The Many Functions of STN1 in Telomere Regulation and DNA Replication
- Author(s): Caridi, Christopher Patrick
- Advisor(s): Nugent, Connie
- et al.
Telomeres protect linear chromosomes and serve a critical function maintaining genomic stability. Telomeres serve three main functions that include preventing inappropriate chromosome fusions, blocking nuclease activity and restoring sequence lost from the end replication problem. In Saccharomyces cerevisiae, three end binding proteins, Cdc13, Stn1 and Ten1, are responsible for some of the functions of telomeres. All three proteins appear to serve a protective role as all three CST proteins are essential. Loss of any of the components of CST leads to increased single stranded telomeric DNA and activation of the DNA damage checkpoint. Cdc13 recruits telomerase, a reverse transcriptase that extends the terminal 3’ single stranded overhang. Cdc13 and Stn1 also interact with DNA polymerase α, which is the primary polymerase responsible for synthesizing the complementary strand of the template made by telomerase. Telomerase activity is probabilistic and increases with decreasing telomere length. Telomerase is a unique enzyme that lacks in vitro processivity but can obtain in vivo processivity at short telomeres through a Tel1 dependent process. Yet, evidence indicates the regulation of telomerase activity is tightly coupled with DNA polymerase α. How DNA polymerase α and telomerase action are coordinated is currently unknown. This dissertation sheds some light on the effect of DNA polymerase α on telomerase processivity and activity. It provides evidence that DNA polymerase α has a complex interaction with telomerase, affecting both telomerase processivity and activity.
This dissertation also sheds light on the exciting possibility that Stn1 facilitates semiconservative DNA replication. A fascinating observation presented in this dissertation is the non-telomere associated damage that accumulates in stn1 loss of function mutants. Prior observations show that stn1 mutants are synthetic with temperature sensitive ddk mutants. Conversely, over expression of STN1 can alleviate some of the phenotypes of temperature sensitive ddk mutants. This dissertation shows that the DDK physically interacts with Stn1. Furthermore, the target of this interaction appears to be the MCM helicase, the essential target of the DDK. Over production of STN1 enhances Mcm4 phosphorylation and loss of function stn1 mutants reduce Mcm4 phosphorylation. Importantly, alleles of MCM4 that allow for DNA replication in the absence of the DDK alleviate some of the defects in stn1 mutants. Thus, evidence strongly indicates that STN1’s role in DNA replication is to facilitate DDK dependent phosphorylation of Mcm4.