Calibration of Pulsed Field Gel Electrophoresis for Measurement of DNA Double-strand Breaks

Abstract

The pulsed field gel electrophoresis (PFGE) assay was calibrated for the measurement of X-ray-induced DNA double-strand breaks in Chinese hamster ovary (CHO) cells. Calibration was conducted by incorporating [125I]deoxyuridine into DNA, which induces one double-strand break for every disintegration that occurs in frozen cells. Based on the percentage of the DNA migrating into the gel, the number of breaks/dalton/Gy was estimated to be (9.3 +/- 1.0) x 10(-12). This value is close to (10 to 12) x 10(-12) determined by neutral filter elution using similar cell lysis procedures at 24 degrees C and at pH 8.0. Also, the estimate is in good agreement with the value of (11.7 +/- 2) x 10(-12) breaks/dalton/Gy as measured in Ehrlich ascites tumour cells using the neutral sucrose gradient method (Blöcher 1988), and (6 to 9) x 10(-12) breaks/dalton/Gy as measured in mouse L and Chinese hamster V79 cells using neutral filter elution (Radford and Hodgson 1985).

Evidence for a G2 Checkpoint in p53-Independent Apoptosis Induction by X-Irradiation

“…3), even though both of these cell lines are p53 deficient. The level of DNA repair observed in these cell lines was very similar to that observed in large variety of p53 wild-type cell lines (1,2,16). In addition, no DNA repair defect was observed following UV irradiation of a p53-deficient mouse cell line (24).…”

“…Under our electrophoresis conditions, intact genomic DNA remains in the gel well, whereas smaller (X-ray-broken) DNA migrates into the gel as a tight smear that appears as a band (36,54). Importantly, the amount of DNA released from the well is directly proportional to the number of unrepaired DSBs in the cell (1,54). Exponentially growing cultures of each cell line were irradiated with increasing doses of X rays and immediately processed for PFGE so that no repair could take place (see Materials and Methods).…”

“…To prepare cells for pulsed-field gel electrophoresis (PFGE) analyses, cells were washed in phosphate-buffered saline (PBS) and resuspended at 10 7 /ml in 1.0% low-melting-point agarose at 42ЊC (1). The cell-agarose slurry was then cast into agarose plugs (80 l), which were subsequently lysed in proteinase K, washed, and treated with RNase A as described previously (1). The plugs were then cast into a 1.0% horizontal agarose gel in 0.5ϫ TBE (45 mM Tris [pH 8.2], 45 mM boric acid, 1.25 mM EDTA) with 0.5 g of ethidium bromide per ml and subjected to PFGE for 28 h in a homemade (50) pulsed-field gel chamber at 10ЊC and 150 V. Under these electrophoresis conditions, chromosomal DNA which had broken into 3-to 5-Mbp fragments migrated as a discrete band on the gel.…”

“…Therefore, using DNA DSB induction by X rays as a parameter, we assessed the radiosensitivities of HL-60 and HCW-2 cells at the biochemical level. We first performed a dose-response experiment using PFGE to quantitate the number of DSBs in chromosomal DNA following X-ray exposure without DNA repair (1,2,36). Under our electrophoresis conditions, intact genomic DNA remains in the gel well, whereas smaller (X-ray-broken) DNA migrates into the gel as a tight smear that appears as a band (36,54).…”

Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

“…Therefore, we hypothesized that the X-ray hypersensitivity of scid cells in the G 1 and early S phases may have arisen from a reduced ability to carry out DNA DSB repair at those specific phases of the cell cycle. To test this experimentally, we examined DNA DSB repair activity throughout the cell cycle, using a PFGE assay that has been extensively described before (1,26,43,68). Briefly, scid and wild-type pre-B cells were synchronized with nocodazole as shown in Fig.…”