Alu-mediated detection of DNA damage in the human genome.
A new approach to monitoring UV damage and repair in the human genome has been developed. The proposed approach is based on a combination of features unique to interspersed repetitive Alu elements, and the ability of certain DNA lesions to block Taq polymerase-mediated DNA synthesis: namely, the extraordinary abundance of Alu repeats throughout the human genome in conjunction with distinct sequence motifs comprising long runs of T residues which are likely targets for formation of UV lesions. Hence, Taq polymerase-mediated extension synthesis with Alu specific primers was employed to visualize formation of discrete predicted adducts within the element. Several variations of the Alu-primer driven amplification protocol were developed to monitor the following aspects of damage: (i) induction of UV-photoproducts at predicted sites within the Alu sequence, (ii) modification of extension synthesis profiles, (iii) UV dose dependent, quantitative inhibition of Alu-primer driven amplification. The assays reveal sites of predicted Taq polymerase blockage within the Alu sequence, a global decrease in the mean length of extension products, and a measurable reduction in the quantity of extension products that is inversely proportional to UV dose. Thus, the exceptional abundance of Alu repeats and their primary sequence features, in combination with the ability of UV lesions to block elongation by Taq polymerase, provide a novel and sensitive system for detecting UV damage in the human genome. The system detects UV damage at levels that are compatible with cellular DNA repair, and provides a unique amplification-based protocol for probing the overall integrity of human DNA.
Englander
E W
EW
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. eengland@sbi.utmb.edu
Howard
B H
BH
D002460:Cell Line; D004247:DNA / Q000528:radiation effects; D018736:DNA Adducts / Q000032:analysis; D004249:DNA Damage; D005347:Fibroblasts; D016678:Genome; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D014466:Ultraviolet Rays
D018736:DNA Adducts; D004247:DNA