Biochemical analysis of proteins in the telomere maintenance pathway

Dezwaan, Diane C.

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https://hdl.handle.net/2142/18345 Copy

Description

Title

Biochemical analysis of proteins in the telomere maintenance pathway

Author(s)

Dezwaan, Diane C.

Issue Date

2011-01-14T22:47:00Z

Director of Research (if dissertation) or Advisor (if thesis)

Freeman, Brian C.

Doctoral Committee Chair(s)

Belmont, Andrew S.

Committee Member(s)

• Ceman, Stephanie S.
• Nardulli, Ann M.
• Gillette, Martha U.
• Freeman, Brian C.

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Telomere
• Telomerase
• Chaperone
• Cellular dynamics

Abstract

Eukaryotic linear chromosomes culminate in nucleoprotein structures designated telomeres. The terminal telomeric DNA consists of tandem repeats of a G-rich motif that is established and maintained by the action of the specialized reverse transcriptase called telomerase. In addition to the function of telomerase, the telomere environment requires an efficient means to assemble and disassemble a multitude of structures to operate correctly and to help achieve cellular homeostasis. Distinct protein assemblies are nucleated at telomeric DNA to both guard the ends from damage and lengthen the DNA after replication. In yeast, Cdc13 recruits either Stn1-Ten1 to form a protective cap or the telomerase holoenzyme to extend the DNA. I have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13. The telomere length and telomerase telomere occupancy also appear to be yeast Hsp90 dependent. Taken together, my data show that the Hsp82 chaperone facilitates telomere DNA maintenance by promoting transitions between two operative complexes and by reducing the potential for binding events that would otherwise block the assembly of downstream structures. The first telomerase cofactor identified was the budding yeast protein Est1, which is conserved through humans. While it is evident that Est1 is required for telomere DNA maintenance, understanding its mechanistic contributions to telomerase regulation has been limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNA extension. Although Est1 displayed specific DNA and RNA binding, neither activity iii contributed significantly to telomerase stimulation. Rather Est1 mediated telomerase upregulation through direct contacts with the reverse transcriptase subunit. My studies provide insights into the molecular events used to control the enzymatic activity of the telomerase holoenzyme.

Graduation Semester

2010-12

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http://hdl.handle.net/2142/18345

Copyright and License Information

Copyright 2010 Diane C. Dezwaan

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