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 abil ity of certain DNA lesions to block Tag polymerase-mediated DNA synthe sis: 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 l esions. Hence, Tag polymerase-mediated extension synthesis with Alu sp ecific primers was employed to visualize formation of discrete predict ed adducts within the element. Several variations of the Alu-primer dr iven amplification protocol were developed to monitor the following as pects of damage: (i) induction of UV-photoproducts at predicted sites within the Alu sequence, (ii) modification of extension synthesis prof iles, (iii) UV dose dependent, quantitative inhibition of Alu-primer d riven amplification. The assays reveal sites of predicted Tag polymera se blockage within the Alu sequence, a global decrease in the mean len gth 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 f eatures, in combination with the ability of UV lesions to block elonga tion by Tag polymerase, provide a novel and sensitive system for detec ting UV damage in the human genome. The system detects UV damage at le vels that are compatible with cellular DNA repair, and provides a uniq ue amplification-based protocol for probing the overall integrity of h uman DNA. (C) 1997 Elsevier Science B.V.