10.1021/bi801115g.s001 Ross A. Edwards Ross A. Edwards Megan S. Lee Megan S. Lee Susan E. Tsutakawa Susan E. Tsutakawa R. Scott Williams R. Scott Williams John A. Tainer John A. Tainer J. N. Mark Glover J. N. Mark Glover The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50 American Chemical Society 2015 phosphopeptide binding pocket WD CstF p 53 stabilization BARD 1 architecture DNA damage signals protein BARD 1 complexes BRCA BRCT domains DNA damage BARD 1 BRCT domain uncovers BARD 1 deletion mutants BARD 1 RNAP II stability 2015-12-16 15:23:31 Journal contribution https://acs.figshare.com/articles/journal_contribution/The_BARD1_C_Terminal_Domain_Structure_and_Interactions_with_Polyadenylation_Factor_CstF_50/2010990 The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.