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Title: Structural studies on proteins involved in human telomere maintenance
Authors: Ufuk Borucu
Keywords: DRNTU::Science::Chemistry::Crystallography::Electron microscopy
DRNTU::Science::Chemistry::Crystallography::X-ray crystallography
DRNTU::Science::Chemistry::Analytical chemistry::Proteins
Issue Date: 2017
Source: Ufuk Borucu. (2017). Structural studies on proteins involved in human telomere maintenance. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: In order to sustain genomic stability and cellular viability, the ends of linear eukaryotic chromosomes are capped and protected by telomeres. The mechanism by which the length of telomeric DNA is maintained involves a specialized reverse transcriptase, called telomerase. This thesis focuses on the structural investigation of two proteins that play essential roles in the regulation of different stages of telomerase activity: TCAB1 and the CST complex. TCAB1 participates in the maturation process of the telomerase RNA subunit and is critical for telomerase trafficking in vivo. Mutations in TCAB1 lead to defects in telomere maintenance and give rise to severe forms of dyskeratosis congenita (DC). The CST complex, consisting of Ctc1, Stn1 and Ten1, limits telomerase activity in the late stages of telomere elongation. CST also initiates the fill-in synthesis of the complementary strand by recruiting the Polα/Primase complex. Mutations in CST are associated with Coat Plus, DC and related diseases. In this thesis, I present various strategies for the expression and purification of TCAB1 and the CST complex and structural analysis using negative stain electron microscopy (EM). Using primary sequence analysis and computational methods, I demonstrated that TCAB1 contains seven putative WD40 repeats within its central domain rather than the five published. Using various strategies for expression of TCAB1 in E.coli, that lead to aggregated protein, it was published that TCAB1 folding requires an elaborate machinery including the TCP-Ring Complex (TRiC) that is only present in higher eukaryotes. For the CST complex, I found that expression of full length Ctc1 in E.coli was not feasible and that it necessitated co-expression with Stn1 and Ten1. This was achieved by setting up MultiBac expression in insect cells. Results are presented for a purification strategy for obtaining a pure complex that contains stoichiometric amounts of the three proteins in mg quantities. As an initial stage to determining the cryo-EM structure, I present a low-resolution threedimensional structure (25Å) of the CST complex obtained using negative stain EM and single-particle reconstruction. Finally, I describe strategies to obtain a higher resolution structure bound to telomeric DNA.
DOI: 10.32657/10356/72778
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Theses

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