Development of quantitative DNA cleavage assay for XPG endonuclease activity using endogenous nuclear proteins in human cell lines

Abstract

XPG, a structure-specific DNA endonuclease responsible for the 3' incision of DNA lesions during nucleotide excision repair (NER), is associated with high risk of skin cancer as well as skeletal, neurological and developmental abnormalities when functionally defective. These observations have led to the model wherein the endonuclease activity of XPG is important for NER. Herein, we first demonstrate a sensitive assay of XPG cleavage activity using direct nuclear extracts as an XPG source. This method provided quantitative evaluation of the activity of endogenous XPG endonuclease derived from cells with high reproducibility. Our new assay takes advantage of 3'-end oligolabeling of the bubble-shaped substrate. Our results demonstrate efficient cleavage of the model substrate in two XPG wild-type cell lines (human fibroblasts and RKO colon cancer cells) in a time- and dose-dependent manner. In addition, XPG-deficient cells manifested lower cleavage activity relative to normal XPG cells, indicating that the incision activity of XPG was intrinsic in our methodology. It was also found that 7 mM MgCl2 and buffer pH 6.8 resulted in optimal endonucleolytic activity. Based on these results, our modified methodology has potential for quantitative monitoring of XPG cleavage activity in any cell type or tissue of interest.