Nucleic Acids Research Advance Access originally published online on January 3, 2008
Nucleic Acids Research 2008 36(4):1260-1272; doi:10.1093/nar/gkm866
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2008, Vol. 36, No. 4 1260-1272
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Multiple DNA-binding sites in Tetrahymena telomerase
Children's Medical Research Institute, 214 Hawkesbury Road, Westmead NSW 2145 and University of Sydney, NSW 2006, Australia
*To whom correspondence should be addressed. Tel: +61 2 9687 2800; Fax: +61 2 9687 2120; Email: tbryan{at}cmri.usyd.edu.au
Received July 26, 2007. Revised September 13, 2007. Accepted September 24, 2007.
Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre (‘anchor sites’) is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a Kd of 
8 nM. Both the Km and Kd increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.