Unscheduled DNA synthesis: a functional assay for global genomic nucleotide excision repair.
The unscheduled DNA synthesis (UDS) assay measures a cell's ability to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique in living cells by creating 6-4 photoproducts and pyrimidine dimers using UVC irradiation, then allowing for their repair. Repair is quantified by the amount of radioactive thymidine incorporated after this insult, and the length of time allowed for this incorporation is specific for repair of particular lesions. Radioactivity is evaluated by grain counting after autoradiography. The results are used to diagnosis repair-deficient disorders clinically and provide a basis for investigation of repair deficiency in human tissues or tumors. At the present time, no other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation, as discussed in Chapter 28, is not an equivalent technique, as it specifically measures transcription-coupled repair at active genes, a subset of total NER.
Kelly
Crystal M
CM
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Latimer
Jean J
JJ
D001345:Autoradiography; D020137:Base Pair Mismatch / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000528:radiation effects; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D016678:Genome; D006801:Humans; D011740:Pyrimidine Dimers / Q000032:analysis / Q000378:metabolism; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; D004247:DNA