DNA repair patch mediated double-strand DNA break formation in human cells

doi:10.1074/jbc.M003126200

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Papers In Press, published online ahead of print May 25, 2000
J. Biol. Chem, 10.1074/jbc.M003126200
Submitted on April 12, 2000
Revised on May 19, 2000
Accepted on May 24, 2000

DNA repair patch mediated double-strand DNA break formation in human cells

Stéphane Vispé and Masahiko S Satoh

Sante et Environnement, CHUL, Ste-Foy, Quebec G1V 4G2

Corresponding Author: Masahiko.Sato{at}crchul.ulaval.ca

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 bp 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 less than 5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only negligible difference was found between the amount of double-strand 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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