Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems

Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been studied in vitro using cell extracts or highly purified repair factors and radiolabeled DNA of known sequence with DNA damage at a defined site. This chapter describes procedures for preparation of DNA substrates designed for analysis of damage recognition, either the 5' or the 3' incision event, excision (resulting from concerted dual incisions), and repair synthesis. Excision in Escherichia coli is accomplished by the three-subunit Uvr(A)BC excision nuclease and in humans by six repair factors: XPA, RPA, XPC(.)hR23B, TFIIH, XPF-ERCC1, and XPG. This chapter outlines methods for expression and purification of these essential repair factors and provides protocols for performing each of the in vitro repair assays with either the E. coli or the human excision nuclease.