Abstract
To investigate the mechanism of double strand DNA break formation in mammalian cells, an in vitro assay was established using closed circular DNA containing two uracils on opposite DNA strands (18 and 30 base pairs apart) and extracts prepared from human cells. In this assay, formation of double strand breaks was detected by the conversion of circular DNA to linear DNA. Approximately 4-fold more double strand DNA breaks were produced by extracts from cells deficient in DNA ligase I (46BR) relative to those produced by extracts from control cells (MRC5, derived from a clinically normal individual). In parallel with the amount of double strand DNA breaks, extracts from 46BR cells produced longer repair patches (up to 24 bases in length) than those from MRC5 cells (typically <5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only a negligible difference was found between the amount of doublestrand DNA breaks or the repair patch size generated in the assay relative to MRC5 extracts. Together, our data demonstrate that double strand DNA breaks are produced through formation of DNA repair patches. We refer to this process of double strand break formation as the "DNA repair patch-mediated pathway."
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