Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End Joining

Abstract

Stenerlöw, B., Karlsson, K. H., Cooper, B. and Rydberg, B. Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells Without Including Heat-Labile Sites. Results for Cells Deficient in Non-homologous End Joining. Radiat. Res. Ionizing radiation induces prompt single-strand breaks and double-strand breaks in DNA. In addition, labile sites are induced that can convert to breaks by heat or mild alkali. When such labile lesions are present within multiply damaged sites, additional double-strand breaks can form. Current protocols for measurement of DNA double-strand breaks involve a lysis step at elevated temperature, and consequently breaks from heat-labile sites will be generated during lysis and will be included in the measurement. However, such sites may not develop into breaks within the cell and may not need DNA double-strand break repair processes for elimination. We present here a new lysis and pulsed-field gel electrophoresis protocol that is carried out entirely at 0-4°C and thus avoids inclusion of heat-labile sites into the measurement. The new recommended lysis procedure involves two steps: the first step includes proteinase-K, which has sufficient activity at 0°C to support lysis, and the second step includes a high salt buffer to strip the DNA from histones and other proteins. By various tests we conclude that lysis is sufficient with this procedure to allow accurate determination of double-strand breaks by pulsed-field gel electrophoresis. Using the new protocol, it was found that heat-labile sites account for 30% of the initial number of double-strand breaks measured by conventional protocols after low LET radiation. In addition we show that heat-labile sites that can convert to double-strand breaks are repaired with fast kinetics and are nearly completely eliminated after 1 hr at 37°C. A study of cells deficient in non-homologous end joining reveals that the residual fast repair response typically seen in such cells is solely due to repair at heat-labile sites and is not due to repair of prompt DSBs. 2

SU11752 inhibits the DNA-dependent protein kinase and DNA double-strand break repair resulting in ionizing radiation sensitization

“…However, initial experiments using X-ray-induced DSBs revealed only a modest inhibition of DSB repair by SU11752. A recent report shows that the FAR assay overestimates the number of X-ray-induced DSBs, since other types of DNA damage are converted to strand breaks during high-temperature protease treatment of chromatin (Stenerlow et al, 2003). In agreement with this report, we observed a significant increase of X-ray-induced DSBs using protease treatment at 501C compared to low-temperature protease treatment.…”

Gamma-H2AX foci counting: image processing and control software for high-content screening

“…The obtained result that ~30 γH2AX foci were present in human 48BRhtert fibroblasts 0.5 h after exposure to 1 Gy Xrays was similar to the yield of DSBs that is induced per Gy of X-rays in a human cell 9,10 . This indicates a one-to-one correlation between the number of X-ray-induced γH2AX foci and DSBs.…”

Extreme resistance of bdelloid rotifers to ionizing radiation

“…The average molecular sizes at 560, 840, and 1,120 Gy are 350, 250, and 190 kbp, respectively, with an average of 0.005 double-strand breaks (DSBs) Gy Ϫ1 ⅐Mbp Ϫ1 , a value within the range of 4 to 7 ϫ 10 Ϫ3 DSBs Gy Ϫ1 ⅐Mbp Ϫ1 measured by pulsedfield gel electrophoresis or sucrose sedimentation analysis for Escherichia coli, Saccharomyces cerevisiae, and diverse mammalian cell lines (9)(10)(11)(12). Some of the DSBs we observed may have arisen from non-DSB lesions as a result of high-temperature digestion, an effect estimated to cause about a 30% overestimate in ␥-irradiated mammalian cells (13).…”