Further progress has been made in the past year in our research program aimed at understanding the functions of the RAG1-RAG2 protein complex in molecular detail. In 2015, we reported the first crystal structure of the RAG1/RAG2 complex. This was a major advance, making it possible for the first time to give an explanation of the so called 12/23 rule in V(D)J recombination, which forces it to work on two different DNA sites. But this was a structure of the protein complex alone, without DNA, which left many questions about mechanism unanswered. It was necessary to obtain detailed information on structures containing DNA, and to study them at all stages of the cleavage reaction. As the DNA cleavage occurs in several defined steps, at least four distinct species of the RAG-DNA complex can be obtained. Crystals of most of these species were obtained and their structures were refined. In addition, we used the technique of cryo-electron microscopy to obtain further information. Combining these techniques, we found that an essential step in RAG-mediated DNA cleavage is the unwinding of several base pairs of DNA adjacent to the recombination signal sequence. This unexpected step may also be found in related enzymes of the transposase class. This work has been published. We are also continuing our work on longer versions of the RAG proteins, which contain regulatory elements controlling RAG activity.
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| 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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| Gupta, Shikha; Gellert, Martin; Yang, Wei (2011) Mechanism of mismatch recognition revealed by human MutS? bound to unpaired DNA loops. Nat Struct Mol Biol 19:72-8 |
| 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 |
| Grundy, Gabrielle J; Yang, Wei; Gellert, Martin (2010) Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proc Natl Acad Sci U S A 107:22487-92 |
| Grundy, Gabrielle J; Ramón-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 |
