In partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology

In the

School of Biological Sciences

Wenying Guo

Will defend her dissertation

Multiple roles of the telomeric CST complex in preserving genome stability at sub-telomeric and difficult-to-replicate chromosomal regions

Apr. 29. 2022

10 am EST

In-person: EBB Children’s Healthcare of Atlanta (CHOA) Seminar Room

Via Zoom:

https://gatech.zoom.us/j/5710176492?pwd=dTZ5V3ltb3Z1YWh4OWVpZ1BBRXlXQT09

 Thesis Advisor:

Kirill Lobachev, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Francesca Storici, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Matthew Torres, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Yury Chernoff, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Loren Williams, Ph.D.

School of Chemistry and Biochemistry

Georgia Institute of Technology

ABSTRACT:

The CST is an essential multiprotein complex composed of Cdc13, Stn1, and Ten1 in budding yeast and CTC1, STN1, and TEN1 in vertebrates. The complex is involved in telomere maintenance which is critical to genome stability. The complex binds to the telomeric single-stranded (ss) DNA overhangs and ensures proper telomere capping. Recent evidence suggests that the mammalian CST performs non-telomeric functions during replication stress. In my work, using budding yeast, I investigated how CST deficiency can affect genome instability at sub-telomeric and non-telomeric regions. In the first part, I used the CDC13 defective allele I identified, cdc13-707fs. I found that upon Cdc13 deficiency, stretches of ssDNA can reach up to 55 kb. I further characterized the consequences of ssDNA exposure and found that exposed ssDNA is prone to hyper-mutability mediated by the translesion polymerase ζ during restoration synthesis. I determined the mode of restoration synthesis and found that it proceeds from the telomere towards the centromere. In the second part, I found that ssDNA generated as a result of telomere uncapping and 5’ → 3’ resection favors the cis interaction between inverted repeats (IR) located 43 kb away from the telomere. Using a set of different inverted repeats, I showed that the parameters that are known to constrain their fragility, namely divergency and long spacers, are not effective in cdc13-707fs. Importantly, I showed that the IR-mediated chromosomal rearrangements upon CST deficiency do not result from an increase in DNA breakage but rather from a DNA fold-back mediated by the annealing of the ssDNA followed by dicentric chromosome formation, its breakage in anaphase and subsequent repair events. I characterized the genetic dependency of IR-mediated fragility in cdc13-707fs and found that the ssDNA annealing proteins Rad52 and Rad59, as well as the structure-specific nucleases Slx4 and Rad1, are required for intrastrand interaction and inverted dicentric dimer formation. In the third part, I explored the non-telomeric functions of CST, which has never been studied in budding yeast. I found that CST deficiency leads to an increase in chromosomal breakage and recombination at inverted repeats located 343 kb away from the telomere. This suggests that the CST complex plays an important role at difficult-to-replicate chromosomal regions. These findings in yeast have implications for understanding mechanisms that shape eukaryotic genomes during evolution and development and mechanisms of hereditary diseases and tumorigenesis.