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Title: Structural and functional analysis of proteins that regulate fission yeast telomerase activity
Author: Barrow, Jonathan Edward
ISNI:       0000 0004 9350 707X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Shelterin is a protein-DNA complex found at chromosome ends, which prevents their recognition by the double strand break surveillance function of the DNA damage response and plays important roles in regulation of telomere assembly and telomerase activity. In Schizosaccharomyces pombe, the shelterin complex consists of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1. In spite of its biological importance in maintaining genomic integrity, relatively little is known about the structures of the proteins of the shelterin complex and how their interactions coordinate their regulatory functions. This thesis describes experiments to investigate the structural and biochemical basis of shelterin assembly and regulation. Using a combination of approaches, Ccq1 was shown to behave as an elongated, modular and somewhat flexible molecule, dimerising tightly through a C-terminal coiled-coil region. Binding experiments showed that Ccq1 interacts with Tpz1 with micromolar affinity, whereas Poz1 and Tpz1 were found to bind more tightly with an apparent Kd in the low nanomolar range. This suggests a highly stable Poz1-Tpz1 interaction occurs in vivo in keeping with the proposed role for this sub-complex as a structural bridge within the shelterin assembly. The minimal binding motifs for Poz1 and Ccq1 were identified within the C-terminal Tpz1 region and, importantly, shown to not overlap, allowing each protein to bind to Tpz1 non- competitively to form a stable three component sub-complex. Negative stain electron microscopy of this tripartite assembly containing full-length Poz1-Tpz1-Ccq1, co-expressed in insect cells, generated a low resolution envelope in which the three components are arranged to form an interrupted shell-like structure surrounding a central void. Most strikingly, the structure shows a markedly asymmetric architecture in spite of the 2: 2: 2 stoichiometry of Ccq1, Tpz1 and Poz1 in the complex. The molecular basis for this asymmetry remains unclear, but may have important implications for telomerase regulation.
Supervisor: Smerdon, Steve Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available