Measurement of cellular repair activities for oxidative DNA damage

Two related assays capable of determining cell extract repair activities fo r different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of ox idative damage present in the DNA. In one, singlet oxygen is used to genera te predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl ra dicals are used to generate a broad spectrum of damage including oxidized b ases and strand breaks. Assay conditions were adjusted to ensure that radio label incorporation was directly proportional to cell extract repair activi ty. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage. (C) 2000 Elsevier Science Inc.