Abstract
DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.
Highlights
It has been demonstrated that the most lethal type of DNA damages, double-strand DNA breaks (DSBs),3 are produced by exposure of DNA to ␥- or x-rays [1, 2]
Base Damage and Double-strand DNA Break Formation duced on opposite DNA strands and separated by an intervening sequence that can be denatured at 37 °C, “spaced” damages can be defined as damages produced on opposite DNA strands and separated by an intervening sequence that has a Tm value above 37 °C (Fig. 1A)
46BR cell extracts were required to be used or excess amounts of flap endonuclease-1 (FEN-1) were needed to be added to assays with extracts prepared from normal fibroblasts, MRC5, to experimentally detect DSB formation [26], these results suggest that repair of “spaced” uracils by long patch base excision repair (BER) introduces a risk of DSB formation
Summary
Cells and Cell Extract Preparation—Lymphoblastoid cell lines, GM00621 (clinically normal), GM01953 (clinically normal), GM06351 (clinically normal), GM03334 (AT heterozygote), GM09588 (AT heterozygote), GM09585 (AT heterozygote), GM02782 (AT homozygote), and GM03189 (AT homozygote), were purchased from the NIGMS Human Genetic Cell Repository (Camden, NJ). Irradiated pBS—MNNG-treated or ␥-ray-irradiated pBS (25 DSB formation reaction was carried out with 200 ng of circung/l) was incubated with 0.1 units/l of endo III for 30 min at larized genomic DNA and 50 g of protein extracts as 37 °C. Cleaved non-damaged CATGCGTGATAAGTCCTCGACTGTGCCTTCTAAAAdipBS and either MNNG-treated or ␥-ray-irradiated pBS were deoxy[32P]A-3Ј) was ligated to DSB ends by 5 units of T4 DNA mixed and incubated at 96 °C for 5 min. Endo III–Endo IV Treatment of Circularized Genomic DNA— a 20-l reaction mixture containing 20 mM Tris-HCl, pH 8.0, 1 Circularized genomic DNA (250 ng) or pBS (250 ng) exposed to mM EDTA, 1 mM dithiothreitol, and 100 g/ml bovine serum either MNNG or ␥-rays were treated with 2 units of endo III in albumin for 30 min at 37 °C. After purification of DNA by Nucleospin Extract II kit, DNA was treated with alkaline phosphatase, labeled with 32P, and fractionated on agarose gel as described under “DSB formation assay with circularized genomic DNA.”
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