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Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay

Lookup NU author(s): Joanna GÓRNIAK, Dr Kerry Cameron, Dr Kevin WaldronORCiD, Professor Thomas von Zglinicki, Professor John Mathers, Dr Sabine Langie

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Abstract

DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

Publication metadata

Author(s): Gorniak JP, Cameron KM, Waldron KJ, von Zglinicki T, Mathers JC, Langie SAS

Publication type: Article

Publication status: Published

Journal: Mutagenesis

Year: 2013

Volume: 28

Issue: 6

Pages: 673-681

Print publication date: 01/11/2013

Online publication date: 04/10/2013

Acceptance date: 28/08/2013

ISSN (print): 0267-8357

ISSN (electronic): 1464-3804

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/mutage/get047

DOI: 10.1093/mutage/get047

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