Abstract
Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER) in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which 12 reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analyzed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.
Highlights
The ability of cells to protect against a large variety of DNA disruptions is a vital process for living organisms
Base excision repair (BER) and nucleotide excision repair (NER) belong to the subgroup of DNA repair mechanisms that are active on structurally modified DNA bases
The inter-experimental variability given by the 12-minigel format was low, with the coefficient of variation between 7 independent experiments being 7.7% for both BER and NER
Summary
The ability of cells to protect against a large variety of DNA disruptions is a vital process for living organisms. Gene expression has been shown to be a misleading source of information, because changes in mRNA levels do not necessarily reflect changes in enzyme activity and vice versa (Damia et al, 1998; Vogel et al, 2000; Paz-Elizur et al, 2007; Stevens et al, 2008; Hanova et al, 2011; Slyskova et al, 2012a,b) This is due to extensive post-transcriptional and post-translational modifications and protein–protein interactions that take part in regulating the activity of repair proteins (Fan and Wilson, 2005; Hu and Gatti, 2011; Nouspikel, 2011). Measuring the true phenotypic endpoint seems in this context to be the most informative, straightforward, and perhaps the most reliable way of characterizing the DNA repair processes
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