Probing interactions of the BRCT protein family with damaged chromatin

• Author / Creator

  Kulepa, Ayodeji N

• The DNA damage response (DDR) is critical for maintaining genomic integrity and prevention of genotoxic consequences leading to carcinogenesis. The DDR signaling cascade results either in the activation of the repair pathway, or initiation of apoptosis if the repair is not possible. Many hereditary and sporadic forms of human cancers have been linked to mutations in key DDR genes such as tumor protein p53, Ataxia telangiectasia-mutated kinase (ATM) and Breast cancer1 (BRCA1). In the early stages of DDR, the histone variant H2AX is phosphorylated at serine 139 to form γH2AX. This modification leads to the recruitment of Mediator of DNA damage checkpoint protein 1 (MDC1) which specifically interacts with the pSxxY-COOH motif of γH2AX through its BRCT domain. The structural dynamics underlying the interaction of MDC1 with the chromatin remains to be deciphered. We have recombinantly expressed and purified histone octamers with H2AX and aimed to phosphorylate the C-terminal tail. This will help provide insights into the MDC1-nucleosome interaction.
  Another BRCT domain protein, BRCA1 is a widely known tumor suppressor protein involved in the homologous recombination (HR) pathway for repair of DNA double-strand breaks (DSB). Mutations in the phosphopeptide binding domain of BRCA1 are associated with increased breast cancer risks and lead to defects in the DDR, sensitizing cells to radiation and many DNA-targeting cancer therapies. The finding that BRCA1 mutations impact HR and sensitize cells to the single strand break repair enzyme poly (ADP) ribose polymerase (PARP) has led to promising approaches to develop BRCA1 BRCT inhibitor for combinatorial therapy with PARP inhibitors. This approach can be used to target cancerous cells with the wild-type BRCA1 and prevent HR repair of DNA lesions from radio and chemotherapy treatments. Our collaborators recently developed a potential BRCA1 BRCT inhibitor that might lead to new avenues for anticancer therapy development. The inhibitor is a peptide which binds specifically to the BRCA1 BRCT domain on the same surface as the phosphorylated peptides. We tested the ability of this inhibitor to block binding of BRCA1 BRCT to a model binding peptide containing the pSer-x-x-Phe motif. Specificity is also tested by the ability of this inhibitor to block interactions between a related BRCT protein MDC1 and its peptide target pSer-x-x-Tyr-COO-. We performed in vivo experiments to determine the permeability of this inhibitor in U2OS cells. The ability of the inhibitor to disrupt BRCA1 interaction with the protein Abraxas was visualized. The peptide was expressed in U2OS cells, and its kinetics visualized by Fluorescence recovery after photobleaching (FRAP). Overall, we prove the specificity of our inhibitor to BRCA1 BRCT and confirm its permeability in U2OS cells.

• Subjects / Keywords

  • BRCT
  • BRCA1
  • Chromatin
  • DNA
  • DNA damage

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-n46f-5c52

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.