Date of Award
Doctor of Philosophy (PhD)
The cell’s ability to maintain genome integrity is largely dependent on a complex network of proteins that respond to DNA damage and replication stress. Consequently, loss-of-function mutations in DNA repair genes are common drivers of oncogenesis and hereditary diseases. Tumor suppressors BRCA1 and BRCA2 exemplify this phenomenon, as inherited mutations in either gene confer elevated lifetime risks of breast and ovarian cancer. BRCA1/2 deficient tumors exhibit defects in double strand break (DSB) repair and replication fork protection (RFP), which renders them sensitive to PARP inhibitors and DNA damaging chemotherapeutics. These characteristics describe the “BRCAness” phenotype, which can be recapitulated when other repair proteins and signaling pathways are inhibited. Therefore, the discovery of druggable “BRCAness” factors harbors tremendous therapeutic potential. In this dissertation I show that TPX2/Aurora A, a mitotic kinase complex, promotes BRCA1 functions by binding and negatively regulating 53BP1. Loss of TPX2/Aurora A causes defects in BRCA1 and RAD51 foci formation, homologous recombination repair (HRR), and replication fork protection, and confers sensitivity to hydroxyurea (HU) treatment. These BRCAness phenotypes are reversed upon loss of 53BP1, and occur through a distinct pathway from BRCA1. Additionally, this work identifies novel roles for chromatid cohesion factors in regulating replication fork progression and fork restart. Loss of Pds5 or Wapl causes significant slowing of the replication fork, a failure to restart replication following replication stress, and a loss of cohesin loading behind active replication forks. Depletion of the core cohesin-ring component Rad21 rescues these defects, suggesting that faithful DNA replication and genome stability requires the proper re-distribution of cohesin rings by Pds5 and Wapl. Finally, I describe a novel mass spectrometry- based method for analyzing replication fork dynamics called MS-BAND (Mass Spectrometry-Based Analysis of Nascent DNA). This technique may provide an easier, faster, and more objective replacement for traditional DNA fiber analysis. Future work may use MS-BAND to screen compounds and gene knockouts for replication fork defects, leading to the discovery of novel chemotherapeutic targets. All together, this dissertation identifies novel BRCAness modulators and lays the groundwork for developing more effective cancer treatments.
Chair and Committee
Jeffrey Bednarski, Douglas Chalker, Nupam Mahajan, James Skeath,
Byrum, Andrea Karl, "Identifying Novel Regulators of Double Strand Break Repair and Replication Fork Protection" (2019). Arts & Sciences Electronic Theses and Dissertations. 1992.
Available for download on Friday, December 15, 2119