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Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily and specifically measured by detecting the 24-32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern blot hybridization. The second method is to 3′- endlabel the excised oligonucleotide directly with radiolabeled dNTP and a DNA polymerase, using a complementary oligonucleotide with a 5′-overhang that serves as a template. This protocol is fast and sensitive, but relies on accurate foreknowledge of the site of 3′-incision for the particular lesion being used. [ABSTRACT FROM AUTHOR]

Cells remove a wide array of potentially toxic and mutagenic lesions from their genomes by a major repair pathway called nucleotide excision repair (NER). This repair process involves a multiprotein nuclease complex that incises a damaged DNA strand on the 5′- and 3′-sides of a lesion (1). In humans, defects in the genes that participate in NER can lead to a rare recessive disorder, xeroderma pigmentosum (XP). Hypersensitivity to sunlight and a predisposition to skin cancer are the most characteristic traits of XP patients. NER-defective XP cells belong to one of seven genetic complementation groups (A-G). Although the NER process in mammalian cells consistently leads to the excision of damaged DNA fragments 24-32 nucleotides in length, the exact positions of the 5′- and 3′-incisions depend on the lesion being repaired. For instance, during the removal of a thymine dimer, incisions are made at the 22nd-24th phosphodiester bond on the 5′-side of the lesion and at the 5th phosphodiester bond on the 3′-side of the lesion (2). The main sites of incision during repair of DNA containing a 1,3-intrastrand d(GpTpG)-cisplatin crosslink are at the 16th-20th phosphodiester bond 5′- and the 8th-9th phosphodiester bond on the 3′-side of the lesion (3). [ABSTRACT FROM AUTHOR]