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 doub le-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs li near plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleo mycin. The bleomycin-induced DSB possesses a complex structure similar to t hat produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Re gardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-end s 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 tempe rature, time, ionic strength, ATP utilization and extract protein concentra tion were examined. Similar reactions were performed with the bleomycin-lin earized substrate. In all cases, end-joined molecules ranging from dimers t o higher molecular weight forms were produced and observed directly in agar ose gels stained with Vistra. Green and imaged with a Fluorlmager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of less than or equal to0.25 ng double-stranded DNA p er band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires less than or equal to 100 ng substrate DNA a nd greater than or equal to 50% conversion of substrate to product is achie ved with simple substrates such as restriction enzyme-cleaved DNA. Using ou r assay we have observed a 6-fold lower repair rate and a lag in reaction i nitiation 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.