Renfrew, Kyle Brayton (2014-12). Telomere Dynamics in Arabidopsis thaliana. Doctoral Dissertation. Thesis uri icon

abstract

  • Telomeres are the physical ends of eukaryotic chromosomes. Because chromosome ends resemble double-strand breaks, telomere binding proteins mask telomeres from DNA damage response machinery. Consequently, telomere protection physically blocks telomere replication by the unique ribonucleoprotein (RNP) reverse transcriptase, telomerase. Telomerase access to telomeres is strictly regulated in the cell, and thus telomeres vacillate in status from telomerase accessible and telomerase inaccessible states. Here, I report the mechanistic contributions of the telomerase accessory protein POT1a (Protection Of Telomeres 1) in Arabidopsis telomere dynamics. POT1a, one of three POT1 paralogs in Arabidopsis, is essential for telomere replication. My work revealed POT1a is an activator of telomerase and stimulates its enzymology. POT1a physically binds two telomere proteins, CTC1 and STN1, and all three proteins can associate with active telomerase in vivo. In contrast, POT1a competed with TEN1, a capping protein shown to negatively regulate telomerase activity, for an interface on STN1. Thus, POT1a contributes to telomere dynamics though its interactions with telomerase and telomere binding proteins. Additionally, I examined the function of TER2, a non-canonical telomerase RNA that negatively regulates the TER1 (canonical) telomerase RNP. Null mutations in ter2 result in mild telomere phenotypes. However, when ter2 mutation was combined with the loss of POT1a, pot1a ter2 double mutants exhibited severe rates of telomere shortening and early onset defects in plant morphology and development. Thus, POT1a and TER2 represent two distinct regulators of telomere maintenance in Arabidopsis thaliana. Lastly, I observed the consequences of POT1 gene duplication. Evolutionary analysis revealed POT1a post-duplication was under Darwinian selection pressure for non-synonymous changes in three amino acid sites. Reversion of these residues back to the ancestral (POT1b) state resulted in a reduced ability of these mutants to genetically complement the telomere maintenance defect of pot1a mutants. In addition, these mutants had a reduced affinity for CTC1 in vitro. Therefore, POT1a is under positive evolutionary selection for its role in telomere maintenance and its association with CTC1. In summary, my work has elucidated the contributions of POT1a to Arabidopsis telomere dynamics, and how these functions contribute to its role in promoting telomere maintenance.
  • Telomeres are the physical ends of eukaryotic chromosomes. Because chromosome ends resemble double-strand breaks, telomere binding proteins mask telomeres from DNA damage response machinery. Consequently, telomere protection physically blocks telomere replication by the unique ribonucleoprotein (RNP) reverse transcriptase, telomerase. Telomerase access to telomeres is strictly regulated in the cell, and thus telomeres vacillate in status from telomerase accessible and telomerase inaccessible states.

    Here, I report the mechanistic contributions of the telomerase accessory protein POT1a (Protection Of Telomeres 1) in Arabidopsis telomere dynamics. POT1a, one of three POT1 paralogs in Arabidopsis, is essential for telomere replication. My work revealed POT1a is an activator of telomerase and stimulates its enzymology. POT1a physically binds two telomere proteins, CTC1 and STN1, and all three proteins can associate with active telomerase in vivo. In contrast, POT1a competed with TEN1, a capping protein shown to negatively regulate telomerase activity, for an interface on STN1. Thus, POT1a contributes to telomere dynamics though its interactions with telomerase and telomere binding proteins.

    Additionally, I examined the function of TER2, a non-canonical telomerase RNA that negatively regulates the TER1 (canonical) telomerase RNP. Null mutations in ter2 result in mild telomere phenotypes. However, when ter2 mutation was combined with the loss of POT1a, pot1a ter2 double mutants exhibited severe rates of telomere shortening and early onset defects in plant morphology and development. Thus, POT1a and TER2 represent two distinct regulators of telomere maintenance in Arabidopsis thaliana.

    Lastly, I observed the consequences of POT1 gene duplication. Evolutionary analysis revealed POT1a post-duplication was under Darwinian selection pressure for non-synonymous changes in three amino acid sites. Reversion of these residues back to the ancestral (POT1b) state resulted in a reduced ability of these mutants to genetically complement the telomere maintenance defect of pot1a mutants. In addition, these mutants had a reduced affinity for CTC1 in vitro. Therefore, POT1a is under positive evolutionary selection for its role in telomere maintenance and its association with CTC1.

    In summary, my work has elucidated the contributions of POT1a to Arabidopsis telomere dynamics, and how these functions contribute to its role in promoting telomere maintenance.

publication date

  • December 2014