TRF1 inhibits telomere C-strand DNA synthesis in vitro

E. J. Smucker, J. J. Turchi

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomere length in vivo, but the mechanism of regulation remains largely undefined. We have developed an in vitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and synthetic DNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesis catalyzed by DNA polymerase α/primase (pol α). Inhibition required that TRF1 be bound to duplex telomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibition was shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of the DNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol α-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstream of the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but not necessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, we assessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem ga14 operators. In these experiments, the addition of the purified Ga14-DNA binding domain (Ga14-DBD) protein has no effect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition was observed on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, when bound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussed with respect to the role of TRF1 in telomere length regulation.

Original languageEnglish (US)
Pages (from-to)2426-2432
Number of pages7
JournalBiochemistry
Volume40
Issue number8
DOIs
StatePublished - Feb 27 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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