Nucleic Acids Research Advance Access originally published online on November 29, 2008
Nucleic Acids Research 2009 37(2):354-367; doi:10.1093/nar/gkn830
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Nucleic Acids Research, 2009, Vol. 37, No. 2 354-367
© 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 |
Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p
1Howard Hughes Medical Institute and 2Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA
*To whom correspondence should be addressed. Tel: +1 410 516 8749; Fax: +1 410 516 5213; Email: zappulla{at}jhu.edu
Correspondence may also be addressed to Deborah S. Wuttke. Tel: +1 303 492 4576; Fax: +1 303 492 5894; Email: deborah.wuttke{at}colorado.edu
Received July 18, 2008. Revised September 16, 2008. Accepted October 14, 2008.
Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3'-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3'-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel ‘action at a distance’ inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3'-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.
Present address: David C. Zappulla, Department of Biology, Johns Hopkins University, Baltimore, MD 21218-2685, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.