RNA aptamers selected against DNA polymerase beta inhibit the polymerase activities of DNA polymerases beta and kappa

Nucleic Acids Res. 2006 May 17;34(9):2579-86. doi: 10.1093/nar/gkl326. Print 2006.

Abstract

DNA polymerase beta (polbeta), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polbeta from a variable pool of 8 x 10(12) individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polbeta activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polbeta active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polbeta also inhibited DNA polymerase kappa, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polbeta and kappa, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase activity in cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Aptamers, Nucleotide / pharmacology*
  • Binding, Competitive
  • DNA Polymerase beta / antagonists & inhibitors*
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / metabolism
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism
  • Humans
  • Nucleic Acid Synthesis Inhibitors*
  • Oligonucleotides / metabolism
  • SELEX Aptamer Technique

Substances

  • Aptamers, Nucleotide
  • Nucleic Acid Synthesis Inhibitors
  • Oligonucleotides
  • DNA Polymerase beta
  • DNA-Directed DNA Polymerase
  • POLK protein, human