UC San Diego

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.