A Structural Approach to Unveil the Role of BRCA1 in the Context of Transcription

The research presented in this thesis aims to uncover the intricate manner in which BRCA1 interacts with RNAP II during mRNA production utilizing a unique microchip system developed in our lab. We were first able to prove the effectiveness of our tunable system using a breast cancer model of patient derived triple negative breast cancer (TNBC) cells. Here we switched out different mammalian antibodies and collected images of the same structure from different angles. This served many purposes: (1) it proved the system could be tuned for specific uses; (2) it demonstrated that all subunits were present in the complex; (3) it eliminated the need for the tilt function allowing for a less intensive computational procedure. In the BRCA1 wild type cell line we were able to incorporate into our 3D reconstruction, atomic models of the BRCA1-BARD1 heterodimer and the RNAP II core in regions of major unoccupied density. Other areas of minor missing density were overlaid with a short strand of DNA and ubiquitin moieties, which proved agreement with Co-IPs. Next we sought to compare the wild type structure with a BRCA1 mutant variety. Using these techniques, we determined the 3D structure of the mutated complex. After further analysis slight differences were detected between the two complexes, especially in the placement of the atomic models. Overall we were able to determine structural abnormalities that occur when a mutation is present in BRCA1 that may have future applications for targetable therapy in TNBC patients. Moreover, by using TNBC as the disease model, we have created a platform that can be used to evaluate other human diseases due to the tunable nature of our microchip system.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Biomedical Engineering

Department dc:contributor.department

Biomedical Engineering

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2016

Author dc:creator

• Winton, Carly Elizabeth

Chair dc:contributor.committeechair

• Kelly, Deborah F.

Committee members dc:contributor.committeemember

• Poelzing, Steven
• Sheng, Zhi

• Breast cancer
• BRCA1
• electron microscopy
• transcription

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

vt_gsexam:6966

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/64477