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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000374-06
Application #
8736547
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$380,661
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Ishii, Haruhiko; Du, Hansen; Zhang, Zhaoqing et al. (2009) Mi2beta shows chromatin enzyme specificity by erasing a DNase I-hypersensitive site established by ACF. J Biol Chem 284:7533-41
Du, Hansen; Ishii, Haruhiko; Pazin, Michael J et al. (2008) Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription. Mol Cell 31:641-9