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Updated: May 29, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
Development of quantitative DNA cleavage assay for XPG endonuclease activity using endogenous nuclear proteins in
Preeyaporn Koedrith1, Young Rok Seo
1Department of Life Science, Dongguk University-Seoul, Seoul 100-715, Republic of Korea.
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.
Insights
We developed a sensitive assay to measure the DNA repair function of Xeroderma Pigmentosum Group D (XPG) endonuclease activity. This method accurately quantifies XPG
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- Xeroderma Pigmentosum Group D (XPG) is a DNA endonuclease crucial for nucleotide excision repair (NER).
- Defects in XPG are linked to significant health issues, including skin cancer and developmental disorders.
- The endonuclease activity of XPG is considered vital for its role in DNA repair.
Purpose of the Study:
- To develop and validate a sensitive assay for quantifying XPG endonuclease activity.
- To enable quantitative monitoring of endogenous XPG activity in various cell types and tissues.
Main Methods:
- Utilized direct nuclear extracts as a source of XPG.
- Employed a novel assay involving 3'-end oligolabeling of a bubble-shaped DNA substrate.
- Optimized reaction conditions, identifying 7 mM MgCl2 and pH 6.8 as optimal.
Main Results:
- Demonstrated efficient cleavage of the model substrate by XPG in wild-type human fibroblasts and RKO colon cancer cells.
- Showcased time- and dose-dependent cleavage activity.
- Confirmed that XPG-deficient cells exhibit significantly lower cleavage activity, validating the assay's specificity.
Conclusions:
- The developed assay provides a reproducible and quantitative method for assessing XPG endonuclease activity.
- This methodology can be applied to monitor XPG function in diverse cellular contexts.
- The findings support the importance of XPG's endonuclease function in DNA repair and cellular health.
