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

Abstract

Purpose: The signal model proposes that all chromatid breaks arise from a single DNA double strand break (dsb) via a recombinational exchange mechanism. Here the prediction that chromatid breaks arise from a single dsb is tested. Method: The genetically engineered Chinese hamster cell line GS19-43 containing a unique yeast I-Sce I recognition site was treated with I-Sce I endonuclease (Meganuclease) in the presence of the porating agent streptolysin O. Chromatid breaks were scored at 4h, chromosome breaks at 18 and 22h following treatment (cells used for a 4h fixation were prelabelled with BrdU over two cell-cycles). Positive controls were treated with the restriction endonuclease Pst 1. Results: I-Sce I endonuclease produced chromatid breaks and at higher enzyme concentrations isochromatid breaks but no chromatid interchanges. About 16% of the chromatid breaks had a 'colour-switch' between the sister-chromatids at the site of breakage, as revealed by FPG staining. At the longer fixation times (18 and 22h) chromosome breaks were observed, but again no interchanges were seen. Chromatid and chromosome breaks always appeared on the same chromosome. Conclusions: The production of chromatid breaks from a single dsb fulfils the prediction of the signal model. Moreover, the production of colour-switch breaks at a similar frequency to that for ionizing radiation indicates that chromatid breaks are produced via recombinational exchanges, a significant proportion of which occurs between sister chromatids. The majority is intrachromatid, not involving strand-switches. The absence of interchromosomal exchanges at all fixation times indicates a requirement of two dsb in two different chromosomes for their formation.