Charting the Landscape of Tandem BRCT Domain–Mediated Protein Interactions

Abstract

Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes a large number of proteins responsible for detection of DNA damage, promotion of repair, and coordination with cell cycle progression. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The BRCT domain is a modular protein domain critical for relaying signals in the DDR. We performed a systematic analysis of protein-protein interactions involving tandem BRCT domains (tBRCT) in the DDR by combining literature curation, yeast two hybrid (Y2H) screens, and tandem affinity purification coupled to mass spectrometry (TAP-MS). We identified one previously unrecognized BRCT protein and generated human protein-protein interaction network for this type of modular domain. This study also reveals several novel components in DNA damage signaling such as COMMD1 and mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.

BRCA1 recruitment to damaged DNA sites is dependent on CDK9

“…1B), confirming the interaction detected using epitope-tagged tBRCT baits. 14 We also conducted reciprocal coimmunoprecipitation of endogenous BRCA1 and BARD1 in HEK293FT cell extracts and confirmed this interaction (Supplementary Figs 1A and 1B).…”

“…12,13 In a previous work, our group generated a human proteinprotein interaction network centered on interactions mediated by the tBRCT domain and identified CDK9 (cyclin-dependent kinase 9) as a putative interaction partner of BRCA1 and BARD1 tBRCTs. 14 The cyclin-dependent kinases (CDKs) participate in many DDR-related processes, such as signal transduction and cell cycle arrest. 15,16 CDK9 was originally characterized as the catalytic subunit of the positive elongation complex (PTEFb), which is responsible for promoting transcription through the phosphorylation of the C-terminal domain (CTD) of the holoenzyme RNA polymerase II.…”

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage

“…Focused analysis has identified such BRCA1 and BRCA2 interactors as BARD1 and PALB2 (Wu et al 1996;Xia et al 2006), while network analysis (Pujana et al 2007), an immunoprecipitation-based study (Wang et al 2000), and a yeast two-hybrid (Y2H) and mass spectrometry (MS)-based analysis of a BRCA1 functional motif have identified yet other BRCA1-interacting proteins (Woods et al 2012). However, gaps in the BRCA1 network likely remain, given the limited understanding of BRCA1 function.…”

An RNA interference screen identifies new avenues for nephroprotection

“…It is conserved between mammals and arthropods. Its product reportedly associates with a mediator of the cell stress response, PAXIP1, and a Rho GTPase regulator, epithelial cell transforming 2 (ECT2), 71 but its biochemical function is unknown. Considering our findings, we propose to name the gene Regulator of Ischemia and Stress Tolerance 1 (RIST1).…”