In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.
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.
Pastwa
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Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Neumann
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Winters
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TA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D001761:Bleomycin / Q000494:pharmacology; D002457:Cell Extracts; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009994:Osmolar Concentration; D018384:Oxidative Stress / Q000187:drug effects; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature
D002457:Cell Extracts; D001761:Bleomycin; D000255:Adenosine Triphosphate