Telomere Capping Protein Stn1p Require the RAD6-Mediated Ubiquitination Pathway for DNA Repair and Function in Global DNA Replication
- Author(s): Tran, Tim Quoc
- Advisor(s): Nugent, Connie
- et al.
Current models suggested that the CST complex has a dual role in the budding yeast. First, it has a physical capping mechanism that prevent exonucleolytic degradation, and recombination of telomeres. Secondly, it facilitates the efficient replication of chromosome ends by controlling the activities of telomerase and Pol-α primase. The work in this thesis shows that RAD6 is require for DNA post replication repair at the telomere when STN1 is compromised. Rad6p is an E2 ubiquitin conjugating enzyme that, through its interactions with E3 ubiquitin ligases, promotes DNA post-replication repair via Rad18p as well as histone modification via Bre1p. Histone modification via Bre1p is also require for telomere maintenance and recombination repair. Our objective is to design experiments to show that both E3 ubiquting ligases. through its interaction with Rad6p, promote DNA post-replication repair and recombination repair. In addition, Stn1p has additional role in facilitating DNA replication at non-telomeric sites in the genome (Stewart et al, 2012). The overall hypothesis in this thesis is that Stn1p require Rad6p for post replication repair of single stranded gaps left behind by loss of function of STN1 and it may have non-telomere role.
I have shown that the genetic interaction between STN1 and RAD6 is synthetically lethal by constructing the double mutant combinations. The percentage of viable spores from the double mutant stn1-281 rad6∆ is extremely low and synthetic. I have shown that the double mutants stn1-281 rad6 ∆have a synthetic phenotype caused by the replicative problems arising at repetitive sequences, such as the rDNA locus. The double mutants stn1-281 rad18∆ show that cells are very sick, suggesting that post-replication repair at the telomere is essential under defective STN1 background. Interestingly, the double mutant stn1-281 bre1∆ revealed a greater synthetic phenotype, suggesting that histone modification is essential for repair when STN1 is compromised. Beside STN1, defects in Ten1 also showed synthetic interaction. The double mutant ten1-105 rad6∆ showed negative synthetic interaction. The genetic analysis has revealed that both Stn1p and Ten1p mutants require all three arms of the RAD6 pathways involving post-replicative repair (PRR) and homologous recombination repair (HRR) pathways for cell viability. This suggested that Stn1p and Ten1p may have a role in facilitating DNA repair at damaged sites, presumably single stranded gaps. Another portion of this thesis is trying to show that Stn1p may have nontelomeric role elsewhere in the genome.
The question of whether Stn1p has nontelomeric functions in DNA repair and maintenance have also been explored. Pulse field gel electrophoresis has shown that Stn1p mutants have fragmented or shattered chromosomes when treated with S1 endonuclease, suggesting that there are single-stranded DNA gaps outside of telomere. In addition, the double mutants stn1-281 rad18∆ and stn1-281 bre1∆ have fragmented chromosomes when treated with S1 endonuclease. The Klenow synthesis assay has detected single stranded DNA gaps in the Stn1p mutants at repetitive regions. These data have suggested that Stn1p may have nontelomeric function outside of the telomere such as DNA replication and repair.