Production of chromatid breaks by single dsb: evidence supporting the signal model

Purpose: The signal model proposes that all chromatid breaks arise from a s ingle DNA double strand break (dsb) via a recombinational exchange mechanis m. Here the prediction that chromatid breaks arise from a single dsb is tes ted. Method: The genetically engineered Chinese hamster cell line GS19-43 contai ning a unique yeast I-SceI recognition site was treated with I-SceI endonuc lease (Meganuclease) in the presence of the porating agent streptolysin O. Chromatid breaks were scored at 4 h, chromosome breaks at 18 and 22 h follo wing treatment (cells used for a 4 h fixation were prelabelled with BrdU ov er two cell-cycles). Positive controls were treated with the restriction en donuclease Pst 1. Results: I-SceI endonuclease produced chromatid breaks and at higher enzyme concentrations isochromatid breaks but no chromatid interchanges. About 16 % of the chromatid breaks had a 'colour-swirch' between the sister-chromati ds at the site of breakage, as revealed by FPG staining. At the longer fixa tion times (18 and 22 h) chromosome breaks were observed, but again no inte rchanges were seen. Chromatid and chromosome breaks always appeared on the same chromosome. Conclusions: The production of chromatid breaks from a single dsb fulfils t he prediction of the signal model. Moreover, the production of colour-switc h breaks at a similar frequency to that for ionizing radiation indicates th at chromatid breaks are produced via recombinational exchanges, a significa nt proportion of which occurs between sister chromatids. The majority is in terchromatid, not involving strand-switches. The absence of intel-chromosom al exchanges at all fixation times indicates a requirement of two dsb in tw o different chromosomes For their formation.