Idol, Rachel A (2005-08). Telomeres and their associated factors in Arabidopsis thaliana. Doctoral Dissertation. Thesis uri icon

abstract

  • Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary for genome integrity. Telomeres are maintained by intermittent action of telomerase. I explored the kinetics of telomere length homeostasis in the model plant Arabidopsis thaliana by crossing wild type plants to different generations of telomerase deficient plants, and then analyzing telomere length in the resulting progeny. Unexpectedly, I found plants lacking telomerase for seven generations can lengthen telomeres when telomerase is reintroduced, but one generation is not sufficient to reestablish the telomere set point. Est1 is a non-catalytic component of the Saccharomyces cerevisiae telomerase holoenzyme. To investigate the role of Est1 in higher eukaryotes, I identified two putative Est1 homologues in Arabidopsis, AtEST1a and AtEST1b. Plants deficient in AtEST1a displayed no vegetative or reproductive defects. However, plants deficient for AtEST1b were sterile and had severe vegetative and reproductive irregularities. Surprisingly, no defects in telomere maintenance were observed in any single or double mutant line. This suggests that the Est1- like proteins in plants have evolved new functions outside of telomere length maintenance and end protection.One consequence of telomere dysfunction is end-to-end chromosome fusion. In mammals, telomere fusion is mediated through NHEJ and requires DNA Ligase IV (Lig4). Lig4 is an essential component of the NHEJ pathway along with the Ku70/Ku80 heterodimer and DNA-PKcs. To address the mechanism of chromosome fusion in Arabidopsis, we investigated the role of Lig4 in mutant combinations lacking TERT, the catalytic subunit of telomerase, and Ku70. Surprisingly, telomere end-to-end fusions were observed in ku70 tert lig4 triple mutants, suggesting that neither Lig4 nor Ku70 are required for the fusion of critically shortened telomeres in Arabidopsis. To investigate the origin of genome instability, terminal restriction fragment analysis was performed on triple mutants. Strikingly, telomeres diminished five to six-fold faster than in a tert single mutant. Moreover, in the triple mutants, telomere tracts were extremely heterogeneous, suggesting that the telomeres were exposed to catastophic nucleolytic attack. These data provide the first evidence that Lig4 contributes to telomere maintenance and chromosome end protection.
  • Telomeres are important protein-DNA structures at the ends of linear
    eukaryotic chromosomes that are necessary for genome integrity. Telomeres
    are maintained by intermittent action of telomerase. I explored the kinetics of
    telomere length homeostasis in the model plant Arabidopsis thaliana by crossing
    wild type plants to different generations of telomerase deficient plants, and then
    analyzing telomere length in the resulting progeny. Unexpectedly, I found plants
    lacking telomerase for seven generations can lengthen telomeres when
    telomerase is reintroduced, but one generation is not sufficient to reestablish the
    telomere set point.
    Est1 is a non-catalytic component of the Saccharomyces cerevisiae
    telomerase holoenzyme. To investigate the role of Est1 in higher eukaryotes, I
    identified two putative Est1 homologues in Arabidopsis, AtEST1a and AtEST1b.
    Plants deficient in AtEST1a displayed no vegetative or reproductive defects.
    However, plants deficient for AtEST1b were sterile and had severe vegetative
    and reproductive irregularities. Surprisingly, no defects in telomere maintenance
    were observed in any single or double mutant line. This suggests that the Est1-
    like proteins in plants have evolved new functions outside of telomere length
    maintenance and end protection.One consequence of telomere dysfunction is end-to-end chromosome
    fusion. In mammals, telomere fusion is mediated through NHEJ and requires
    DNA Ligase IV (Lig4). Lig4 is an essential component of the NHEJ pathway
    along with the Ku70/Ku80 heterodimer and DNA-PKcs. To address the
    mechanism of chromosome fusion in Arabidopsis, we investigated the role of
    Lig4 in mutant combinations lacking TERT, the catalytic subunit of telomerase,
    and Ku70. Surprisingly, telomere end-to-end fusions were observed in ku70 tert
    lig4 triple mutants, suggesting that neither Lig4 nor Ku70 are required for the
    fusion of critically shortened telomeres in Arabidopsis. To investigate the origin of
    genome instability, terminal restriction fragment analysis was performed on triple
    mutants. Strikingly, telomeres diminished five to six-fold faster than in a tert
    single mutant. Moreover, in the triple mutants, telomere tracts were extremely
    heterogeneous, suggesting that the telomeres were exposed to catastophic
    nucleolytic attack. These data provide the first evidence that Lig4 contributes to
    telomere maintenance and chromosome end protection.

publication date

  • August 2005