This year our studies on the detailed structure of the RAG1-RAG2 protein complex have made considerable progress. We succeeded in preparing crystals of a complex of the catalytically essential core portions of both proteins, together with their DNA substrate, specifically the complex after completion of the DNA cleavage reaction. A structure sufficiently detailed to enable the identification of the catalytically active site, the probable DNA-binding regions, and many other features, including the locations of important mutations, has been obtained. The structure generally confirms the outlines that were visualized five years ago by electron microscopy, but now with much higher resolution and specificity. Current work is aimed at clarifying the position of DNA in the complex, and also at obtaining structures of other stages of the reaction. We have also continued our studies of the effects of auto-ubiquitylation of RAG1 on a particular lysine residue (K233) within the RAG1 N-terminal region. Having found that the in vitro DNA cleavage activity of RAG1/2 is stimulated several-fold by this modification, we have now extended this work to assess the complete V(D)J recombination reaction inside cells. RAG1 auto-ubiquitylation is found also to be important for this recombination, both for DNA cleavage and for a later stage of the repair process that completes the rejoining of the DNA products. Together with our recent work showing stimulation of recombination by phosphorylation of RAG1, these results make it evident that V(D)J recombination is modulated by several metabolically significant protein modifications.
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| Dayal, Sandeep; Nedbal, Jakub; Hobson, Philip et al. (2011) High resolution analysis of the chromatin landscape of the IgE switch region in human B cells. PLoS One 6:e24571 |
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