Comet sensitivity in assessing DNA damage and repair in different cell cycle stages

DG McArt, George McKerr, Kurt Saetzler, Vyvyan Howard, Stephen Downes, GR Wasson

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.
LanguageEnglish
Pages299
JournalMutagenesis
Volume25
Issue number3
DOIs
Publication statusPublished - 2010

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DNA Repair
DNA Damage
Cell Cycle
Repair
Cells
DNA
Ionizing radiation
Comet Assay
Ionizing Radiation
Assays
S Phase
Cell Division
Radiation Dosage
G1 Phase
HeLa Cells
Experiments
Cell Count
Population

Cite this

McArt, DG., McKerr, G., Saetzler, K., Howard, V., Downes, S., & Wasson, GR. (2010). Comet sensitivity in assessing DNA damage and repair in different cell cycle stages. Mutagenesis, 25(3), 299. https://doi.org/10.1093/mutage/geq006
McArt, DG ; McKerr, George ; Saetzler, Kurt ; Howard, Vyvyan ; Downes, Stephen ; Wasson, GR. / Comet sensitivity in assessing DNA damage and repair in different cell cycle stages. In: Mutagenesis. 2010 ; Vol. 25, No. 3. pp. 299.
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abstract = "The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.",
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McArt, DG, McKerr, G, Saetzler, K, Howard, V, Downes, S & Wasson, GR 2010, 'Comet sensitivity in assessing DNA damage and repair in different cell cycle stages', Mutagenesis, vol. 25, no. 3, pp. 299. https://doi.org/10.1093/mutage/geq006

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages. / McArt, DG; McKerr, George; Saetzler, Kurt; Howard, Vyvyan; Downes, Stephen; Wasson, GR.

In: Mutagenesis, Vol. 25, No. 3, 2010, p. 299.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Comet sensitivity in assessing DNA damage and repair in different cell cycle stages

AU - McArt, DG

AU - McKerr, George

AU - Saetzler, Kurt

AU - Howard, Vyvyan

AU - Downes, Stephen

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N2 - The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

AB - The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

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