Assay for Nucleotide Excision Repair Protein Activity Using Fractionated Cell Extracts and UV-Damage

Mammalian cells remove carcinogenic damage caused to DNA by ultraviolet (UV) light and certain other mutagens mainly by using the pathway known as nucleotide excision repair (NER). This involves damage recognition, unwinding of the DNA around the site of damage, incision on either side of the lesion, removal of a fragment containing the lesion, and finally DNA synthesis and ligation to form a repair patch of ∼30 nucleotides. The use of purified proteins and reconstituted systems has revealed the protein components that are essential for the core dual-incision reaction (1 ). XPA and the single-strand binding protein replication protein A (RPA) associate with each other and preferentially bind to damaged DNA. XPC, which usually exists bound to its partner protein hHR23B, is also involved in damage recognition. TFIIH, a transcription initiation complex, which includes the XPB and XPD helicases as subunits (2 ), is involved in local opening of DNA around the site of damage (3 ). The incisions are made by two structure-specific endonucleases, XPG and the ERCC1-XPF complex (4 –6 ). These proteins cleave the damaged strand of the unwound DNA 3′ and 5′ to the lesion respectively. A fragment of ∼24–32 nucleotides containing the lesion is released and the gap is filled by a DNA repair synthesis reaction involving the proliferating cell nuclear antigen-(PCNA) dependent DNA polymerase δ or ε holoenzyme (7 ) using the undamaged strand as a template, and the patch is joined by a DNA ligase.