A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

DNA Repair (Amst). 2015 Mar;27:9-18. doi: 10.1016/j.dnarep.2014.12.006. Epub 2015 Jan 6.

Abstract

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

Keywords: AP endonuclease; Abasic sites; Aldehyde-reactive probe; Base excision repair; Click chemistry; Methoxyamine.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry*
  • Alkynes / pharmacology
  • Biotin / analogs & derivatives
  • Biotin / chemistry
  • Cell Death
  • DNA / metabolism
  • DNA Damage*
  • DNA Mutational Analysis / methods*
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / antagonists & inhibitors
  • HeLa Cells
  • Humans
  • Hydroxylamines / chemistry*
  • Hydroxylamines / pharmacology
  • Methyl Methanesulfonate / pharmacology
  • Sensitivity and Specificity

Substances

  • Alkynes
  • Hydroxylamines
  • O-2-propynylhydroxylamine
  • O-(biotinylcarbazoylmethyl)hydroxylamine
  • Biotin
  • DNA
  • methoxyamine
  • Methyl Methanesulfonate
  • APEX1 protein, human
  • DNA-(Apurinic or Apyrimidinic Site) Lyase