New Perspectives on Unscheduled DNA Synthesis: Functional Assay for Global Genomic DNA Nucleotide Excision Repair.

Abstract

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than 3H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the 3H thymidine-based assay.