Detection and Measurement of Nucleotide Excision Repair Synthesis by Mammalian Cell Extracts in Vitro

Nucleotide excision repair of damaged DNA can be carried out in a cell-free environment, making the process amenable to biochemical study and manipulation. In this article, methods for a versatile repair synthesis assay are described. Protocols are given for preparing plasmid DNA substrates, preparing whole cell extracts, performing repair synthesis reactions, and analyzing the data obtained. Methods are also described for in vitro complementation of repair synthesis defects and for carrying out two-stage incision/synthesis reactions.