Student Theses and Dissertations

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

de Lange Laboratory

Abstract

Telomeres are the terminal nucleo protein components of eukaryotic chromosomes that safeguard genome integrity. Human telomeric DNA consists of double-stranded (ds) 5’-TTAGGG-3’ repeats terminating in a 3’ single-stranded (ss) overhang of the G-rich strand.The sequence and structure of the telomeric DNA is recognized and bound by the six-subunit shelterin complex, which plays critical roles in telomere protection and maintenance. First, shelterin protects the genome by repressing aberrant activation of the DNA damage response at telomeres, which resemble broken DNA ends. Second, due to their location at the end of the chromosome, telomeres present a unique challenge to the canonical DNA replication machinery. The replisome cannot fully copy the terminal repeats, resulting in gradual telomere shortening over successive cell divisions. Once telomeres become too short, they lose protection, triggering the DNA damage response and downstream apoptosis or senescence. Unchecked excessive telomere shortening is pathological and can manifest as a number of telomere biology disorders, but routine telomere shortening insomatic cells is also a tumor suppressor pathway. Thus, telomere length must be carefully controlled and shelterin recruits two maintenance enzymes to the telomere to do so: telomerase and DNA Polymerase α (Polα)/primase bound to Ctc1/Stn1/Ten1 (CST). Decades of research have focused on telomerase, which extends the G-rich telomeric strand to counteract telomere shortening in germline and stem cells. More recently, CST–Polα/primase,which synthesizes the complementary C-rich strand, has emerged as a second critical regulator of telomere length. The body of work contained in this thesis describes our efforts to understand the molecular mechanisms underlying C-strand maintenance by this enigmatic complex.Chapter 1 serves as an introduction to telomere maintenance by shelterin and CST–Polα/primase.I contextualize the work presented in this thesis alongside commentary on findings from other groups and the field at large, discussing recent studies on the evolution, function, and regulation of CST–Polα/primase. I also introduce the telomere biology disorder Coats plus syndrome (CP), which is caused by mutations in CST,and in one case, the shelterin subunit POT1.This thesis provides a molecular basis for the pathogenesis of a subset of these CP mutations.In Chapter 2,I set out to determine the structure of CST–Polα/primase using cryogenic electron microscopy (cryo-EM), a state-of-the-art structural technique well suited to large and flexible macromolecular complexes. Surprisingly, the structure I obtained was of an inactive complex where the Polα/primase bound to CST is in an auto-inhibited conformation in competent for enzymatic activity. Evolutionary conservation analysis showed that the interface between CST and Polα/primase in this structure is unique to metazoans and lost in unicellular eukaryotes. A CP mutation maps to the primary interface observed, supporting a physiological role for this conformation.I propose that this inactive state may represent a “recruitment state” that forms during recruitment of CST–Polα/primase to the telomere and exists prior to activation of the enzyme. Concurrent to my finding of the CST–Polα/primase recruitment complex (RC), another group determined the structure of CST–Polα/primase in a pre-initiation complex (PIC) competent for enzymatic activity. The existence of these two drastically different conformations raised the question of how this complex might be regulated.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Download

Included in

Life Sciences Commons

Share

COinS