TRF1 Inhibits Telomere C-Strand DNA Synthesis in Vitro
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- 作者：Elizabeth J. Smucker and John J. Turchi
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：February 27, 2001
- 年：2001
- 卷：40
- 期：8
- 页码：2426 - 2432
- 全文大小：142K
- 年卷期：v.40,no.8(February 27, 2001)
- ISSN：1520-4995
文摘
Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomerelength in vivo, but the mechanism of regulation remains largely undefined. We have developed an invitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and syntheticDNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesiscatalyzed by DNA polymerase 
/primase (pol ). Inhibition required that TRF1 be bound to duplextelomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibitionwas shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of theDNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstreamof the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but notnecessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, weassessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators.In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has noeffect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition wasobserved on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, whenbound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussedwith respect to the role of TRF1 in telomere length regulation.
/primase (pol ). Inhibition required that TRF1 be bound to duplextelomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibitionwas shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of theDNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstreamof the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but notnecessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, weassessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators.In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has noeffect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition wasobserved on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, whenbound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussedwith respect to the role of TRF1 in telomere length regulation.