New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair

Abstract

Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30kb. The single cell gel electrophoresis (Comet) assay allows detection of single- or double-strand breaks at a 10-100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

Figure 1. Diagram showing the structure of a Comet with FISH

Duplex DNA is arranged in loops between matrix-attachment sites (MAS). Intact loops are supercoiled and remain within the head; loops containing one or more strand breaks are relaxed and extend forming the tail. In undamaged cells containing a diploid set of chromosomes, the two loops containing each of the pair of alleles for each gene will usually result in two separate spots; each loop can sustain damage, potentially increasing the number of spots. For simplicity we represent only one allele for each gene, thus the total number of spots could be higher than that given here. For alkaline Comet-FISH, two possible models are proposed for the distribution of domain-specific signals: in panel A, the complementary DNA strands in each loop migrate separately, thus the double-stranded probes annealing to each DNA strand appear in two or more spots regardless of the location of the break; however, a nick within the probed sequence would result in an additional spot (gene W). In panel B, the denatured strands migrate close to each other up to the nick, then the nicked strand unwinds away from its partner strand, thus signals could appear in one or more spots, depending on the location and number of nicks. Gene Z, near a MAS, is always found in heads. Panel C depicts a neutral Comet-FISH. Although the presence of a single nick is sufficient to relax the loop, the complementary DNA strands remain annealed. Intact sequences and those containing single-strand breaks appear in one spot (genes X and Y); genes containing a double strand break may appear in two spots (gene W).

Figure 2. Representative image from a Comet-FISH assay

Primary human skin fibroblasts were treated with 0.1 J/m² UVC, harvested, processed for alkaline Comet essentially as described in [33] and for FISH as described in [53]. Fluorescent probes were obtained from commercial sources, labeled with SpectrumOrange for a 145 kb domain containing the p53 gene, and with Direct-Green for the centromeric region of chromosome 7. One XP-C cell is shown from a slide prepared with cells collected immediately after UV-irradiation; the cells were treated with T4 endonuclease V to cut the DNA at the sites of CPD. The Comet head shows more intense DAPI staining than the tail. The signals from the FISH probes are shown as captured by the software, without enhancements. Red and green arrows indicate the positions of the signals for the p53 and chromosome 7 centromeric regions respectively.