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A qPCR-based assay to quantify oxidized guanine and other FPG-sensitive base lesions within telomeric DNA
Nathan O'Callaghan, Natalie Baack, Razinah Sharif, and Michael Fenech
Nutritional Genomics and DNA Damage Research Group, Food and Nutritional Sciences, Commonwealth Scientific and Industrial Research Organization (CSIRO), Adelaide, Australia
BioTechniques, Vol. 51, No. 6, December 2011, pp. 403–412
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Telomere shortening is an important risk factor for cancer and accelerated aging. However, it is becoming evident that oxidatively damaged DNA within the telomere sequence may also cause telomere dysfunction. Here we describe a reliable, cost-effective quantitative PCR (qPCR)-based method to measure the amount of oxidized residues within telomeric DNA that are recognized and excised by formamidopyridine DNA glycosylase (FPG). We also report that in an in vitro model of oxidative stress oxidized base lesions measured using this method are more prevalent within telomeric sequences. Furthermore, this method is sufficiently sensitive to detect changes in oxidative stress induced by zinc deficiency and hydrogen peroxide within the physiological range.

Telomeres are hexamer (TTAGGG) repeats that protect the genome against chromosomal instability and accelerated cellular senescence (1,2). However, the number of telomeric repeats decreases with each cell division in somatic cells, and this process is accelerated as a result of base damage or strand breaks induced within the telomere sequence (3,4). Excessively short telomeres have been shown to lead to chromosomal instability via telomere end-fusion and the generation of breakage-fusion-bridge cycles (5,6). These changes in genome stability are important initiating events in aging-related disorders such as cancer (5,6). Furthermore, there is increasing evidence that telomere shortening and dysfunction are associated with obesity, psychological stress, immune dysregulation, cancer, cardiovascular disease, and neurodegenerative disorders such as Alzheimer disease (7-12). In telomeres, guanine is particularly sensitive to oxidation, and strand breaks within the telomere sequence leads to accelerated telomere shortening (3,4).

Reactive oxygen species (ROS) have been implicated in numerous disease states (13). Means of controlling ROS-induced damage to DNA, lipids, and proteins include antioxidants (such as vitamins C and E), as well as enzymatic mechanisms including superoxide dismutase, catalase, and glutathione peroxidase (13). In the case of oxidized DNA bases, these lesions must be rapidly identified and repaired to avoid the formation of point mutations in DNA, which could lead to pathological phenotypic changes. This is affected by the coordinated recruitment of components of the base excision repair (BER) pathway, including glycosylase, endonuclease, polymerase, and ligase proteins (13). Under certain conditions, resulting in increased levels of free radicals and/or reduced levels of antioxidants, an imbalance can occur, and the capacity for prevention and repair can become compromised, which can lead to the accumulation of damaged bases in the DNA sequence.

Both the bases and the ribose components of DNA have been identified as being susceptible to oxidative damage, however guanine residues have been shown to be particularly sensitive, with 7,8-dihydro-8-oxyguanine (8oxoGua) or the deoxyribonucleoside form of this lesion (8oxodG) being a common biomarker of oxidative stress (13). It has been estimated that 100–500 8oxoGua lesions are formed per cell per day (14). Telomere shortening has been observed in cell lines under hyperoxic stress conditions (40% oxygen partial pressure), where the rate of telomere attrition increased from 90 bp per population doubling (PD) to 500 bp per PD (14). Importantly, this study also showed that telomere lengths following hyperoxic treatment were as short as those of senescent cells (∼4 kb) (14). The mechanism for attrition was shown to be an accumulation of single-strand breaks, probably due to damage to the deoxyribose component in the phosphodiester backbone of DNA (14). The high incidence of guanine residues in telomeric DNA sequences increases the probability of the accumulation of 8oxoGua in telomeres during oxidative stress. The presence of 8oxoGua inhibits telomerase activity and diminishes binding of TRF1 and TRF2 proteins to the telomere sequence, leading to disruption of telomere length, maintenance, and function (15).

A wide range of methods have been developed to measure global base damage, including oxidation of DNA, the most common being antibody-based detection methods or analytical techniques involving HPLC or mass spectrometry. The measurement of base damage in specific DNA sequences, such as the telomere, is much more challenging, because the specific DNA sequences or repeats are usually in small abundance and need to be specifically identified and assayed at the molecular level. Kruk et al. (1995) described the use of a Southern blot–based method to identify oxidative base lesions within the telomere, however this method is laborious and requires relatively large amounts of DNA (16). A more practical approach is to use quantitative PCR (qPCR) with telomere-specific primers that uses fluorescent signal detection as the PCR proceeds and is now routinely used to measure telomere length (17,18). qPCR is an extremely sensitive method of analysis, which allows initial template levels to be precisely and accurately quantified. Recent studies have capitalized on this sensitivity, using reduced amplification efficiency to detect loss of amplicon integrity; the efficiency and specificity of this system depends on the use of base lesion-specific glycosylase treatment of DNA prior to the PCR to convert unique base lesions to abasic sites and strand breaks at that site (19). Here we describe a qPCR-based method to measure the amount of oxidized residues, as well as other base lesions within telomeric DNA that are recognized and excised by the bacterial enzyme, formamidopyrimidine DNA-glycosylase (FPG). FPG is specific for oxidized purines, including 8oxoGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FaPyGua), and 4,6-diamino-5-formamidopyrimidine (FaPyAde) and other ring-opened purines (20,21). This method is based on differences in PCR kinetics between DNA template exhaustively digested by FPG and undigested DNA, i.e., change cycle threshold (ΔCT = CT treated-CT untreated). We describe the validation of this method in in vitro models of oxidative stress and nutrient deficiency (leading to oxidative stress) and demonstrate that oxidation-induced base lesions are more prevalent within telomeric sequences relative to the total genome.

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