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Title: Studies into the structure of human telomerase and the utilisation of its DNA substrate
Author: Alves, D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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Human telomerase is minimally composed of protein (hTERT) and RNA (hTR) components, although information regarding the stoichiometry of these moieties within the active enzyme remains undetermined. The low natural abundance of human telomerase, coupled with poor levels of in vitro expression has limited the use of classical biophysical approaches to gain such structural insights. Initial work within this thesis describes the use of a single-molecule fluorescence technique employing two-colour coincidence detection (TCCD) to directly observe and characterise the complex formed between fluorescently-labelled hTERT, hTR and telomeric substrate DNA components. It was found that catalytically active human telomerase comprises a kinetically-stable complex between one hTERT and one hTR component, which in turn recruits just one substrate DNA moiety. The interaction between telomerase and its DNA substrate is dependent upon a number of factors. Chapter 3 describes the substrate’s potential to form non-classical four-stranded DNA structures called G-quadruplexes, and the use of agents that stabilise such structures to perturb the substrate’s utilisation by telomerase. A method for directly assessing telomerase activity in vitro was developed and used to demonstrate that a quinoline-based G-quadruplex-binding macrocycle was capable of regulating telomerase activity. Other factors that have potential roles in the control of substrate-binding are the chaperone proteins Hsp90 and p23. Initial efforts to elucidate any such effects are described in Chapter 4, with the demonstration that telomerase activity in wheat germ lysate could be stimulated through the addition of p23 forming a firm basis for future study.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available