Telomeres stabilize the genome by distinguishing natural chromosome ends from DNA damage (end protection) and by facilitating replication of terminal DNA tracts via telomerase (telomere maintenance). These functions are executed through a dynamic molecular switch that converts the chromosome terminus from a fully protected (telomerase non-extendable) conformation into an open (telomerase-extendable) state. The molecular basis of the switch is unknown, but it must be strictly controlled as inappropriate exposure of the terminus or failure to fully replicate it leads to stem cell disease and cancer. In this renewal application we exploit the model eukaryote Arabidopsis to reveal new insight into the interplay between telomerase and the CST (CTC1/STN1/TEN1) telomere protein complex. The application builds on important discoveries from the previous funding period that include: 1) an alternative telomerase RNP complex with protein binding partners implicated in chromosome end protection; 2) a dynamic switch between a telomerase accessory factor POT1a, which positively regulates telomerase, and TEN1 a core component of CST, which negatively regulates telomerase; and 3) a non-canonical telomerase RNA (TER2) that negatively regulates telomerase enzyme activity in response to DNA damage. The central hypothesis of this application is that telomerase components together with CST orchestrate the assembly and dynamic exchange of end protection and replication complexes to promote telomere stability. This hypothesis will be tested through two specific Aims that employ a combination of biochemical, genetic and cell biological approaches.
Aim 1 will investigate how blunt-end telomeres are stabilized. This work will focus on the roles of Ku, POT1b and POT1c in telomere protection, and will test the hypothesis that a processed form of TER2, TER2s, serves as an RNA scaffold to cap blunt-end telomeres.
Aim 2 will define the dynamic interactions between CST, telomerase accessory proteins and the TER2 regulatory RNA to elucidate how these factors control telomere synthesis by telomerase.
Aim 2 focuses on the roles of TEN1 and TER2 in terminating telomerase-mediated telomere elongation. The impact of these studies is that they will establish new paradigms for telomere protection and telomerase regulation, and yield insight into the emerging roles of long noncoding RNA in promoting genome integrity. In a broader sense, the results will explain how rapid evolution of telomere components can be used to achieve common mechanisms of telomere homeostasis as well as new modes of telomere regulation. Since it is likely that the sophisticated mechanisms of telomerase regulation discovered in Arabidopsis are conserved, completion of these aims will provide important new information about telomerase and CST related human disease.
Telomeres are essential for genome integrity and as a consequence, understanding how the telomere complex safeguards genome stability is crucial for elucidating the fundamental mechanisms that promote stem cell survival and impede carcinogenesis. Studies in model organisms established the paradigms for human telomere biology, and continue to uncover novel telomere components and regulatory mechanisms. In this tradition, we will exploit the genetic tractability of Arabidopsis and its extreme tolerance t telomere dysfunction to investigate mechanisms of telomere protection and telomerase regulation in multicellular organisms.
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