Biochemical reconstitution of antigen receptor gene regulation

National Institute On Aging

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Abstract

During FY15 we accomplished the following: 1) We continued studies of 12/23 RSS-dependent RAG 1/2 cleavage using chromatin assembled templates. For these studies we purified all combinations of core and full length RAG1 and RAG2 proteins, as well as PHD domain mutated RAG2. These proteins were used in cleavage assays using plasmids assembled with recombinant (unmodified) core histones or with histone H3 trimethylated at position 4. 2) We generated RAG1- and RAG2-Halo fusion proteins for visualization of recombination centers in living cells. These RAG derivatives were tested for function by expression in 6312 pro-B cells followed by analysis of DH recombination. Visualization studies have been initiated using spinning disk confocal microscopy.