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. 2013 Sep;41(16):7700-12.
doi: 10.1093/nar/gkt524. Epub 2013 Jun 17.

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

Jia Guo  1 , Philip C Hanawalt, Graciela Spivak
Affiliations

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

Jia Guo et al. Nucleic Acids Res. 2013 Sep.

Abstract

Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

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Figures

Figure 1.
Figure 1.
Comet-FISH with strand-specific probes. (a) After DNA-damaging treatment, cells are lysed, incubated with endonucleases or glycosylases and subjected to electrophoresis. Hybridization of strand-specific probes to the termini of the DNA segments of interest permits the quantification of TCR; staining the bulk of the DNA allows the analysis of GGR. (b) Schematic representation of comet-FISH. Adjacent green and red probe signals indicate an intact DNA strand with no lesions within the segment; separated green and red probe signals suggest a damaged DNA strand with a lesion within the segment. Representative comet-FISH images of a cell damaged with UV showing (c) the bulk DNA stained with DAPI, (d) Alexa 488-labeled probes targeting the 3′ regions of the transcribed ATM strands, (e) Alexa 594-labeled probes targeting the 5′ regions of the transcribed ATM strands and (f) an overlay of Figure 1c, d and e (scale bars, 5 µm); (g), (h) and (i) show enlargements of the probes signals from panel f.
Figure 2.
Figure 2.
Design and synthesis of strand-specific fluorescent probes for comet-FISH. (a) The 3′ and 5′ regions of the ATM gene are amplified by PCR using biotinylated forward primers, natural reverse primers and aminoallyl-dUTP. Subsequently, the biotinylated strands and the non-biotinylated strands of the PCR products are separated by streptavidin-coated beads. The probes targeting the 3′ and 5′ regions of the ATM gene are labeled with Alexa 488 and Alexa 594, respectively. Finally, all the biotinylated probes are combined as probes for the ATM TS, whereas all the non-biotinylated probes are mixed as probes for the ATM NTS. (b) Agarose gel containing the double-stranded PCR product (lane 1), single-stranded DNA with biotin (lane 2), single-stranded DNA without biotin (lane 3) and annealed double-stranded DNA (lane 4). (c) Agarose gel with fluorescently labeled strand-specific probes stained with ethidium bromide (left) or unstained (right). Probes for the 3′ region of the ATM TS (lanes 1 and 5), probes for the 3′ region of the ATM NTS (lanes 2 and 6), probes for the 5′ region of ATM TS (lanes 3 and 7), probes for the 5′ region of the ATM NTS (lanes 4 and 8). (d) Absorption spectra of probes targeting the 3′ region (green) and the 5′ region (red) of the ATM TS. (e) Absorption spectra of probes targeting the 3′ region (green) and the 5′ region (red) of the ATM NTS. The peaks centered at 260, 492 and 588 nm correspond to DNA, Alexa 488 and Alexa 594, respectively.
Figure 3.
Figure 3.
Requirement for CSB and actively transcribing RNAP II for TCR of low levels of CPD. Left panels: 24 h time course of repair of CPD in the TS (solid lines) and NTS (dashed lines) of the ATM gene in (a) GM00037F (wild-type) cells, (b) GM00739 (CSB) cells, (c) GM16684 (XPC) cells and (d) α-amanitin-treated XPC cells. The results shown represent the numbers of ATM strands with CPD per 30 cells. The averages and standard deviations were obtained from three independent experiments. Right panels: 24 h time course of repair of CPD in the genome overall in (e) wild-type cells, (f) CSB cells, (g) XPC cells and (h) α-amanitin-treated XPC cells. The results shown represent the percentages of DNA in comet tails. The averages and standard deviations are calculated from 30 cells treated with T4 endonuclease V.
Figure 4.
Figure 4.
CSB, UVSSA and actively transcribing RNAP II are required for preferential repair of 8-oxoG in ATM. Left panels: 24 h time course of repair of 8-oxoG in the TS (solid lines) and NTS (dashed lines) of the ATM gene in (a) GM00037F (wild-type) cells, (b) GM00739 (CSB) cells, (c) Kps3 UVSS cells and (d) α-amanitin-treated wild-type cells. The results shown represent the average number of ATM strands with 8-oxoG per 30 cells. The averages and standard deviations were obtained from three independent experiments. Right panels: 24 h time course of repair of 8-oxoG in the genome overall in (e) wild-type cells, (f) CSB cells, (g) UVSS cells and (h) α-amanitin-treated wild-type cells. The results shown represent the percentages of DNA in comet tails. The averages and standard deviations were calculated from 30 cells treated with hOGG1.
Figure 5.
Figure 5.
Model for TCR of oxidized base damage, initiated by BER and resolved by NER. A damaged base in a DNA segment (1) is recognized by a glycosylase and converted to an abasic site (2). This intermediate product, or the subsequent single-strand break generated by an AP endonuclease (3), is repaired via BER unless an elongating RNAP encounters either structure. Upon transcription arrest and recruitment of TCR factors, the lesion is repaired via NER and transcription may resume.
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