 is modified only in the presence of RAG1, implying some significant specificity in the process. In cellular experiments, we found that inhibitors of PARP-1 interfere with some but not all V(D)J recombination reactions. These observations are now being pursued.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$518,716
Indirect Cost
City
State
Country
Zip Code
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
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
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