INVESTIGATING THE UBIQUITIN LIGASE ACTIVITY OF BRCA1 FROM C. ELEGANS
Herrington, Lauren
Herrington, Lauren
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2025-05-19
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BRCA1 is a tumor suppressor protein that normally acts with its partner, BARD1, to facilitate DNA repair, regulation of the cell cycle, and regulation of gene expression. The Caenorhabditis elegans homologs of BRCA1 and BARD1, BRC-1 and BRD-1, respectively, retain these key functions and thus make C. elegans a suitable model organism for studying the functions of BRCA1. While the functions of BRCA1 and BRC-1 are well characterized, the molecular mechanisms by which these functions are carried out are still unclear. For example, BRCA1 and BRC-1 possess E3 ubiquitin ligase activity towards histone H2A in nucleosomes, but it is unknown how this contributes to tumor suppression. While inherited mutations in BRCA1 that disrupt tumor suppression lack E3 ubiquitin ligase activity, they also interfere with other critical molecular functions, such as BARD1 binding. To pinpoint the role of ligase activity, we aim to characterize a mutant construct of BRC-1 in C. elegans that lacks E3 ubiquitin ligase activity towards histone H2A but retains the ability to bind BRD-1. In vitro ubiquitination assays demonstrate that our candidate for this mutant of BRC-1, Trip A, is ligase-dead towards histone H2A in nucleosomes. Co-purification of BRC-1 and BRD-1 in which only BRC-1 contained the histidine tag revealed that BRC-1:BRD-1 binding is retained in the Trip A mutant. While these results demonstrate that Trip A meets in vitro requirements for a ligase-dead mutant, further in vivo experiments are needed to confirm its suitability. If confirmed as a suitable ligase-dead mutant through in vivo experiments, Trip A can be expressed in C. elegans to identify which functions of BRC-1 depend on E3 ubiquitin ligase activity towards histone H2A in nucleosomes.
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Biology