Hybridization of G-quadruplex-forming peptide nucleic acids to guanine-rich DNA templates inhibits DNA polymerase η extension

Biochemistry. 2014 Aug 19;53(32):5315-22. doi: 10.1021/bi5006859. Epub 2014 Aug 5.

Abstract

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridines / pharmacology
  • DNA / chemistry*
  • DNA / genetics
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • G-Quadruplexes
  • Guanine / chemistry*
  • Humans
  • Nucleic Acid Synthesis Inhibitors
  • Peptide Nucleic Acids / chemistry
  • Peptide Nucleic Acids / metabolism
  • Protein Binding

Substances

  • Acridines
  • Nucleic Acid Synthesis Inhibitors
  • Peptide Nucleic Acids
  • Guanine
  • DNA
  • DNA-Directed DNA Polymerase
  • Rad30 protein
  • BRACO-19