TY - JOUR
AU - Collins, Andrew, R.
AB - Abstract The comet assay (single-cell gel electrophoresis) is now the most popular method for measuring low levels of damage in cellular DNA. Cells are embedded in agarose on a microscope slide and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. The % of DNA in the tail reflects the break frequency. Digestion of nucleoid DNA with lesion-specific endonucleases extends the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with a genotoxic agent, incubating them and using the comet assay to follow the removal of the damage. An important feature of the assay is that damage is detected at the level of individual cells. The comet assay can be combined with fluorescent in situ hybridization, using labelled probes to particular DNA sequences, and DNA damage and repair can be examined at an even finer level of resolution. Here, we provide a general review of the technique, answer some technical and theoretical questions and give examples of applications of the method. Introduction The alkaline comet assay (single-cell gel electrophoresis) is now the method of choice, if low levels of DNA damage are to be measured—for example in lymphocyte samples from human population or intervention studies or in in vitro or in vivo genotoxicity testing or investigations into the mechanisms of DNA damage and DNA repair. Cells are embedded in low-melting point agarose on a microscope slide and lysed in a solution containing Triton X-100, to break down membranes, and high salt, to remove histones and other soluble proteins. This leaves supercoiled DNA attached to a nuclear matrix—a structure known as a nucleoid (1). On electrophoresis, at high pH, DNA is attracted towards the anode but migration occurs only if breaks (or alkali-labile abasic sites) are present. The migrating DNA from each cell forms a comet-like structure when viewed by fluorescence microscopy with a suitable stain, and the proportion of DNA in the tail indicates the frequency of breaks (Figure 1A). The advantages of the assay include simplicity, speed, sensitivity, applicability to different cell types and kinds of DNA damage, avoidance of radioactive labelling and—perhaps most significantly—the fact that damage is assessed at the level of individual cells. Fig. 1 Open in new tabDownload slide General principles of the comet assay combined with FISH. Fig. 1 Open in new tabDownload slide General principles of the comet assay combined with FISH. As an example of this assay's versatility, it has been modified to study different kinds of damage. At the nucleoid stage, an incubation with a lesion-specific endonuclease takes place to reveal types of damage other than simple strand breaks. Formamidopyrimidine DNA glycosylase (FPG) is used to detect oxidized purines (mainly 8-oxoGua), endonuclease III for oxidized pyrimidines, T4 endonuclease V for ultraviolet (UV)-induced cyclobutane pyrimidine dimers and AlkA for alkylation damage. Another application of the comet assay is the study of repair of different kinds of DNA damage. Cells are incubated after treating them with a specific damaging agent, and the damage remaining (e.g. strand breaks or enzyme-sensitive sites) is measured at intervals during the incubation (2). It is commonly believed that the neutral version of the assay (electrophoresis performed at pH of around 7) detects only double-strand breaks, while the alkaline version, at pH >12, detects also single-strand breaks. Some history is helpful here. Thirty years ago, Cook and Brazell (1,3) described the nucleoids produced by lysing cells in high salt and detergent and produced a model of DNA structure in which the DNA is attached at intervals to a scaffold or matrix. The linear DNA molecule is in effect constrained as a series of loops of on average 100 kb (4), which retain the supercoiling of the nucleosomes because they are not free to rotate. One strand break in the loop is enough to relax the supercoiling. Cook et al. (5) observed the relaxed DNA spilling out into a ‘halo’ when breaks were introduced. The first example of the comet assay (not referred to by that name) was published by Ostling and Johanson in (6). They damaged DNA with ionizing radiation and detected a tail at a dose as low as 0.25 Gy. They ascribed the creation of the tail to relaxation of supercoils, referring to the early nucleoid studies. In effect, the halo was pulled towards the anode and became the tail. A few years later, the alkaline comet assay was devised by Singh et al. (7). They also used ionizing radiation to damage DNA. The range of doses used was similar to that employed by Ostling and Johanson—and damage was detected at a dose of 0.25 Gy. Thus, the neutral and alkaline versions of the assay appear to be equally good at detecting ionizing radiation damage. Since the yield of double-strand breaks is ∼5–10% of that of single-strand breaks, it is evident that under neutral conditions, single as well as double-strand breaks are responsible for the tail and relaxation of the supercoiled DNA loops seems to be the obvious explanation. There is another, more elaborate and uncommon version of the neutral assay, involving removal of proteins by prolonged digestion with protease at 50°C, which is credibly claimed to detect only double-strand breaks (8). The special feature of the comet assay is the ability to study DNA damage in individual cells. By combining comets with fluorescent in situ hybridization (FISH) and applying labelled probes to particular DNA sequences, an even finer level of resolution could be achieved (Figure 1B). Although this possibility was recognized early on, relatively few FISH-comet papers have been published until now. FISH probes Various probes have been developed and applied in standard FISH techniques and then with comets. The type of probe depends on which target sequences are to be detected. Centromere, telomere and ribosomal DNA repeats, as well as short interspersed repetitive elements (SINEs) and long interspersed repetitive elements (LINEs), are the repetitive sequences most widely used as FISH probes. They give strong, easily detected signals and so are an attractive choice, but the range of applications is limited. Strong signals are seen also with other non-gene-specific probes: chromosome arm- or band-specific painting probes (DNA from microdissected chromosomes), as well as whole-chromosome painting probes (DNA from flow-sorted chromosomes). All these types of probes are available commercially. Probes for specific DNA sequences can consist of polymerase chain reaction products, cDNAs or genomic DNA cloned in cosmids, P1 artificial chromosomes, bacterial artificial chromosomes (BACs) or yeast artificial chromosomes. Large unique probes that are not commercially available can be prepared for FISH using standard molecular biology techniques. Genomic DNA clones can be obtained from various laboratories dealing with genome mapping and sequencing. Of these genomic probes, BAC clones are especially valuable as they were used to construct well-characterized and sequenced genomic libraries (9) and so specific sequences can be precisely targeted. The insert size in such libraries is generally in the 100–400 kb size range (10). These clones are easy to work with, but—as large genomic DNA fragments—they contain repetitive sequences, which may result in a high background. To suppress these repetitive sequences, blocking DNA containing a high fraction of repeats can be added to the hybridization reaction. Another possible explanation of high background signals may be the need to amplify the signals with fluorescent antibodies, which also amplifies ‘noise’. In some cases, these technical obstacles can be avoided by using chemically synthesized oligonucleotide probes, which are rapidly delivered to a target, any unbound probe subsequently being thoroughly eliminated during the washing steps. Peptide nucleic acid (PNA) probes are synthetic analogues of DNA in which, instead of a phosphodiester backbone, the bases are attached via methylene carbonyl bonds to repeating units of N-(2-aminoethyl) glycine (11). Because there are no charged phosphate groups, PNA–DNA duplexes have enhanced stability when used as FISH probes. Another useful design of probe is the ‘padlock probe’—a linear oligonucleotide designed so that the two end segments, connected by a linker region, are complementary—in opposite orientations—to adjacent target sequences (12). On hybridization, the two juxtaposed probe ends can be joined by a DNA ligase, circularizing the padlock probe and leaving it physically catenated to the target sequence. The reaction requires a perfect match between the probe ends and the target sequence and therefore, it is stable and extremely specific. By amplifying circularized padlock probes through in situ rolling circle DNA synthesis (Figure 2), specifically reacted probes are readily distinguished from non-specifically bound probe molecules. The crucial feature of these probes when applied to comets is that the reaction steps are performed at 37°C so that there is no tendency for the agarose to melt or become unstable. Fig. 2 Open in new tabDownload slide Target-primed rolling-circle amplification (RCA) of padlock probes in situ (reproduced from reference 20, Shaposhnikov, S., Larsson, C., Henriksson, S., Collins, A. and Nilsson, M. (2006) Detection of Alu sequences and mtDNA in comets using padlock 620 probes. Mutagenesis, 21, 243–247. Oxford University Press). (A) Overview of procedure. The target DNA is made single stranded and available for hybridization using specific restriction digestion and 5′ exonucleolysis. Target-matched padlock probes are circularized by sequence-specific ligation and reacted probes are amplified using RCA. (B) Image of comet after hybridization with padlock probe to an Alu sequence. The RCA products are visualized by hybridization of fluorescence-labelled oligonucleotides and form sub-micron-sized objects that can be observed using fluorescence microscopy. Fig. 2 Open in new tabDownload slide Target-primed rolling-circle amplification (RCA) of padlock probes in situ (reproduced from reference 20, Shaposhnikov, S., Larsson, C., Henriksson, S., Collins, A. and Nilsson, M. (2006) Detection of Alu sequences and mtDNA in comets using padlock 620 probes. Mutagenesis, 21, 243–247. Oxford University Press). (A) Overview of procedure. The target DNA is made single stranded and available for hybridization using specific restriction digestion and 5′ exonucleolysis. Target-matched padlock probes are circularized by sequence-specific ligation and reacted probes are amplified using RCA. (B) Image of comet after hybridization with padlock probe to an Alu sequence. The RCA products are visualized by hybridization of fluorescence-labelled oligonucleotides and form sub-micron-sized objects that can be observed using fluorescence microscopy. Visualization and scoring The visualization and scoring of FISH comets are not easily automated. The signals are complex and vary from cell to cell: scoring is not simply a matter of recording the proportion of comets with signals, the distribution between head and tail or even the number of signals per comet, as individual attention to each comet is required to identify and quantitate the signals. Furthermore, the position and orientation of the signals within the comet and in relation to each other are important observations that rely on human interpretation. However, automated systems for detection of various objects at the microscopic level are rapidly evolving, and it is likely that scoring of at least certain kinds of FISH signals will be automated in the near future. An important difference between standard chromosomal FISH experiments and FISH comets, that affects the ease of scoring, is the spatial distribution of the preparations and, in consequence, of the observed signals. In standard FISH studies, DNA preparations are normally fixed or adsorbed onto microscope slides, and so the objects observed are two-dimensional. Comet preparations, however, exist in three-dimensional (3-D) space. The original organization of the chromatin is of course altered by electrophoresis, but the signals from FISH are still in a 3-D matrix. This 3-D organization of the DNA in comet preparations is a positive feature, as it certainly reflects to some degree the real organization of chromatin in the living cell; but on the other hand, it leads to serious difficulties in the process of visualizing and scoring the signals. Also, it is impossible to obtain realistic images in photographs that represent only one plane. How many signals to expect? Our understanding of comet DNA organization is based on the hypothesis that the topology of DNA in comets reflects the topology of DNA in living cells. It is therefore expected that the number of signals observed in comets will be comparable with the number of signals detected in living cells or chromosome spreads, the only limitation being detection efficiency. Our results with chromosome 16 probes have shown that the number of signals in neutral and alkaline electrophoresis is comparable with the number expected; denaturation of DNA in the alkaline version of the assay doubles the number of signals relative to the number seen under neutral conditions since each strand of DNA will act as a target for the FISH probe. The number of signals is an issue that recurs later in this article. FISH provides insight into the structure of chromatin FISH was first combined with the comet assay by Santos et al. (13), who investigated the behaviour of centromeric and telomeric DNAs under electrophoresis. They hybridized comets from neutral electrophoresis with probes to all centromeres, all telomeres, chromosome-specific centromere and telomere DNA and three segments of the gene MGMT, which codes for the DNA repair enzyme O6-methylguanine methyltransferase. Rather than using a conventional DNA stain such as 4′,6-diamidino-2-phenylindole, they visualized comet tails by hybridization with labelled whole human DNA. It is not stated whether the cells had been treated with a DNA-breaking agent or whether breaks arose during the RNase/proteinase K digestion that followed cell lysis. This information is relevant because it is known that chromatin organization in living cells is dynamic and depends on the status of DNA replication, repair and expression (14). All these can be affected by treatment with DNA-breaking agents. Telomere probes were predominantly seen over the head of the comet, consistent with their known association with the nuclear membrane. The size of telomeric sequences is ∼15–50 kb. Centromere DNA is much longer (with a size of several megabases) and gave signals best described as long strings of dots, extending into the comet tail. The signals from MGMT probes were found in both head and tail of comets and generally formed a linear array corresponding to the three gene segments. The MGMT gene is located close to the telomere and therefore was expected to be found near the nuclear membrane. But proximity is relative; it is now known from the complete sequence of the human genome that MGMT is ∼4 Mb from the end of chromosome 10—several loop lengths away from the telomere. The length of a typical comet tail—around 100 μm—is equivalent to 400 kb of (fully extended) DNA. The MGMT gene has a total length of 170 kb. In our experiments with this same gene, using the alkaline comet assay, we found that one end was indeed almost always located in or near the comet head, but the other end appeared in the tail with a frequency consistent with the number of strand breaks (relaxed loops) present (15). Our tentative explanation is that the MGMT DNA sequence may include a ‘matrix-associated region’ (MAR) that causes it to remain associated with the comet head. Arutyunyan et al. (16,17) were also interested in telomeric DNA and its sensitivity to damage induced by drugs used in cancer therapy. They used telomere-specific PNA probes with comets developed from human leukocytes by the alkaline method. After bleomycin treatment, appearance of telomere probes in the tail parallelled total DNA migration, i.e. there was no evidence for preferential damage to telomere DNA (16,17). Mitomycin C (MMC)-treated cells showed a similar pattern, i.e. parallel dose response curves for % of telomere probes and % of total DNA in the tail, although at higher doses of MMC (>25 μg/ml) migration decreases again—as expected in view of the ability of MMC to create DNA–DNA crosslinks which hamper unwinding (16). In contrast, cisplatin—another cross-linking reagent—reduced bleomycin-induced migration to the tail to a greater extent for telomeric DNA than for total DNA, the implication being that cisplatin has a preferential tendency to attack telomeric DNA (17). Probes for 12 of the human chromosome set were applied by Rapp et al. (18) to comets from UV(A)-irradiated lymphocytes. They found that chromosomes with the highest density of active (expressed) genes were most likely to be found in the head. The authors suggested, as a working hypothesis, that chromosomes with a high gene density are more resistant to DNA-damaging agents and/or better able to repair the damage. An alternative explanation would be simply in terms of the association of active gene-rich regions of DNA with the sites of transcription, which are located on the surface of the nuclear matrix (14,19), i.e. in the head of the comet. There is a tendency to refer to DNA in the comet tail as damaged DNA and DNA in the head as undamaged. FISH experiments indicate that this is an over-simplification; clearly, there are other factors, apart from the presence of lesions, determining where a particular sequence of DNA will be located, i.e. whether it is able to ‘escape’ into the tail. These factors include the nature of the damage and the organization of the chromatin. FISH helps to explain how comets are formed Certain questions relating to the behaviour of DNA within the comet, and the principles underlying the assay, can be addressed using FISH. It is sometimes asked whether damage to mitochondrial DNA (mtDNA) can be detected. There are a priori reasons why not since mtDNA is not attached to the nuclear matrix—or even located in the nucleus. It is a small molecule (<17 kbp) compared with the size of loops of genomic DNA. In an empirical test, we identified mtDNA using specific padlock probes (20). Prior to lysis of cells embedded in agarose, numerous signals were seen, clustered close to the counter-stained nucleus. After a few minutes of lysis, signals had moved away from the nucleus, and this dispersal continued during subsequent stages of the comet assay, until after electrophoresis, they appeared to be randomly distributed over the gel. In contrast, FISH with probes to Alu sequences, which occur throughout the genomic DNA, colocalized with tail DNA, showing no tendency to disperse. Recently, we compared the FISH signals obtained by applying genomic probes from human chromosome 16q to the DNA of comets from either neutral or alkaline electrophoresis of cells damaged with H2O2, which induces single-strand breaks with very few double-strand breaks (21). The sequence probed—with a length of ∼100 kb—was equivalent to a substantial fraction of the tail length. After neutral electrophoresis, signals appeared as linear chains of dots, entirely consistent with the idea that the tail consists of DNA molecules extending out from the nucleoid core towards the anode (Figure 3A–C). The sequence includes a MAR (S. Shaposhnikov, B. Salenko, E. Frengen, P. D. Thomsen, V. Zverev, H. Prydz and A. R. Collins, in preparation) and indeed we noted that the chain of dots typically remained associated at one end with the comet head. After alkaline electrophoresis, signals typically appeared as single dots, with very few chains (Figure 3D–F). Although this seems to indicate that the DNA has undergone further fragmentation, when probes to adjacent target regions of human chromosome 16q (with a combined length of ∼200 kb) were hybridized to DNA from alkaline comets, the two colours used to identify the probes tended to appear close together (Figure 3F). If further fragmentation had occurred, the signals would have been separated. Fig. 3 Open in new tabDownload slide Different types of signal seen in FISH experiments with comet DNA after neutral and alkaline electrophoresis (reproduced from reference 21, Shaposhnikov, S. A., Salenko, V. B., Brunborg, G., Nygren, J. and Collins, A. R. (2008) Single-cell gel electrophoresis (the comet assay): loops or fragments? Electrophoresis 29, 3005–3012; with permission, Copyright Wiley-VCH Verlag GmbH & Co. KGaA). (A) Probe RPCI-1 213H19 labelled with biotin, with neutral comets from cells treated with 0.1 mM H2O2; (B) Probes RPCI-1 213H19 and RPCI-6 32H24 labelled with digoxigenin (green) and biotin (red), respectively, with neutral comets from cells irradiated with UV(C) at 0.2 Jm−2; (C) Probe RPCI-6 32H24 labelled with biotin, with neutral comets from cells treated with 0.1 mM H2O2; (D) Probe RPCI-1 213H19 labelled with two colours (digoxigenin, green and biotin, red), with alkaline comets from cells irradiated with UV(C) at 0.2 Jm−2; (E) Probe RPCI-1 213H19 labelled with biotin, with alkaline comets from cells treated with 0.1 mM H2O2; (F) Probes RPCI-1 213H19 and RPCI-6 32H24 labelled with digoxigenin (green) and biotin (red), respectively, with alkaline comets from cells irradiated with UV(C) at 0.2 Jm−2. Fig. 3 Open in new tabDownload slide Different types of signal seen in FISH experiments with comet DNA after neutral and alkaline electrophoresis (reproduced from reference 21, Shaposhnikov, S. A., Salenko, V. B., Brunborg, G., Nygren, J. and Collins, A. R. (2008) Single-cell gel electrophoresis (the comet assay): loops or fragments? Electrophoresis 29, 3005–3012; with permission, Copyright Wiley-VCH Verlag GmbH & Co. KGaA). (A) Probe RPCI-1 213H19 labelled with biotin, with neutral comets from cells treated with 0.1 mM H2O2; (B) Probes RPCI-1 213H19 and RPCI-6 32H24 labelled with digoxigenin (green) and biotin (red), respectively, with neutral comets from cells irradiated with UV(C) at 0.2 Jm−2; (C) Probe RPCI-6 32H24 labelled with biotin, with neutral comets from cells treated with 0.1 mM H2O2; (D) Probe RPCI-1 213H19 labelled with two colours (digoxigenin, green and biotin, red), with alkaline comets from cells irradiated with UV(C) at 0.2 Jm−2; (E) Probe RPCI-1 213H19 labelled with biotin, with alkaline comets from cells treated with 0.1 mM H2O2; (F) Probes RPCI-1 213H19 and RPCI-6 32H24 labelled with digoxigenin (green) and biotin (red), respectively, with alkaline comets from cells irradiated with UV(C) at 0.2 Jm−2. This two-colour experiment was performed with comets from UV(C)-irradiated cells; the pyrimidine dimers produced by the UV were converted to breaks by T4 UV endonuclease V digestion. In another experiment with UV(C), we used a single probe to the 100 kb sequence and counted the number of signals per cell. The dose of 0.2 Jm−2 produces 1.2 UV endonuclease-sensitive sites per 106 bases or 0.12/100 kb, and so the chance of a break occurring within the probed region was only one in eight (21). Approximately twice as many signals (mostly dots) were seen after alkaline electrophoresis as after neutral electrophoresis (mostly chains). This is to be expected since the denatured DNA provides two strands for hybridization. The average numbers of signals seen per cell corresponded closely with the numbers expected in an exponentially growing culture of cells. Thus, the dots and chains seem to contain equal lengths of DNA, dependent simply on the breaks induced by UV + endonuclease. If the signals after alkaline electrophoresis are not from further-fragmented DNA, why do they appear as dots rather than chains? We assume that DNA coalesces into condensed foci under alkaline electrophoresis or during subsequent neutralization. It is true that under conventional staining, alkaline comets have a granular appearance. In a recent report, condensed foci are also seen with an immunostaining technique (22). Whether the coalescence is accompanied by shearing of the loops from the matrix is not evident from our experiments. However, Klaude et al. (23) found that the DNA in the tail after electrophoresis in one direction behaved during subsequent electrophoresis at 90°C to the original direction as if it was detached from the matrix. FISH comets and human disease FISH has been employed with different kinds of probes to attempt to link the aetiology of different cancers to particular DNA susceptibility. The whole-chromosome approach employs chromosome-painting probes (developed from chromosome-specific DNA and available for each human chromosome) to identify DNA from particular chromosomes within the comet. A higher level of resolution is achieved by using probes to chromosome regions. Probes to individual genes give the highest level of resolution. Harréus et al. (24) obtained biopsies of apparently healthy mucosa from patients with oropharyngeal carcinoma and from patients undergoing tonsillectomy. Tissue was enzymically disaggregated, and the cells were treated with benzo[a]pyrene diolepoxide to induce damage before performing the alkaline comet assay. As they chose to use tail moment to assess damage, it is not clear how extensive tail migration was, but there was no difference between comets from the two patient groups. FISH was performed with whole-chromosome paints for chromosomes 1, 3, 5 and 8. In undamaged comets, fluorescent signals were typically two small, closely associated spots—surprisingly localized, in view of the large amount of DNA in even the smallest chromosomes. When damage was present, resulting in a comet tail, the appearance of specific chromosome signals as a series of spots in the tail was taken as an indication of fragmentation within that chromosome. In cells from tonsillectomy patients, signals from all four chromosome probes were equally likely to appear in the comet tail, whereas in cells from cancer patients, chromosomes 3, 5 and 8 appeared more frequently in the tail than did chromosome 1. In a useful review article, Glei et al. (25) describe several studies of the relative susceptibility of cancer-related genes (APC, KRAS and TP53) to damage by a variety of genotoxic model compounds known to induce oxidative stress. In general, these genes showed higher sensitivity—indicated by a preferential location in the comet tail. Acute myeloid leukaemia is associated with previous occupational exposure to benzene or exposure to chemotherapeutic drugs such as melphalan, an alkylating agent, or the topoisomerase II inhibitor etoposide. Different exposures are typified by different patterns of chromosome aberration in the leukocytes; melphalan commonly causes deletion of chromosome 5q31, while translocations involving 11q23 are associated with etoposide. Benzene is associated with damage to various chromosomes, and its mechanism of action (via metabolites hydroquinone and 1,4-benzoquinone) is unclear. Escobar et al. (26) treated human lymphoblastoid cells TK6 with hydroquinone, melphalan or etoposide. The cells were developed as comets and hybridized with probes specific for 5q31 and 11q23. For each damaging agent, appearance of specific fluorescence signals in the tail was compared for the two probes. Perhaps surprisingly, melphalan and etoposide did not show significant associations with damage to either probe, while hydroquinone produced a significant excess of 5q31 signals in the tail. Again, it is not clear why certain DNA sequences should have a greater tendency to incur damage than others. Kumaravel and Bristow (27) irradiated normal and breast cancer cell lines with 10 Gy of γ-radiation to give ∼30–40% tail DNA after neutral electrophoresis. (The neutral method included a protease digestion and is probably comparable with that of Olive et al. (8), which is claimed to detect only double-strand breaks.) FISH was carried out with probes for chromosome loci associated with breast cancer—HER-2/neu, ZNF217 and p53. ZNF217 signals rarely appeared in the tail in normal or cancer cell lines. However, HER-2/neu and p53 signals were more likely to appear in the comet tails from cancer cell lines compared with those from normal cell lines. This may indicate a disruption of the normal organization of these DNA sequences so that they are no longer associated with the nuclear matrix in the cancer cell lines—a pattern referred to as genetic instability by the authors; the alternative explanation that these sequences are preferentially damaged by γ-radiation is unlikely, in view of the nature of this physical damaging agent. Whether the suggested genetic instability is relevant to the aetiology of breast cancer cannot be answered since these cell lines are already transformed. It is also unclear whether the instability is specifically linked with breast cancer, as cell lines representing other kinds of cancer were not examined. Identifying specific genes and following their repair If a probe for a particular gene gives a signal in the tail of the comet, the implication is that there is a break (or enzyme-sensitive site) in the region of that gene. According to the ‘loop model’ of comet formation, there is a break in the loop containing the gene (not necessarily in the gene itself). Different regions of the DNA may be more or less susceptible to damage, and so the likelihood of signals appearing in the tail after a given dose of damage might vary from gene to gene. With the conventional comet assay, we can monitor cellular DNA repair by treating cells with a damaging agent, incubating them and measuring the tail intensity (equivalent to lesion frequency) at intervals. Typically, in cultured animal cells (including human), strand breaks induced by H2O2 or ionizing radiation are rejoined with a t½ of ∼10 min. Oxidized bases and UV-induced pyrimidine dimers are repaired over a period of hours. (For reasons that are not clear, freshly isolated human lymphocytes rejoin breaks and other lesions more slowly.) While the decrease in tail intensity with incubation represents repair of the genome overall, it is possible to compare the kinetics of repair of a specific gene (or, more accurately, of the DNA region containing the gene) by following the ‘retreat’ of the gene-specific signals from tail to head over the incubation period. McKenna et al. (28) examined the repair of the TP53 gene in human tumour cells, following γ-irradiation (with 5 Gy). FISH was performed with a probe spanning a 200-kb sequence including the TP53 gene. McKenna et al. reported an immediate increase in the number of signals, mostly in the comet tails, immediately after irradiation, which they attributed to breaks induced in the probed DNA sequence. They found a decrease in the number of signals over the first 15 min, after which time most were in comet heads. By 60 min, the number of signals was back to the normal level, while the % tail DNA (representing total DNA and its repair) was still elevated. They claimed that TP53 repair was faster than repair of the DNA overall. Given that 5 Gy produces breaks at a rate of 1.5 per 109 Da (6) (or ∼1 per 2 Mb), the chance of a ‘direct hit’ within the probed region is very low; therefore, the initial increase in number of signals is unlikely to represent actual breakage within that region, but probably results from the separation of DNA strands under alkaline conditions, revealing two targets for the probe for each copy of the gene. (In the undamaged comet, although alkaline treatment denatures the DNA, it readily renatures upon neutralization because the DNA loops are still constrained by supercoiling, and strand separation is impossible. This applies also to the DNA that remains in the head of a damaged comet.) Still, there is a clear indication that repair of the chromatin domain containing TP53 is faster than overall repair. Using a different approach, we studied the repair of three genes—DHFR which encodes dihydrofolate reductase, MGMT from Chinese hamster and the human TP53 gene (15). Probes were designed for each end of the gene and detected using antibodies linked to different coloured dyes so that the two ends of the gene were distinguished as red or green signals after hybridization. Chinese hamster ovary (CHO) cells were treated with H2O2, resulting in comets with about half the DNA in the tail. We expected DHFR signals to appear in the tail with an average frequency of 50%. In fact, almost all signals were in comet heads. This is unlikely to represent an extreme resistance of the DNA region containing DHFR to oxidation; this gene, which is relatively small, contains an MAR, and so we assumed that the gene was attached to the nuclear matrix. For the MGMT gene, CHO cells were treated either with H2O2 or with the photosensitizer Ro 19-8022 and light to oxidize guanines in the DNA to 8-oxoGua, detected as FPG-sensitive sites. In contrast to the DHFR experiment, signals did now appear over tail DNA—though they were predominantly green dots, while almost all red dots were located over the head. After 20 min incubation following treatment with H2O2, or 2 h incubation following exposure to photosensitizer plus light, almost all green as well as red signals were in the head, and the % of total DNA in the tail was close to the background level, indicating similar time-courses for total DNA repair and repair of MGMT. Next, we treated human lymphocytes with H2O2 and hybridized using oligonucleotide probes to the TP53 gene. Signals of both colours appeared in the tail. After 20 min incubation, tail % DNA had decreased by about one-third but virtually all TP53 signals were in the head. Thus, the region of DNA containing this gene was evidently repaired significantly more quickly than the bulk of the DNA. Our results with these three genes are schematically represented in Figure 4. Fig. 4 Open in new tabDownload slide Diagrammatic representation of DNA in a comet (reproduced from reference 15, Horvathova, E., Dusinska, M., Shaposhnikov, S. and Collins, A. R. (2004) DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis, 19, 269–276. Oxford University Press). Four extended, relaxed DNA loops are shown forming a comet tail. They are relaxed because a break is present somewhere in the loop. Loops are anchored to the nuclear matrix (indicated by the net of light grey lines). DNA loops with intact DNA remain supercoiled and within the head (indicated by the two tangles). The p53 gene is wholly in the tail. The DHFR gene is held close to the matrix by the presence of an MAR. It is speculated that an MAR is also present in the MGMT gene, near one end, which is held in the head, while the other end extends into the tail. Fig. 4 Open in new tabDownload slide Diagrammatic representation of DNA in a comet (reproduced from reference 15, Horvathova, E., Dusinska, M., Shaposhnikov, S. and Collins, A. R. (2004) DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis, 19, 269–276. Oxford University Press). Four extended, relaxed DNA loops are shown forming a comet tail. They are relaxed because a break is present somewhere in the loop. Loops are anchored to the nuclear matrix (indicated by the net of light grey lines). DNA loops with intact DNA remain supercoiled and within the head (indicated by the two tangles). The p53 gene is wholly in the tail. The DHFR gene is held close to the matrix by the presence of an MAR. It is speculated that an MAR is also present in the MGMT gene, near one end, which is held in the head, while the other end extends into the tail. Kumaravel et al. (29) studied damage and repair in the TP53 gene, after ionizing radiation or H2O2 treatment of normal and breast cancer cell lines. They also reported preferential repair of TP53 in both cell types. Preferential repair of UV-induced damage was reported for actively transcribing genes and the transcribed strand within these genes in the 1980s (30,31), using a technique that required high doses of UV and operated at the level of resolution of a restriction fragment of the gene. With the comet assay, very low-damage doses are employed, but the level of resolution is the DNA loop containing the gene. McKenna et al. (32) also investigated repair of damage induced by MMC, a DNA cross-linking agent. After MMC treatment, embedding in agarose and lysis, nucleoids were γ irradiated with 5 Gy. MMC cross-links retard movement of DNA and so decrease the intensity of the comet tail. As repair of the cross links proceeds, the % tail DNA increases, until at 24 h it is at the level seen in control nucleoids not treated with MMC. Again, the TP53 gene was probed by FISH. The % of nucleoids showing spots in the tail increased during the first 4 h at a greater rate than the increase in the % of nucleoids showing a tail (representing overall genome repair). Thus, preferential repair of cross links in the TP53 gene region is demonstrated. Conclusions Combining FISH with comets is potentially very informative. Current practical problems are likely to be solved by developments in probe technology. There is no universal agreement over how comets are formed, and indeed, our understanding of the underlying processes is incomplete. It is therefore not surprising that difficulties arise in interpreting the results of FISH experiments. 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TI - Increasing the resolution of the comet assay using fluorescent in situ hybridization—a review
JO - Mutagenesis
DO - 10.1093/mutage/gep021
DA - 2009-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/increasing-the-resolution-of-the-comet-assay-using-fluorescent-in-situ-cnIf7FtzoL
SP - 383
EP - 389
VL - 24
IS - 5
DP - DeepDyve
ER -