Telomeres, linear chromosome termini composed of long tracts of G-rich repetitive DNA, are crucial for long-term proliferation of cells. Telomere-dedicated binding factors protectagainst DNA damage response that would result in end-to-end fusions and genomic instability The importance of telomere length regulation is demonstrated by the impact of reductions in telomere length regulators on age-related disease phenomena. In cells that rely on active proliferation, the reverse transcriptase telomerase, replenishes eroded telomeric DNA to maintain average length through RNA templated sequence addition, thereby telomeres will not become critically short. Although the subject of intense investigation for over 25 years, the mechanism by which telomerase is regulated has not been determined. A long-standing model proposes that telomere length is maintained by a length-sensing mechanism that regulates telomerase to ensure that shortened telomeres are restored to an average length. This presumably relies on the ability of telomeres to interconvert between telomerase extendible and non-extendible states, but decades later, the field has not yet identified a length-sensing mechanism or how a “telomerase extendible” state is established. This model also assumes that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of chromosomes, formed after replication of telomeric DNA is complete. Our recent work has shown that in wild type cells, there is a second substrate for telomerase, created as the result of spontaneous fork collapse during duplex telomeric DNA replication. The work in this dissertation explores a novel pathway for telomere length homeostasis through regulation of replication fork collapse and the subsequent response by telomerase. Two questions have largely driven this work: how does replication fork collapse affect telomere length heterogeneity, and how does this heterogeneity affect phenotype? The work presented in this dissertation shows that replication fork collapse is a major driver of telomere length homeostasis. Fork collapse and the subsequent response by telomerase are coordinated by the dual activities of t-RPA, facilitating replisome progression through duplex telomeric DNA and recruiting telomerase to the fork. Lastly, my work reveals a potential novel pathway of telomere regulation through regulation of telomerase interaction with collapsed forks.

The S-phase checkpoint, developed by the cell in response to DNA damage, is critical to maintaining cellular function. The S-phase checkpoint operates through two separate pathways, the DNA damage checkpoint and the DNA replication checkpoint. Both pathways of the S-phase checkpoint respond to DNA damage in Saccharomyces cerevisiae, or budding yeast, through separate mechanisms both relying on two critical kinases, Mec1 and Rad53. My goal in my master’s thesis research was to work toward elucidating the details of these two kinases’ roles in both pathways of the S-phase checkpoint. To do so, I subjected highly conserved, hydrophobic residues on the surfaces of Mec1 and Rad53 to extensive mutagenesis using a novel technique designed to identify rare separation-of-function mutations, as well as traditional loss-of-function screens. By conducting a thorough genetic screen of the two kinases, I sought to aid efforts in uncovering additional protein factors involved in the S-phase checkpoint and in clarifying how the two different pathways work in conjunction to respond to DNA damage throughout the cell. Determining specifically how Mec1 and Rad53 work within the two pathways can lead to similar discoveries about the DNA damage response pathways in mammalian cells, facilitating the development of future treatments of cancer and other debilitating genetic diseases.

The ends of linear chromosomes are composed of distinctive structures that maintain chromosome stability and cellular viability. Both sequence and structure of telomeres are important to their function. They are composed of TG-rich, repetitive, non-coding sequences and present unique challenges for genomes. A balance of both shortening and lengthening pathways maintains telomere length homeostasis. Telomere length dysregulation has been linked to human aging, cancer, and disease (telomeropathies), so studying the details of how this regulation occurs is vital to our understanding of telomere biology and human health.

Telomeres have been proposed to serve two distinct functions; end protection and end replication. As end protection factors, they help distinguish the natural ends of linear chromosomes from double stranded breaks of DNA that need to be repaired by DNA repair mechanisms and prevent end-to-end fusions. Telomeric DNA is lost at every round of replication due to incomplete replication. This loss is compensated by de novo synthesis of telomeric repeats by the enzyme telomerase. A lot has been learned about the factors implicated in both these functions and how they contribute to telomere length regulation.

In the budding yeast, Saccharomyces cerevisiae, a trimeric complex composed of Cdc13-Stn1-Ten was initially proposed to be the main players in telomere “capping” and serving an end-protection function. More recently, this complex has been shown to have homology to the RPA complex and function in the efficient replication of duplex telomeric DNA, and so was aptly named t-RPA. This discovery led to multiple questions about the role of replication at telomeres and how that contributes to telomere length regulation.

This dissertation describes my efforts in identifying a new model for telomere length regulation, which revolves around the significant effects of efficient replication to telomere length homeostasis. Errors that lead to replication fork collapse within the genome can have detrimental and even lethal results for the cell. At chromosome ends, that are recognizably difficult to replicate regions of the genome, replication fork collapse has been exploited by the cell to regulate telomere length. By following the events that occur immediately after a replication fork collapse, I found that collapsed forks at telomeres are recognizes by telomerase with high efficiency and subject to extensive elongation. Moreover, this process is under genetic control. Strikingly, when I studied the effects of known telomere length regulators, I found that they were modulating telomere length through their effects on replication fork collapse. This illustrated the previously unappreciated role of replication, and supported a new model for telomere length homeostasis that is driven by replication fork collapse and the subsequent response by telomerase.

A leading objective in biology is to identify the complete set of activities that each gene performs in vivo. For many years, the telomere biology field has sought define the functional regulatory surface of telomerase, a goal that eluded many researchers due to the lack of structural data for any of the protein subunits. A major goal of this thesis work has been to develop of a rapid genetic approach that can identify amino acids on the surfaces of proteins (even in the absence of structural data) that, when mutated, surgically eliminate single biochemical activities. I used over-expression dominant negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain. This approach is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are therefore able to compete with the endogenous protein (Herskowitz 1987). To test these assumptions, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy (in collaboration with the Wuttke lab), which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts.

Using reverse mutagenesis to target highly conserved charged residues in the ODN assay has been efficient and extremely fruitful. By applying this strategy to all three protein subunits of telomerase, I have been able to define multiple biochemically distinct activities, including identifying two novel surfaces (one in Est3 and one in Est1) required for unknown regulatory functions.

I also supervised several undergraduate students and summer interns to apply the ODN strategy to various essential genes involved in DNA replication and repair, which led to the discovery of EST5. This is the first identification in over twenty years of a gene involved in the telomerase pathway that causes progressive telomere shortening and cellular senescence.

The ends of linear chromosomes are composed of a long array of highly conserved repeat sequences that are essential for genome stability. These chromosome ends, called telomeres, act as a molecular clock that dictates cell division potential. With every cell division, a small amount of DNA erodes from each chromosome terminus, and while this erosion process is initially insignificant, the cumulative effect after many cell divisions can be catastrophic, ultimately resulting in cell death or cancerous transformations. At a certain point, so much telomeric DNA is lost that a signal is sent to halt further cell division. This block to indefinite cell division can be alleviated by the enzyme telomerase which adds telomeric DNA to chromosome ends, thereby preventing telomere shortening. Telomerase has been the subject of intensive studies since its discovery, as the contribution of telomere length regulation to human health, with regard to both cancer biology and human aging, is substantial. A major goal of the Lundblad lab is to achieve a full understanding of how this enzyme is regulated, in order to uncover novel insights into how chromosome ends can make such wide-spread contributions to human biology. In budding yeast, a telomeric dedicated RPA complex (the t-RPA complex), formed by the three essential proteins Cdc13, Stn1 and Ten1 is central to ensuring telomere homeostasis as it ensures the elongation of the telomeres by recruiting the cellular retro-transcriptase telomerase. Furthermore, it promotes efficient replication of the telomere as a telomeric specific complex that becomes part of the replisome as the replication fork advances through the duplex telomeric DNA. This dissertation describes my efforts to uncover how a third complex, the canonical RPA complex, performs a role in telomere maintenance with t-RPA and telomerase.