Assaying for the dual incisions of nucleotide excision repair using DNA with a lesion at a specific site.
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 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'- end-label 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.
Shivji
Mahmud K K
MK
Hutchison/MRC Research Centre, MRC Cancer Cell Unit, Cambridge, UK.
Moggs
Jonathan G
JG
Kuraoka
Isao
I
Wood
Richard D
RD