Lymphocytes are white blood cells that are part of the adaptive immune system. The adaptive immune response is triggered by the invasion of pathogens, such as bacteria and viruses. When this occurs, special receptors called immunoglobulins and T-cell receptors are made. A tremendous number of different types of pathogens exist so it is necessary for the immune system to be able to produce thousands of different receptors that recognize different invaders. However, there is not enough genetic material in the human genome to account for the wide diversity of these receptors that fight infection. Nature has created a way to diversify this adaptive immune system using a process called V(D)J recombination. In V(D)J recombination, there are two proteins known as RAG1 and RAG2 that interact with DNA recognition sequences (RS) in the pool of V, D, and J genes that are used to make immunoglobulins and T-cell receptors produced by the different lymphocytes. The main objective of this study is to correlate the binding, cleavage, and structural responses of the RS to the RAG proteins to understand the nonrandom usage of V, D, and J gene segments for recombination.
These aims will be accomplished by measuring the thermodynamic parameters of RAG-RS binding to determine relative specificities. Protein-DNA binding will be determined from fluorescence anisotropy and isothermal titration calorimetry. The kinetics of binding and cleavage will also be determined to understand the properties that govern these interactions. In addition, the role of the HHE Zn(ll) site for recombination will be investigated. Our goal is to understand the nonrandom usage of V, D, and J genes to create a diverse repertoire of immunoglobulin and T-cell receptors in the immune system. Moreover, if RAG proteins interact with DNA that does not contain the normal RS, then any gene can be moved around to other parts of the genome, which in turn can lead to the initiation of lymphomas. The knowledge from this project will be useful in the design of therapies to combat cancers caused by incorrect RAG-RS interactions that lead to aberrant V{D)J recombination. ? ? ?
| Zhao, Shuying; Gwyn, Lori M; De, Pallabi et al. (2009) A non-sequence-specific DNA binding mode of RAG1 is inhibited by RAG2. J Mol Biol 387:744-58 |
| Gwyn, Lori M; Peak, Mandy M; De, Pallabi et al. (2009) A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity. J Mol Biol 390:863-78 |
