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Title: Mechanisms underlying genome maintenance and tumour suppression by BRCA2
Author: Chadha, Saakshi
ISNI:       0000 0004 7426 5597
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Inheritance of germline pathogenic mutations in a single BRCA2 allele predisposes carriers to cancers of the breast, ovaries, pancreas, prostate and other tissues. BRCA2 encodes a complex protein of 3418 residues, with multiple distinct regions implicated in different cellular processes including homologous recombination, replication stress tolerance, maintenance of the G2 and spindle assembly checkpoints, and in completion of cytokinesis. Much evidence suggests that BRCA2 acts as a tumour suppressor by maintaining genome integrity through its functions during DNA replication, repair and chromosome segregation. Neither the mechanisms underlying these functions, nor their significance to tumour suppression, is well understood. In this thesis, I report studies that reveal two novel mechanisms underlying genome maintenance and tumour suppression by BRCA2. The best-studied cellular function of BRCA2 is its role in double strand break repair by homologous DNA recombination, wherein BRCA2 controls the DNA substrate selectivity and activity of the recombinase, RAD51. This control is exerted via evolutionarily conserved BRC repeats motifs, each comprising ~35 residues. BRCA2 homologues in simple organisms such as Caenorhabditis elegans or Ustilago maydis contain only one BRC repeat. By contrast, all known vertebrate BRCA2 orthologues contain 8 BRC repeats whose sequence as well as spacing is conserved. The functional significance of the conserved sequence and spacing of vertebrate BRC repeats in the maintenance of genome integrity is unclear. I report that the BRC1 and BRC4 repeats in chicken BRCA2 are essential for cell viability. Additionally, the BRC6 repeat has an essential, non- redundant role in tolerating replication stress by stabilizing stalled replication forks and preventing the degradation of nascent DNA by MRE11, and the mechanism underlying this function is largely RAD51-independent. Furthermore, my results imply that the BRC repeats in chicken BRCA2 are aligned in the same order as in the human BRCA2, suggesting the BRC6 repeat in chicken BRCA2 may correspond to the BRC6 repeat in human BRCA2. 2 ABSTRACT Although both alleles of BRCA2 must be inactivated to provoke defective homologous DNA recombination, accumulating evidence suggests that BRCA2 heterozygosity suffices for carcinogenesis in murine models and human patients. In a second line of research, I have explored in collaborative studies how heterozygous truncating mutations affecting BRCA2 may promote carcinogenesis. I report that in unchallenged conditions, BRCA2 heterozygous cells appear to be as functionally proficient in key cellular processes as the BRCA2 wildtype cells. However, BRCA2 heterozygosity confers selective sensitivity to replication stress induced by exposure to physiological concentrations of formaldehyde, a known genotoxic agent. Replication fork degradation is observed in BRCA2 heterozygous cells on exposure to formaldehyde, but not hydroxyurea, and this deleterious phenotype has been attributed to the ability of formaldehyde to induce BRCA2 degradation in a proteasome-dependent manner, thereby inducing haploinsufficiency. Overall, my thesis research reveals two novel mechanisms by which BRCA2 maintains genomic integrity. Identification of a novel function for the BRC6 repeat in chicken BRCA2 indicates that individual BRC repeats may have essential functions that are yet to be identified, thereby providing an explanation for the evolutionary conservation of the 8 BRC repeats observed in vertebrate BRCA2. Furthermore, results linking BRCA2 heterozygosity and formaldehyde exposure to increased genomic instability highlights the potential ability of aldehyde exposure in promoting carcinogenesis in heterozygous BRCA2 mutation carriers, which has significant preventive and therapeutic implications.
Supervisor: Venkitaraman, Ashok Sponsor: Cambridge Trust ; Cambridge Philosophical Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: BRCA2 ; cancer ; RAD51