Investigating the Molecular Mechanisms Underlying Ku Essentiality in Human Cells

The Ku heterodimer is composed of two subunits, Ku70 and Ku80, and is well- known for its role in nonhomologous end-joining DNA repair. Ku has also been associated with noncanonical functions in the cell for which its mechanisms are not fully understood such as its roles in telomere maintenance, the cell cycle, and transcriptional regulation. Despite Ku’s conservation across mammalian species, Ku is essential for human cell viability but is dispensable in mice. Essentiality in human cells was previously suggested to be due to Ku’s role in maintaining human telomere length. To study the underlying cause of Ku’s essentiality in humans, a conditional Ku70 knockout TREx-293 cell line was created using CRISPR/Cas9 editing. I observed a loss of cell viability during Ku depletion but did not detect a decrease in telomere length using telomere restriction fragment assays. I used mass spectrometry to investigate proteomic changes caused by a Ku70 knockout and identified that the proteins most dysregulated following Ku depletion were associated with functions in the cell cycle and RNA regulation. These findings suggest that Ku’s essential role is not in maintenance of telomere length, but in other cellular processes. I next investigated Ku’s role in the cell cycle, using flow cytometry to determine the percentage of cells in each cell cycle phase following depletion of Ku for unsynchronised and synchronized cell populations. I found that Ku depleted cells were delayed in progressing through the cell cycle but did not accumulate at specific checkpoints and did not indicate dysregulation of a specific cell cycle phase, suggesting that Ku does not play a direct role in the cell cycle that affects viability. RNA sequencing of Ku depleted cells revealed that Ku-RNA binding with double-stranded RNA and long noncoding RNAs was heavily affected by loss of Ku and implicated the innate immune response among others. Our work may indicate a larger involvement of Ku in modulating gene expression through Ku-RNA interactions. Collectively, this work suggests that Ku-RNA binding plays a prominent role in humans and may underlie Ku essentiality in humans.

Name thesis:degree_name

Ph D

Discipline thesis:degree_discipline

Biochemistry

Grantor dc:publisher

The University of Western Ontario

Year dc:date.issued

2025

Author dc:creator

• Kelly, Rachel

Advisors dc:contributor.advisor

• Schild-Poulter, Caroline
• Edgell, David R

• Ku heterodimer
• gene essentiality
• CRISPR
• conditional knockout
• telomere maintenance
• RNA regulation

Statement dc:rights

• Attribution 4.0 International

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14721/38899

OAI identifier oai:identifier

oai:uwo.scholaris.ca:20.500.14721/38899