Nucleic Acids Research, Vol 25, Issue 18 3698-3704, Copyright © 1997 by Oxford University Press
PW Hammond and TR Cech
We have measured the processivity of telomeric DNA extension by Euplotes
aediculatus telomerase at various concentrations of the nucleotide
substrates dGTP and dTTP. The maximum processivity (approximately 3
repeats) was observed at approximately 100 microM of each dNTP.
Processivity decreased as the dNTP concentrations were reduced and,
surprisingly, as the concentration of dGTP was increased. Also, the
characteristic banding pattern generated by telomerase extension of DNA
primers shifted in response to changes in dGTP concentration. One pattern
with 8 nt periodicity was predominant at dGTP concentrations </=16
microM, while at >/= 250 microM an 8 nt repeat pattern out-of-phase with
the first was observed; at intermediate concentrations the two patterns
coexisted. We propose that two different segments of the RNA subunit can
serve as the template for repeat synthesis; nt 42-49 at low dGTP
concentrations and nt 36-43 at high dGTP concentrations. An alternative
model for the low dGTP pattern involves an internal pause site but no pause
at the end of the template and is, therefore, considered less likely.
Because the effects of dGTP on processivity and banding pattern appear to
be distinct from nucleotide binding in the polymerase active site, we
propose a second dGTP binding site involved in template selection and
processivity.
ARTICLES
dGTP-dependent processivity and possible template switching of euplotes telomerase
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309-0215, USA.
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