Structural investigation of the ubiquitylation of nucleosome core particles by the E3 ligase BRCA1/BARD1
Date of Issue2019-07-12
School of Biological Sciences
Based on the reports from the American Cancer Society, cancer is the leading cause of death in the world, followed by heart diseases. Scientific methods and technology for early detection and intervention would save not only millions of lives but also billions of dollars. Among the types of cancers, according to the World Health Organization, breast cancer is the most common type of cancer in women worldwide. Statistics show that one in eight women is diagnosed with breast cancer, and eight out of 10 women do not have a family history of the disease. Approximately 10% of the breast cancers can be traced back to inherited mutations in the BRCA1 and BRCA2 genes, most of the mutations occurring in BRCA1. Though BRCA1 was found to be the dominant gene involved in breast cancer, only recently insights into how BRCA1 protein-dependent tumor suppression occurs, has started to emerge. A potential tumour suppressor that heterodimerizes with BRCA1 in vitro and in vivo, increasing the stability of BRCA1 is BARD1. The heterodimerization of the BRCA1 and BARD1 tumour suppressor proteins are mediated by the N-terminal RING domains of BRCA1 and BARD1. Ample information is available regarding the diverse functions and characteristics of BRCA1 and BARD1 tumor suppressor proteins, but only in the context of cell biology and biochemistry. Even though structural information on the RING domains and BRCT domains of BRCA1 and BARD1 proteins themselves is available, there is no structural information on the mechanism by which BRCA1-BARD1 heterodimeric complex functions as a chromatin remodeler. Results over the last two decades have provided evidence that histone post-translational modifications (e.g., acetylation, methylation, and ubiquitylation) play a vital role in regulating numerous processes within the eukaryotic cell, such as transcriptional regulation, DNA replication, and repair, leading to genomic instability and tumor progression. In recent years, H2A specific ubiquitin ligases have been discovered and link H2A ubiquitylation (H2AUb) to gene silencing and DNA damage repair. A critical E3 ligase involved in DNA break repair is the tumor suppressor BRCA1. It is well known that mutations in BRCA1 are associated with the occurrence of breast and ovarian cancer. Prominent amongst the many in vivo identified interaction partners of BRCA1 is the interaction of BRCA1 with the BRCA1-associated RING domain protein 1 (BARD1). BRCA1 and BARD1 interact with each other to form a heterodimeric RING-RING complex that ubiquitylates K125, K127, and K129 explicitly in the C-terminal tail of histone H2A in the human nucleosome. Complete understanding of the mechanism by which the chromatin remodelers function and bring about a specific biological outcome is yet to be accomplished. Structurally determining the highly dynamic interactions of E3 ubiquitin ligases with related proteins and their substrates has also proved to be a challenge for a mechanistic investigation into these complex systems, which is further complicated by the technical difficulty in isolating homogenous samples. The latest advancements in the field of cryo-electron microscopy (cryo-EM) are proving helpful. I have used cryo-electron microscopy and biochemical techniques, to understand the mechanism of interaction between BRCA1-BARD1 heterodimer and nucleosome core particle (NCP) to specifically modify K125, K127, and K129 in the C-terminal tail of H2A. Determining the mechanism of interaction at the molecular level would shed light on mechanisms involved to recognize, alter chromatin and how mutations in these proteins are involved in the onset of diseases, cancer. The structural information along with biochemical data and data from functional studies would help in developing novel therapeutic compounds that could target diseases like cancer. In this dissertation, I will describe approaches taken towards the expression and purification of various BRCA1 and BARD1 constructs, characterization of the BRCA1-BARD1 heterodimer in the nucleosomal context, recognition of the NCP, and preliminary structural information using cryo-EM on how the heterodimeric complex binds to the NCP is presented.