Mol. Cells 2016; 39(3): 204-210
Published online February 23, 2016
https://doi.org/10.14348/molcells.2016.2206
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: bbccahn@mail.ulsan.ac.kr
DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In
Keywords
The integrity of DNA is continuously challenged by a variety of exogenous and endogenous DNA-damaging agents. Eukaryotes have evolved to possess a DNA damage response (DDR) that triggers cell cycle arrest, apoptosis, and DNA repair to protect the cell and ameliorate the threat to the organism.
The DDR signaling cascade is highly conserved in
DNA double-strand breaks (DSBs) can be induced either directly by exposure to ionizing irradiation (IR) or indirectly by the topoisomerase I inhibitor camptothecin (CPT), which causes replication fork stalling and collapse in actively cycling cells. DDR proteins for DSBs in mammalian cells are the kinases ATM/ATR, the sensing complex MRE11-RAD50-NBS1, and CHK2 (Langerak and Russell, 2011). The presence of DSBs is indirectly detected by immunostaining the proteins γ-H2AX or RAD51 (Paull et al., 2000; Tarsounas et al., 2004). H2AX is phosphorylated (gamma-H2AX) following exposure to IR and is densely localized around DSBs. Since RAD51 plays an essential role in homologous recombination for DSB repair in mammalian cells, RAD51 foci formation is thought to represent the presence of DSBs. Thus, immunostaining of γ-H2AX and RAD51 is useful for visualizing the localization of DSBs. RAD-51 foci formation has also been used to detect the sites of DSBs following IR in
Another method used to detect DSBs and their repair is the comet assay, a rapid and quantitative technique by which damaged or broken pieces of DNA are measured at the level of individual cells (Olive and Banath, 2006). Increased comet tails indicate the induction of DNA strand breaks. Initially, DNA strand breaks induced by IR or UV irradiation were detected in human blood cells by the comet assay (Lankinen et al., 1996; Olive et al., 1990), and DNA strand breaks induced by other agents such as CPT were subsequently detected (Godard et al., 2002). To date, there is only one report of use of the comet assay in
The identified
The
L4-stage animals on NGM plates were irradiated using a 137Cs source (gamma cell 3000 ELAN) with a rate of 321 rad/min on ice. L4-stage animals were grown on NGM plates containing 0, 1, 2, 5, 10, 20, or 40 μM CPT (Sigma-Aldrich) for 24 h at 20°C
IR-treated worms were allowed to lay eggs. After 24 h, the number of unhatched eggs was counted. L4-stage animals were grown on NGM plates containing 0, 1, 2, 5, 10, 20, or 40 μM CPT for 24 h and then transferred to CPT-free plates with
For the glyoxal-treated comet assay, mitotic germline nuclei were treated with glyoxal to denature the DNA (Hyun et al., 2008). The dissected gonads (30?40 worms) were cut to expose the mitotic tip regions. The mitotic compartments were mixed with glass beads (212?300 μm in diameter) in a micro-centrifuge tube and disrupted in a mini bead-beater (company) at 500 ×
Neutral comet assays were performed by a slight modification of the glyoxal-treated comet assay: after the lysis step, instead of glyoxal treatment, the slides were incubated with RNase A (1 mg/ml, Sigma-Aldrich) and proteinase K (32 U/ml, Roche) for 1 h at 37°C and then electrophoresis was performed at 20 volts for 90 min in TBE buffer. After electrophoresis, the slides were stained and analyzed as above. We used Olive tail moment in this study because it is considered independent of comet shape and is the best-established parameter for this assay, with less variability than tail length and tail DNA content.
The gonads of IR-irradiated worm were dissected 1h after irradiation. The gonads of CPT-treated worms were dissected 24 h after treatment. The gonads were extruded, fixed in 4% paraformaldehyde, and immunostained as described previously (Garcia-Muse and Boulton, 2005; Hyun et al., 2012). Briefly, the gonads were blocked with PBSBT (1X PBS, 0.5% bovine serum albumin, 0.1% Triton X-100) containing 2% non-fat milk at 4°C overnight. The gonads were incubated with primary antibody (1:1,000 dilution for anti-RAD-51, a polyclonal antibody generated with N-terminal 103 amino acids in rabbit) in humid chambers for 16 h at 4°C and then with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 1 h at room temperature in dark conditions. After staining with DAPI, the gonads were mounted on a 1% agarose pad and observed under an epifluorescence microscope (Carl Zeiss
Since
DNA repair defects have been observed in
The neutral comet assay was also performed to specifically examine DSB and repair. Comet tails were observed at 1 h after IR (Fig. 2C), indicating that DSBs were induced by IR. The analysis of tail moments in 100 comets at recovery time of 24 h after IR revealed that 73% of the DSBs were repaired in N2, compared with 30% in
The tail moments in two assays were different. The extent of repair of N2 measured by the glyoxal-comet assay (Figs. 2B and 2D) was lower than that by the neutral comet assay, indicating that unrepaired single-strand breaks reflect the difference. The extent of repair
CPT, a selective inhibitor of topoisomerase I (TOP1), stabilizes TOP1-DNA covalent complexes. Collisions between the replication fork migrating along the DNA and a trapped TOP1-DNA covalent complex result in irreversible replication fork arrest and DSB formation at the fork (Pommier, 2006; Ryan et al., 1991). Since the sensitivity of
We have previously shown that CPT induces DSBs in wild-type N2 by demonstrating an increase in the numbers of germline nucleus showing RAD-51-positive foci (manuscript in preparation). RAD-51 foci were also detected in mitotic nuclei of N2 and
The glyoxal comet assay was first performed to confirm the presence of DNA strand breaks. Representative images are shown (Fig. 4A). There was an increase in CPT-induced DNA strand breaks compared with non-damaged controls in both wild-type N2 and
The neutral comet assay was also performed to assess DSBs induced by CPT. Representative images are shown (Fig. 4C). There was an increase in CPT-induced DSBs compared with non-damaged controls in both wild-type N2 and
The tail moments in two assays were different. The extent of repair of N2 measured by the glyoxal-comet assay (Figs. 4B and 4D) was lower than that by the neutral comet assay, indicating that unrepaired single-strand breaks reflect the difference. Interestingly, DSBs were further generated during recovery times in
In this study, we have investigated DNA DSB induction and repair in wild-type N2 and
We chose a
The comet assay, also known as single-cell gel electrophoresis, can detect DNA damage and repair kinetics at the level of a single cell and has been shown to be a suitable tool for studying the induction and repair of radiation-induced DSBs (Olive and Banath, 2006). The alkaline comet, in which DNA is mobilized under alkaline conditions for DNA denaturation, detects both single-stranded DNA breaks and DSBs, without distinguishing between the two. The neutral comet, in which DNA migrates under neutral conditions, detects only DSBs.
There is only one previous report of use of the comet assay in
We treated germline nuclei with glyoxal instead of alkaline treatment because the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Glyoxal is known to covalently bind to only solution-exposed guanine bases (Broude and Budowsky, 1973). We have previously used glyoxal in DNA hybridization of
To attain high enough sensitivity under the neutral comet assay, germline nuclei were treated with ribonuclease A and proteinase K to obtain relatively pure DNA free from RNA and most proteins (Singh and Khan, 1995). In the wild-type N2 strain, DSBs were detected following treatment with IR or CPT, which is in agreement with studies using the neutral comet assay in human cells (Lankinen et al., 1996; Olive et al., 1990). Comet tails were barely detected under the neutral comet assay without treatments with ribonuclease A and proteinase K (unpublished data). The use of ribonuclease A and proteinase K in this study improved sensitivity of the neutral comet assay. Since DNA strand breaks detected by the glyoxal comet assay come from both single-strand breaks and double-strand breaks, greater tail moments would be expected than those from only double-strand breaks. However, the tail moment detected by the neutral comet assay was greater than that by the glyoxal comet assay. Because sample treatment and the electrophoresis conditions such as volts, currents, and times are different in two assays, it is hard to compare the tail moments directly. Nonetheless, these experiments provided evaluation of the comet assays as a reliable tool to study DNA strand breaks in
DNA damage levels, as measured by tail moment, remained high in
When mammalian cells are exposed to IR, aggregates of repair proteins called nuclear foci appear within their nuclei (Bekker-Jensen et al., 2006). These foci contain proteins involved in HR including RAD51, RAD52, and RAD54, and form in response to DNA damage. Thus, it is thought that they represent sites at which recombinational repair reactions occur (Essers et al., 2002). RAD51 foci formation has served as an indicator of the induction of radiation-induced DSBs and DNA repair (Miyazaki et al., 2004; van Veelen et al., 2005). In the study of
We analyzed RAD-51 foci formation over time after exposure to IR. In wild-type N2, the percentage of germline nuclei showing RAD-51-positive foci decreased with increasing post-irradiation time (Fig. S1). In contrast, the decrease in foci in
IR-induced DSBs in mammalian cells have been also detected by immuno staining of γ-H2AX, which is a marker for DNA strand breaks (Paull et al., 2000). DSB repair kinetics measured by the comet assay correlated with γ-H2AX foci association. Quantitative studies further indicated that the extent of γ-H2AX formation is proportional to the number of DSBs and that their appearance and disappearance correlate very well with the kinetics of DSB repair (Balajee and Geard, 2004; Olive, 2011). Since antibodies against γ-H2AX in
In summary, we have shown that the dissected mitotic germline region can be used for the comet assay in
Mol. Cells 2016; 39(3): 204-210
Published online March 31, 2016 https://doi.org/10.14348/molcells.2016.2206
Copyright © The Korean Society for Molecular and Cellular Biology.
Sojin Park1,2, Seoyun Choi1, and Byungchan Ahn1,*
1Department of Life Sciences, University of Ulsan, Ulsan 44610, Korea
Correspondence to:*Correspondence: bbccahn@mail.ulsan.ac.kr
DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In
Keywords:
The integrity of DNA is continuously challenged by a variety of exogenous and endogenous DNA-damaging agents. Eukaryotes have evolved to possess a DNA damage response (DDR) that triggers cell cycle arrest, apoptosis, and DNA repair to protect the cell and ameliorate the threat to the organism.
The DDR signaling cascade is highly conserved in
DNA double-strand breaks (DSBs) can be induced either directly by exposure to ionizing irradiation (IR) or indirectly by the topoisomerase I inhibitor camptothecin (CPT), which causes replication fork stalling and collapse in actively cycling cells. DDR proteins for DSBs in mammalian cells are the kinases ATM/ATR, the sensing complex MRE11-RAD50-NBS1, and CHK2 (Langerak and Russell, 2011). The presence of DSBs is indirectly detected by immunostaining the proteins γ-H2AX or RAD51 (Paull et al., 2000; Tarsounas et al., 2004). H2AX is phosphorylated (gamma-H2AX) following exposure to IR and is densely localized around DSBs. Since RAD51 plays an essential role in homologous recombination for DSB repair in mammalian cells, RAD51 foci formation is thought to represent the presence of DSBs. Thus, immunostaining of γ-H2AX and RAD51 is useful for visualizing the localization of DSBs. RAD-51 foci formation has also been used to detect the sites of DSBs following IR in
Another method used to detect DSBs and their repair is the comet assay, a rapid and quantitative technique by which damaged or broken pieces of DNA are measured at the level of individual cells (Olive and Banath, 2006). Increased comet tails indicate the induction of DNA strand breaks. Initially, DNA strand breaks induced by IR or UV irradiation were detected in human blood cells by the comet assay (Lankinen et al., 1996; Olive et al., 1990), and DNA strand breaks induced by other agents such as CPT were subsequently detected (Godard et al., 2002). To date, there is only one report of use of the comet assay in
The identified
The
L4-stage animals on NGM plates were irradiated using a 137Cs source (gamma cell 3000 ELAN) with a rate of 321 rad/min on ice. L4-stage animals were grown on NGM plates containing 0, 1, 2, 5, 10, 20, or 40 μM CPT (Sigma-Aldrich) for 24 h at 20°C
IR-treated worms were allowed to lay eggs. After 24 h, the number of unhatched eggs was counted. L4-stage animals were grown on NGM plates containing 0, 1, 2, 5, 10, 20, or 40 μM CPT for 24 h and then transferred to CPT-free plates with
For the glyoxal-treated comet assay, mitotic germline nuclei were treated with glyoxal to denature the DNA (Hyun et al., 2008). The dissected gonads (30?40 worms) were cut to expose the mitotic tip regions. The mitotic compartments were mixed with glass beads (212?300 μm in diameter) in a micro-centrifuge tube and disrupted in a mini bead-beater (company) at 500 ×
Neutral comet assays were performed by a slight modification of the glyoxal-treated comet assay: after the lysis step, instead of glyoxal treatment, the slides were incubated with RNase A (1 mg/ml, Sigma-Aldrich) and proteinase K (32 U/ml, Roche) for 1 h at 37°C and then electrophoresis was performed at 20 volts for 90 min in TBE buffer. After electrophoresis, the slides were stained and analyzed as above. We used Olive tail moment in this study because it is considered independent of comet shape and is the best-established parameter for this assay, with less variability than tail length and tail DNA content.
The gonads of IR-irradiated worm were dissected 1h after irradiation. The gonads of CPT-treated worms were dissected 24 h after treatment. The gonads were extruded, fixed in 4% paraformaldehyde, and immunostained as described previously (Garcia-Muse and Boulton, 2005; Hyun et al., 2012). Briefly, the gonads were blocked with PBSBT (1X PBS, 0.5% bovine serum albumin, 0.1% Triton X-100) containing 2% non-fat milk at 4°C overnight. The gonads were incubated with primary antibody (1:1,000 dilution for anti-RAD-51, a polyclonal antibody generated with N-terminal 103 amino acids in rabbit) in humid chambers for 16 h at 4°C and then with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 1 h at room temperature in dark conditions. After staining with DAPI, the gonads were mounted on a 1% agarose pad and observed under an epifluorescence microscope (Carl Zeiss
Since
DNA repair defects have been observed in
The neutral comet assay was also performed to specifically examine DSB and repair. Comet tails were observed at 1 h after IR (Fig. 2C), indicating that DSBs were induced by IR. The analysis of tail moments in 100 comets at recovery time of 24 h after IR revealed that 73% of the DSBs were repaired in N2, compared with 30% in
The tail moments in two assays were different. The extent of repair of N2 measured by the glyoxal-comet assay (Figs. 2B and 2D) was lower than that by the neutral comet assay, indicating that unrepaired single-strand breaks reflect the difference. The extent of repair
CPT, a selective inhibitor of topoisomerase I (TOP1), stabilizes TOP1-DNA covalent complexes. Collisions between the replication fork migrating along the DNA and a trapped TOP1-DNA covalent complex result in irreversible replication fork arrest and DSB formation at the fork (Pommier, 2006; Ryan et al., 1991). Since the sensitivity of
We have previously shown that CPT induces DSBs in wild-type N2 by demonstrating an increase in the numbers of germline nucleus showing RAD-51-positive foci (manuscript in preparation). RAD-51 foci were also detected in mitotic nuclei of N2 and
The glyoxal comet assay was first performed to confirm the presence of DNA strand breaks. Representative images are shown (Fig. 4A). There was an increase in CPT-induced DNA strand breaks compared with non-damaged controls in both wild-type N2 and
The neutral comet assay was also performed to assess DSBs induced by CPT. Representative images are shown (Fig. 4C). There was an increase in CPT-induced DSBs compared with non-damaged controls in both wild-type N2 and
The tail moments in two assays were different. The extent of repair of N2 measured by the glyoxal-comet assay (Figs. 4B and 4D) was lower than that by the neutral comet assay, indicating that unrepaired single-strand breaks reflect the difference. Interestingly, DSBs were further generated during recovery times in
In this study, we have investigated DNA DSB induction and repair in wild-type N2 and
We chose a
The comet assay, also known as single-cell gel electrophoresis, can detect DNA damage and repair kinetics at the level of a single cell and has been shown to be a suitable tool for studying the induction and repair of radiation-induced DSBs (Olive and Banath, 2006). The alkaline comet, in which DNA is mobilized under alkaline conditions for DNA denaturation, detects both single-stranded DNA breaks and DSBs, without distinguishing between the two. The neutral comet, in which DNA migrates under neutral conditions, detects only DSBs.
There is only one previous report of use of the comet assay in
We treated germline nuclei with glyoxal instead of alkaline treatment because the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Glyoxal is known to covalently bind to only solution-exposed guanine bases (Broude and Budowsky, 1973). We have previously used glyoxal in DNA hybridization of
To attain high enough sensitivity under the neutral comet assay, germline nuclei were treated with ribonuclease A and proteinase K to obtain relatively pure DNA free from RNA and most proteins (Singh and Khan, 1995). In the wild-type N2 strain, DSBs were detected following treatment with IR or CPT, which is in agreement with studies using the neutral comet assay in human cells (Lankinen et al., 1996; Olive et al., 1990). Comet tails were barely detected under the neutral comet assay without treatments with ribonuclease A and proteinase K (unpublished data). The use of ribonuclease A and proteinase K in this study improved sensitivity of the neutral comet assay. Since DNA strand breaks detected by the glyoxal comet assay come from both single-strand breaks and double-strand breaks, greater tail moments would be expected than those from only double-strand breaks. However, the tail moment detected by the neutral comet assay was greater than that by the glyoxal comet assay. Because sample treatment and the electrophoresis conditions such as volts, currents, and times are different in two assays, it is hard to compare the tail moments directly. Nonetheless, these experiments provided evaluation of the comet assays as a reliable tool to study DNA strand breaks in
DNA damage levels, as measured by tail moment, remained high in
When mammalian cells are exposed to IR, aggregates of repair proteins called nuclear foci appear within their nuclei (Bekker-Jensen et al., 2006). These foci contain proteins involved in HR including RAD51, RAD52, and RAD54, and form in response to DNA damage. Thus, it is thought that they represent sites at which recombinational repair reactions occur (Essers et al., 2002). RAD51 foci formation has served as an indicator of the induction of radiation-induced DSBs and DNA repair (Miyazaki et al., 2004; van Veelen et al., 2005). In the study of
We analyzed RAD-51 foci formation over time after exposure to IR. In wild-type N2, the percentage of germline nuclei showing RAD-51-positive foci decreased with increasing post-irradiation time (Fig. S1). In contrast, the decrease in foci in
IR-induced DSBs in mammalian cells have been also detected by immuno staining of γ-H2AX, which is a marker for DNA strand breaks (Paull et al., 2000). DSB repair kinetics measured by the comet assay correlated with γ-H2AX foci association. Quantitative studies further indicated that the extent of γ-H2AX formation is proportional to the number of DSBs and that their appearance and disappearance correlate very well with the kinetics of DSB repair (Balajee and Geard, 2004; Olive, 2011). Since antibodies against γ-H2AX in
In summary, we have shown that the dissected mitotic germline region can be used for the comet assay in
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