The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3′ ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3′ region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3′ end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight.
Skip Nav Destination
Article navigation
January 2015
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
January 06 2015
Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding
Christopher G. Tomlinson;
Christopher G. Tomlinson
*Children's Medical Research Institute, Westmead, NSW 2145, Australia
†University of Sydney, Sydney, NSW 2006, Australia
Search for other works by this author on:
Aaron L. Moye;
Aaron L. Moye
*Children's Medical Research Institute, Westmead, NSW 2145, Australia
†University of Sydney, Sydney, NSW 2006, Australia
Search for other works by this author on:
Jessica K. Holien;
Jessica K. Holien
‡ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Search for other works by this author on:
Michael W. Parker;
Michael W. Parker
‡ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
§Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
Search for other works by this author on:
Scott B. Cohen;
Scott B. Cohen
*Children's Medical Research Institute, Westmead, NSW 2145, Australia
†University of Sydney, Sydney, NSW 2006, Australia
Search for other works by this author on:
Tracy M. Bryan
Tracy M. Bryan
1
*Children's Medical Research Institute, Westmead, NSW 2145, Australia
†University of Sydney, Sydney, NSW 2006, Australia
1To whom correspondence should be addressed (email tbryan@cmri.org.au).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
July 21 2014
Revision Received:
October 27 2014
Accepted:
November 03 2014
Accepted Manuscript online:
November 03 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 465 (2): 347–357.
Article history
Received:
July 21 2014
Revision Received:
October 27 2014
Accepted:
November 03 2014
Accepted Manuscript online:
November 03 2014
Citation
Christopher G. Tomlinson, Aaron L. Moye, Jessica K. Holien, Michael W. Parker, Scott B. Cohen, Tracy M. Bryan; Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding. Biochem J 15 January 2015; 465 (2): 347–357. doi: https://doi.org/10.1042/BJ20140922
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.