RESUMO
We have developed a method for purifying DNA fragments containing excision-repair patches which involves incorporation of biotinated deoxyuridine monophosphate into repair patches followed by isolation of biotin-containing DNA fragments using streptavidin and either isopycnic density gradient centrifugation or gel electrophoresis. Normal human fibroblasts were damaged with UV radiation, rendered permeable and allowed to perform repair synthesis in the presence of ATP, dATP, dGTP, [3H]dCTP and biotinated deoxyuridine triphosphate. The DNA was purified, sonicated to a number-average molecular weight of 150 bp, then incubated with streptavidin, a protein with a high affinity for biotin and with a density of 1.3 g/ml in cesium trifluoroacetate compared to 1.6 g/ml for DNA. Isopycnic centrifugation in cesium trifluoroacetate resulted in the separation of the streptavidin-DNA complex with little or no dissociation. The streptavidin-DNA complex was also separated from free DNA by electrophoresis in 2% agarose. This method is applicable to any type DNA damage repaired by the excision repair pathways in which thymine is present in the repair patches, including damage from chemical carcinogens and ionizing radiation.