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Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

Fung H, Weinstock DM - PLoS ONE (2011)

Bottom Line: Differences in ex vivo cell culture conditions can drastically affect stem cell physiology.DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ).We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

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DNA-PKcs-dependent and -independent repair after low-dose IR.A. Neutral comet assay was performed after irradiation with 2 Gy in the presence of NU7026 (Nu) or vehicle (Veh) and the olive tail moment was calculated for at least 50 cells for each condition at the indicated time points. Error bars indicate one standard deviation. Indicated ratios are between olive tail moments for the same cell line in the presence of NU7026 or vehicle. * indicates p<0.01 compared with same cell line and time point treated with vehicle. B. Comet assays were performed after treatment with etoposide or vehicle (-).
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pone-0020514-g005: DNA-PKcs-dependent and -independent repair after low-dose IR.A. Neutral comet assay was performed after irradiation with 2 Gy in the presence of NU7026 (Nu) or vehicle (Veh) and the olive tail moment was calculated for at least 50 cells for each condition at the indicated time points. Error bars indicate one standard deviation. Indicated ratios are between olive tail moments for the same cell line in the presence of NU7026 or vehicle. * indicates p<0.01 compared with same cell line and time point treated with vehicle. B. Comet assays were performed after treatment with etoposide or vehicle (-).

Mentions: Although site loss in hESCs was predominately mediated by NHEJ, this does not clarify the relative contributions of NHEJ and HR to overall repair, as precise repair of the I-SceI break by either NHEJ or by HR using SceGFP on the sister chromatid will reestablish the I-SceI site. To address this, we treated hESC, MCF7 and U2OS lines with 2 Gy of ionizing radiation, a dose which does not result in significant cell death but can promote G2 cell cycle arrest [14], [16]. At fixed time points, we measured the extent of persistent DNA damage using the neutral comet assay, which quantifies only DSBs (Figure 5A). The extent of damage induced by IR, as measured 5 minutes after irradiation, was reduced in hESCs compared with U2OS and MCF7 cells (Figure 5A). However, the kinetics of repair of IR-induced breaks were similar in hESCs, MCF7 and U2OS cells (Figure 5A). For example, by 4 hour after irradiation, the olive tail moment had decreased 44% in U2OS cells compared with 48–53% in the hESC lines (Figure 5A).


Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

Fung H, Weinstock DM - PLoS ONE (2011)

DNA-PKcs-dependent and -independent repair after low-dose IR.A. Neutral comet assay was performed after irradiation with 2 Gy in the presence of NU7026 (Nu) or vehicle (Veh) and the olive tail moment was calculated for at least 50 cells for each condition at the indicated time points. Error bars indicate one standard deviation. Indicated ratios are between olive tail moments for the same cell line in the presence of NU7026 or vehicle. * indicates p<0.01 compared with same cell line and time point treated with vehicle. B. Comet assays were performed after treatment with etoposide or vehicle (-).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102116&req=5

pone-0020514-g005: DNA-PKcs-dependent and -independent repair after low-dose IR.A. Neutral comet assay was performed after irradiation with 2 Gy in the presence of NU7026 (Nu) or vehicle (Veh) and the olive tail moment was calculated for at least 50 cells for each condition at the indicated time points. Error bars indicate one standard deviation. Indicated ratios are between olive tail moments for the same cell line in the presence of NU7026 or vehicle. * indicates p<0.01 compared with same cell line and time point treated with vehicle. B. Comet assays were performed after treatment with etoposide or vehicle (-).
Mentions: Although site loss in hESCs was predominately mediated by NHEJ, this does not clarify the relative contributions of NHEJ and HR to overall repair, as precise repair of the I-SceI break by either NHEJ or by HR using SceGFP on the sister chromatid will reestablish the I-SceI site. To address this, we treated hESC, MCF7 and U2OS lines with 2 Gy of ionizing radiation, a dose which does not result in significant cell death but can promote G2 cell cycle arrest [14], [16]. At fixed time points, we measured the extent of persistent DNA damage using the neutral comet assay, which quantifies only DSBs (Figure 5A). The extent of damage induced by IR, as measured 5 minutes after irradiation, was reduced in hESCs compared with U2OS and MCF7 cells (Figure 5A). However, the kinetics of repair of IR-induced breaks were similar in hESCs, MCF7 and U2OS cells (Figure 5A). For example, by 4 hour after irradiation, the olive tail moment had decreased 44% in U2OS cells compared with 48–53% in the hESC lines (Figure 5A).

Bottom Line: Differences in ex vivo cell culture conditions can drastically affect stem cell physiology.DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ).We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

Show MeSH
Related in: MedlinePlus