By Philip Hanawalt (Eds.)
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Additional info for DNA Repair Mechanisms
Added T4 endonuclease V, but not pancreatic DNase I, promotes UV dependent repair synthesis. Our results support the view that the incision step in the excision repair sequence is defective in xeroderma pigmentosum (group A) and they demonstrate the utility of permeabilized mammalian cells for probing DNA repair with exogenous enzymes and substrates. INTRODUCTION The variety of pathways that have been proposed for negotiating structural defects, such as pyrimidine dimers, in the DNA of viable systems can be reduced to a small number of essential approaches (1).
Dimers are detected most directly by chromatographic procedures following the acidic hydrolysis of DNA, but such procedures are relatively insensitive at low doses. A more sensitive approach uses endonucleolytic activities from Micrococcus luteus to make single-strand incisions near pyrimidine dimers (1). We have investigated the suitability of a similar technique for the detection of pyrimidine dimers in near-UV-irradiated Chinese hamster ovary (CHO) cells, and have used this technique to examine dimer excision in CHO cells.
The monolayer is scraped down and pipetted against the plate 4 to 5 times in the reserved buffer, and held at 0°. Nuclei are harvested from combined similarly treated plates by centrifugation at 2500 rpm for 3 min at 20° in an International UV centrifuge and then resuspended in fresh buffer by brief vortexing. 1 mM each dATP, dCTP, dGTP, 10 μΜ BrdUTP, 3 30 mM HEPES and 2 μΜ[ Η]-άΤΤΡ at 100 pCi/ml. 8 at 37°. 5% sodium dodecyl sulfate, 50 μg/ml Proteinase Κ and incubated 2 hr at 50°. The digests are sheared by 2 passes through a 25 gauge needle and the DNA is resolved into parental and normally replicated (hybrid) bands by equilibrium sedimentation in neutral CsCl gradients.
DNA Repair Mechanisms by Philip Hanawalt (Eds.)