The comet assay: a method to measure DNA damage in individual cells

Olive, Peggy L.; Banáth, Judit P.

doi:10.1038/nprot.2006.5

Cited by 1,496 publications

(1,130 citation statements)

References 34 publications

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“…Cells for comet analysis were exposed to the indicated drug concentrations at 37°C in the dark and analyzed immediately according to previously published procedures (21,23,42). Image analysis was performed by using Komet 5.5 software (Kinetic Imaging, Nottingham, U.K.).…”

Section: Methodsmentioning

confidence: 99%

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Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743

Soares

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et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

144

111

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Adducts induced by the antitumor alkylator ecteinascidin 743 (ET-743, Yondelis, trabectedin) represent a unique challenge to the DNA repair machinery because no pathway examined to date is able to remove the ET adducts, whereas cells deficient in nucleotide excision repair show increased resistance. We here describe the processing of the initial ET adducts into cytotoxic lesions and characterize the influence of cellular repair pathways on this process. Our findings show that exposure of proliferating mammalian cells to pharmacologically relevant concentrations of ET-743 is accompanied by rapid formation of DNA double-strand breaks (DSBs), as shown by the neutral comet assay and induction of focalized phosphorylated H2AX. The ET adducts are stable and can be converted into DSBs hours after the drug has been removed. Loss of homologous recombination repair has no influence on the initial levels of DSBs but is associated with the persistence of unrepaired DSBs after ET-743 is removed, resulting in extensive chromosomal abnormalities and pronounced sensitivity to the drug. In comparison, loss of nonhomologous end-joining had only modest effect on the sensitivity. The identification of DSB formation as a key step in the processing of ET-743 lesions represents a novel mechanism of action for the drug that is in agreement with its unusual potency. Because loss of repair proteins is common in human tumors, expression levels of selected repair factors may be useful in identifying patients particularly likely to benefit, or not, from treatment with ET-743.cancer therapy ͉ natural products ͉ lesion processing ͉ DNA repair ͉ response prediction

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Section: Methodsmentioning

confidence: 99%

“…Single-cell electrophoresis under neutral conditions (the neutral comet assay), which almost uniquely detects DSBs (22,23), was used to determine whether ET-743 exposure is accompanied by formation of DNA strand breaks. The results (Fig.…”

mentioning

confidence: 99%

Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743

Soares

Escargueil

Poindessous

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

144

111

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“…Single-cell gel electrophoretic comet assays were performed under neutral conditions according to a previous study (Olive and Banath 2006). Briefly, U2OS cells were treated with or without 4 Gy of IR and recovered in normal culture medium for the indicated time at 37°C.…”

Section: Comet Assaysmentioning

confidence: 99%

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

et al. 2013

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Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.

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“…To confirm the occurrence of mutant Htt-induced DSBs further, we directly analyzed cleavage of genomic DNA using the comet assay (Fairbairn et al, 1995;Olive and Banáth, 2006). The assay was performed with rat primary cortical neurons expressing mutant Htt111Q by adenovirus vector at 3 d Figure 2.…”

Section: Mutant Htt Induces Dsbs Of Genomic Dna In Neuronsmentioning

confidence: 99%

“…Comet assay DNA DSBs were evaluated using the neutral single-cell gel electrophoresis assay (comet assay) as described previously (Olive and Banáth, 2006) with minor modifications (CometAssay; Trevigen). Cells were harvested, suspended with ice-cold PBS at 5 × 10 5 /ml, mixed with 1% low melting point agarose at a ratio of 1:10 (vol/vol), and spread gently onto a coverslip to avoid creating bubbles.…”

Section: Quantification Of Immunostain Signalsmentioning

confidence: 99%