Synthesis of Novel poly(dG)-poly(dG)-poly(dC) Triplex Structure by Klenow Exo- Fragment of DNA Polymerase I

Nucleic Acids Res. 2005 Nov 27;33(20):6515-21. doi: 10.1093/nar/gki963. Print 2005.

Abstract

The extension of the G-strand of long (700 bp) poly(dG)-poly(dC) by the Klenow exo(-) fragment of DNA polymerase I yields a complete triplex structure of the H-DNA type. High-performance liquid chromatography analysis demonstrates that the length of the G-strand is doubled during the polymerase synthesis. Fluorescence resonance energy transfer analysis shows that the 5' ends of the G- and the C-strands, labeled with fluorescein and TAMRA, respectively, are positioned close to each other in the product of the synthesis. Atomic force microscopy morphology imaging shows that the synthesized structures lack single-stranded fragments and have approximately the same length as the parent 700 bp poly(dG)-poly(dC). CD spectrum of the polymer has a large negative peak at 278 nm, which is characteristic of the poly(dG)-poly(dG)-poly(dC) triplex. The polymer is resistant to DNase and interacts much more weakly with ethidium bromide as compared with the double-stranded DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Circular Dichroism
  • DNA / chemistry
  • DNA / metabolism*
  • DNA / ultrastructure
  • DNA Polymerase I / metabolism*
  • Ethidium / chemistry
  • Fluorescence Resonance Energy Transfer
  • Microscopy, Atomic Force
  • Polydeoxyribonucleotides / chemistry
  • Polydeoxyribonucleotides / metabolism

Substances

  • Polydeoxyribonucleotides
  • triplex DNA
  • DNA
  • DNA Polymerase I
  • Ethidium