Comet assay-based methods for measuring DNA repair <Emphasis Type="Italic">in vitro</Emphasis>; estimates of inter- and intra-individual variation

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

DNA single-strand breakage in mammalian cells induced by redox cycling quinones in the absence of oxidative stress

Quinone-induced cell death is often attributed to oxidative stress during which the formation of DNA strand breaks is thought to play an important role. In this study, extensive DNA damage was observed in human chronic myelogenous leukemic cells (K562) exposed for 15 minutes to low concentrations (15–100 μM) of the redox cycling quinones 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) and menadione. However, DNA strand breakage and cell death could not be attributed to oxidative stress as the intracellular level and redox status of the reducing equivalents NADP(H) and GSH were unaffected. The intracellular level of NAD⁺ was found to correlate well with the extent of DNA repair (r = 0.93, P < 0.02) and cell proliferation (r = 0.96, P < 0.01) in cells exposed to the quinones. In contrast, a significant decrease in the level of intracellular ATP was only observed in cells exposed to menadione (50–100 μM). These results suggest that redox cycling quinones are capable of inducing DNA damage in mammalian cells by a mechanism that does not involve oxidative stress. Following DNA damage, cell death is dependent on the availability of NAD⁺, which may be key to the rapid repair of strand breaks. © 1995 John Wiley & Sons, Inc.     

Poly(etheno ADP-ribose) blocks poly(ADP-ribose) glycohydrolase activity

Poly(ADP-ribose) is a biopolymer synthesized by poly(ADP-ribose) polymerases. Recent findings suggest the possibility for modulation of cellular functions including cell death and mitosis by poly(ADP-ribose). Derivatization of poly(ADP-ribose) may be useful for investigating the effects of poly(ADP-ribose) on various cellular processes. We prepared poly(etheno ADP-ribose) (poly(epsilonADP-ribose)) by converting the adenine moiety of poly(ADP-ribose) to 1-N(6)-etheno adenine residues. Poly(epsilonADP-ribose) is shown to be highly resistant to digestion by poly(ADP-ribose) glycohydrolase (Parg). On the other hand, poly(epsilonADP-ribose) could be readily digested by phosphodiesterase. Furthermore, poly(epsilonADP-ribose) inhibited Parg activity to hydrolyse ribose-ribose bonds of poly(ADP-ribose). This study suggests the possibility that poly(epsilonADP-ribose) might be a useful tool for studying the poly(ADP-ribose) dynamics and function of Parg. This study also implies that modification of the adenine moiety of poly(ADP-ribose) abrogates the susceptibility to digestion by Parg.     

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: Comparison with quercetin

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells’ capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.     

Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview

There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Poly(ADP-ribose) polymerase activity in intact or permeabilized leukocytes from mammalian species of different longevity

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme which uses NAD as substrate. We have previously tested PARP activity in permeabilized mononuclear blood cells (MNC) from 13 mammalian species as a function of the species-specific life span. A direct and maximal stimulus of PARP activation was provided by including saturating amounts of a double-stranded ollgonucleotide in the PARP-reaction buffer. The data yielded a strong positive correlation between PARP activities and the species' maximal life spans (r=0.84; p0.001). Here, we investigated the formation of poly(ADP-ribose) inliving MNC from two mammalian species with widely differing longevity (rat and man) by immunofluorescence detection of poly(ADP-ribose). The fraction of positive cells was recorded, following -irradiation of intact MNC, as a semiquantitative estimation of poly(ADP-ribose) formation. Human samples displayed a significantly higher percentage of positivity than did those from rats, consistent with our previous results on permeabilized cells. While rat MNC had a higher NAD content than human MNC, the number of radiation-induced DNA strand breaks was not significantly different in the two species. Since poly(ADP-ribosyl)ation is apparently involved in DNA repair and the cellular recovery from DNA damage, we speculate that the higher poly(ADP-ribosyl)ation capacity of long-lived species might more efficiently help to slow down the accumulation of unrepaired DNA damage and of genetic alterations, as compared with short-lived species. (Mol Cell Biochem138: 85–90, 1994)     

Chronic exposure to sublethal doses of radiation mimetic Zeocin selects for clones deficient in homologous recombination

Research indicates that exposure to hypoxia is associated with oxidative stress. In this investigation, healthy subjects were exposed to hypoxia by inhalation of 10% oxygen for 2 h (corresponding to 5500 m above sea level). The levels of strand breaks and oxidatively damaged purine bases, measured by the comet assay, and the expression of genes involved in DNA repair of oxidatively damaged DNA were investigated in mononuclear blood cells (MNBC) at baseline, after 2 h of hypoxia, 2 h of reoxygenation, and 1 day and 8 days after the exposure. The level of strand breaks and oxidized purine bases in MNBC increased following both the 2 h of hypoxia and the 2 h reoxygenation period, whereas this effect was not observed in unexposed subjects. The expressions of oxoguanine DNA glycosylase 1 (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), nei endonuclease VIII-like 1 (NEIL1), and mutY homolog (MUTYH) were unaltered throughout the experiment in both groups of subjects, indicating that DNA repair genes are not up-regulated by the hypoxia and reoxygenation treatment. Taken together, this report shows that inhalation of 10% oxygen for 2 h is associated with increased number of oxidized DNA lesions in MNBC, but acute hypoxia may not inflict upon the regulation of genes involved in repair of oxidized DNA.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Differential Localisation of PARP-1 N-Terminal Fragment in PARP-1+/+ and PARP-1?/? Murine Cells

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 N-terminal fragment encompassing NLS in PARP-1^+/+ and PARP-1^−/− mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-1^+/+ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-1^−/− cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences

Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5′-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5′-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences.