Synthesis of the mammalian telomere lagging strand in vitro

Phillip M. Reveal, Karen M. Henkels, John Turchi

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase α. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase α/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase α on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five- base RNA primer is sufficient for extension with dNTPs by DNA polymerase α. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.

Original languageEnglish (US)
Pages (from-to)11678-11681
Number of pages4
JournalJournal of Biological Chemistry
Volume272
Issue number18
DOIs
StatePublished - May 2 1997
Externally publishedYes

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Telomere
DNA-Directed DNA Polymerase
RNA
DNA
Cell Extracts
DNA Primase
Uridine Triphosphate
Ladders
Neutralizing Antibodies
Complex Mixtures
Thymidine
Nucleotides
In Vitro Techniques
RNA primers
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Synthesis of the mammalian telomere lagging strand in vitro. / Reveal, Phillip M.; Henkels, Karen M.; Turchi, John.

In: Journal of Biological Chemistry, Vol. 272, No. 18, 02.05.1997, p. 11678-11681.

Research output: Contribution to journalArticle

Reveal, Phillip M. ; Henkels, Karen M. ; Turchi, John. / Synthesis of the mammalian telomere lagging strand in vitro. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 18. pp. 11678-11681.
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