Previous Article | Next Article ![]()
Molecular and Cellular Biology, November 2001, p. 7775-7786, Vol. 21, No. 22
Department of Pharmacology and Cancer
Biology, Department of Radiation Oncology, Duke University
Medical Center, Durham, North Carolina 27710
Received 1 May 2001/Returned for modification 4 June 2001/Accepted 7 August 2001
Most tumor cells depend upon activation of the ribonucleoprotein
enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be
reconstituted in vitro with the RNA (hTR) and catalytic (hTERT)
subunits. However, catalytic activity alone is insufficient for the
full in vivo function of the enzyme. In addition, the enzyme must
localize to the nucleus, recognize chromosome ends, and orchestrate
telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel
of 90 N-terminal hTERT substitution mutants into
telomerase-negative cells and assayed the resulting cells for
catalytic activity and, as a marker of in vivo function, for cellular
proliferation. We found four domains to be essential for in vitro and
in vivo enzyme activity, two of which were required for hTR binding.
These domains map to regions defined by sequence alignments and
mutational analysis in yeast, indicating that the N terminus has also
been functionally conserved throughout evolution. Additionally, we
discovered a novel domain, DAT, that "dissociates activities of
telomerase," where mutations left the enzyme catalytically
active, but was unable to function in vivo. Since mutations in this
domain had no measurable effect on hTERT homomultimerization, hTR
binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these
domains provides the first step in dissecting the biological functions
of human telomerase, with the ultimate goal of targeting this
enzyme for the treatment of human cancers.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.22.7775-7786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
N-Terminal Domains of the Human Telomerase Catalytic Subunit
Required for Enzyme Activity in Vivo
*
Corresponding author. Mailing address: LSRC Bldg., Rm.
C225, Research Dr., DUMC, Durham, NC 27710. Phone: (919) 684-9890. Fax:
(919) 684-8958. E-mail: count004{at}mc.duke.edu.
This article has been cited by other articles:
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|