Abstract
Recent advances in the development of functional materials offer new tools to dissect human health and disease mechanisms. The use of tunable surfaces is especially appealing as substrates can be tailored to fit applications involving specific cell types or tissues. Here we use tunable materials to facilitate the three-dimensional (3D) analysis of BRCA1 gene regulatory complexes derived from human cancer cells. We employed a recently developed microchip platform to isolate BRCA1 protein assemblies natively formed in breast cancer cells with and without BRCA1 mutations. The captured assemblies proved amenable to cryo-electron microscopy (EM) imaging and downstream computational analysis. Resulting 3D structures reveal the manner in which wild-type BRCA1 engages the RNA polymerase II (RNAP II) core complex that contained K63-linked ubiquitin moieties—a putative signal for DNA repair. Importantly, we also determined that molecular assemblies harboring the BRCA15382insC mutation exhibited altered protein interactions and ubiquitination patterns compared to wild-type complexes. Overall, our analyses proved optimal for developing new structural oncology applications involving patient-derived cancer cells, while expanding our knowledge of BRCA1’s role in gene regulatory events.
Highlights
Mutations in the breast cancer susceptibility protein (BRCA1) are known to contribute to cancer induction.[1,2] At the molecular level, the intricate details of these events are poorly understood
RNA polymerase II (RNAP II), BRCA1, and BARD1 contained in the nuclear material of HCC70 cells were naturally enriched and co-eluted from Nickel–Nitrilotriacetic acid
In the eluted fractions we found that wild-type BRCA1 associated with BARD1 and the RNAP II large subunit (RPB1) as determined by co-immunoprecipitation experiments
Summary
Mutations in the breast cancer susceptibility protein (BRCA1) are known to contribute to cancer induction.[1,2] At the molecular level, the intricate details of these events are poorly understood. We resolved the position of the BRCA1 C-terminal domain (BRCT) with respect to the RNAP II core, and distinguished the level of structural variability present in the biological samples. We employed computational procedures[11,12] to separately determine the positions of where the BRCA1 structural domains interacted with the RNAP II assemblies, based on antibodylabeling results.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
