Biochemical reconstitution of antigen receptor gene regulation

National Institute On Aging

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Abstract

During FY13, we accomplished the following: 1) Continued with transcriptional pausing experiments using several VH promoters and in vitro transcription reactions in BJAB human B cell extracts. We used pulse-chase protocols to distinguish between polymerase pausing and stalling. 2) To test the model that single-stranded DNA generated as a consequence of transcriptional pausing served as a substrate for the activation-induced deaminase (AID) enzyme, we developed conditions to simultaneously carry out both reactions. However, differences in biochemical optima for transcription and deamination activities ultimately forced us to abandon this experimental route. Instead, we initiated studies to detect single-stranded DNA using potassium permanganate modification of transcription reactions. Similar studies were initiated in B cells undergoing AID-induced rearrangements in our collaborator, Dr. Patricia Gearharts laboratory. 3) We collaborated with Dr. Stephen Desiderio (Johns Hopkins) to examine the effects mutated RAG2 protein on recombination signal sequence recognition and DNA cleavage using nucleosomes assembled with recombinant and specifically modified histones.