In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts

Nucleic Acids Res. 2001 Aug 15;29(16):E78. doi: 10.1093/nar/29.16.e78.

Abstract

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of </=0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires </=100 ng substrate DNA and >/=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Biological Assay / methods*
  • Bleomycin / pharmacology
  • Cell Extracts
  • DNA Damage / drug effects
  • DNA Damage / genetics*
  • DNA Repair / genetics*
  • Dose-Response Relationship, Drug
  • HeLa Cells
  • Humans
  • Kinetics
  • Osmolar Concentration
  • Oxidative Stress* / drug effects
  • Plasmids / genetics*
  • Plasmids / metabolism*
  • Recombination, Genetic / genetics
  • Sensitivity and Specificity
  • Temperature

Substances

  • Cell Extracts
  • Bleomycin
  • Adenosine Triphosphate