Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

• XPA is an essential protein for the repair of bulky DNA lesions through Nucleotide Excision Repair. • XPA works in complex with RPA to orient the DNA for processing. • A site-specific Cy3 fluorophore was incorporated on XPA and reports on ssDNA binding dynamics. • Detailed methods to generate fluorescent XPA and conditions to generate and maintain stable protein are described. Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido- L -phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies. [ABSTRACT FROM AUTHOR]