Date of Award

Spring 5-4-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Regulatory Biology

Department

Biological, Geological and Environmental Sciences

First Advisor

Li, Bibo

Second Advisor

Shukla, Girish

Third Advisor

Severson, Aaron

Subject Headings

Biochemistry, genetics, molecular biology, parasitology

Abstract

Telomeres are a nucleoprotein structure at the end of the chromosome and are essential for genome integrity and chromosome stability. Telomere lengths are primarily maintained by a telomerase-mediated pathway but can be maintained by a homologous recombination-mediated pathway. However, detailed mechanisms of telomere maintenance are still unclear in many eukaryotes, including an important human pathogen, Trypanosoma brucei. Telomeres can be elongated by telomerase in T. brucei, a causative agent of fatal sleeping sickness in humans and nagana in cattle. T. brucei evades host immune response by regularly switching its major surface antigen, variant surface glycoprotein (VSG), a process known as antigenic variation. The telomere structure and telomere proteins play critical roles in T. brucei pathogenesis. In mammalian, yeast, and plant cells, ssDNA binding proteins with OB-fold domains play important roles in coordinating telomere G- and C-strand syntheses. However, no such protein has been described in T. brucei to be specifically associated with the telomere. We identified POLIE, an A-type DNA polymerase, as a crucial telomere complex component in T. brucei and essential in maintaining telomere integrity in T. brucei. Depletion of POLIE in T. brucei leads to an increased amount of DNA damage at telomere/subtelomere, increased frequency of gene conversion-mediated VSG switching, and an increased amount of the telomeric circles (T-circles), suggesting a potential role of POLIE in suppressing DNA recombination at the telomere and the subtelomere. However, I find that telomeric and subtelomeric DNA recombination is unlikely to be mediated by the increased telomeric R-loop level as the telomeric repeat-containing RNA (TERRA) level is significantly lower in POLIE-depleted cells. The telomere G-rich 3’overhangs are dramatically elongated in POLIE-depleted cells, indicating a potential role of POLIE to coordinate telomere G- and C-strand syntheses and suggesting that the long telomere 3’ overhang can induce more telomeric and subtelomeric recombination. In addition, I find that POLIE inhibits telomerase-dependent telomere G-strand extension, identifying POLIE as the first telomere protein that potentially suppresses telomerase in T. brucei. Moreover, depletion of POLIE greatly increases the amount of telomeric C-circles which can be derived from replication stress in the telomere C-strand. Importantly, the elongated telomere 3’ overhang and elevated telomeric C-circle level phenotypes are independent of the telomerase, which suggests that POLIE may promote the telomere C-strand synthesis. Therefore, we identified that POLIE plays a major role in suppressing telomere recombination, coordinating telomerase-mediated telomere G-strand extension, and telomere C-strand synthesis, and maintaining telomere integrity in T. brucei.

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