Fig 1.
The ALC1-/- mutation is epistatic to the PARP1-/- mutation in DT40-cell sensitivity to H2O2 and MMS.
(A) Growth curves corresponding to the indicated cell cultures. (B) DT40 cells of the indicated genotypes were exposed to H2O2 and methyl methanesulfonate (MMS). The dose of the genotoxic agent is displayed on the x-axis on a linear scale, while the percent fraction of surviving cells is displayed on the y-axis on a logarithmic scale. Error bars represent standard deviations for more than three experiments. (C) Schematic of the SNF2N domain in ALC1. The amino acid change in ALC1-/E165Q cells is shown below. (D) The sensitivity of ALC1-/- and ALC1-/E165Q DT40 cells to H2O2 and MMS was very similar. Data as in B. p-value was calculated by a student’s t-test: p (**) <0.01 and n.s. (not significant).
Fig 2.
ALC1 is required for the repair of SSBs, which are BER intermediates, in DT40 cells.
(A) Alkaline-comet assay to detect SSBs. Indicated genotype were tested in untreated cells, immediately after exposure to 25 μM H2O2 on ice, and after a further 30 min incubation (repair period). Error bars represent standard deviations from three independent experiments. Tail-DNA percentage, defined as percentage of damaged DNA (% DNA in tail), was calculated as described in Materials and Methods. Tail-DNA % is displayed on the y-axis on a linear scale. (B) Alkaline elution experiment to detect SSB frequency. The data are expressed as the fraction of DNA remaining on the filter for the indicated genotypes of untreated cells (white circle), pulse H2O2-treated cells (black square), and the H2O2-treated cells incubated for a further 30 min (gray triangle). Elution time is displayed on the x-axis on a linear scale. The percentage of DNA remaining on the filter is displayed on the y-axis on a logarithmic scale. Error bars represent standard deviations from three independent experiments. p-value was calculated by a student’s t-test: p (*) <0.05, and n.s. (not significant).
Fig 3.
ALC1 is required for the repair of SSBs, which are BER intermediates, in human TK6 cells.
(A) DNA-damage sensitivity of TK6 cells carrying the indicated genotype. Data as in Fig 1B. (B–D) Alkaline-comet assay to detect unrepaired SSBs in human TK6 cells. The percentage of DNA strand breaks remaining at the indicated time points is displayed on the y-axis on a linear scale. Error bars represent standard deviations from three independent experiments. (B) Human TK6 cells of the indicated genotype were exposed to 80 μM H2O2 on ice for 30 min. The cells were released in drug-free, pre-warmed culture medium and further cultured for the indicated time. (C) TK6 cells treated with H2O2 as in B were released in culture medium containing 1 μM olaparib then cultured for the indicated time. (D) Wild-type and ALC1-/- TK6 cells were treated with 0.1 and 0.075 mg/ml MMS, respectively, for 15 min. Cells were then released in drug-free, pre-warmed culture medium and further cultured for indicated time. p-value was calculated by a student’s t-test: p (**) <0.01, (*) <0.05, and n.s. (not significant).
Fig 4.
ALC1 is not required for PARylation or recruitment of Polβ or XRCC1 to DNA-damage sites.
(A) DT40 clones of the indicated genotypes were treated with H2O2 or MMS and harvested at the indicated times after treatment. To evaluate the activation of the PARylation event by PARP1 at SSB sites, we measured the cellular concentration of NAD(P)H, the major substrate for PARP. Time after treatment with H2O2 or MMS is displayed on the x-axis, while the relative cellular NAD(P)H concentration is displayed on the y-axis. (B) Knockdown of ALC1 using si-RNA in human HeLa and U2OS cells was verified by western blot analysis using anti-ALC1 and anti-βactin (loading control). (C) H2O2 sensitivity of ALC1-depleted or control si-RNA-treated HeLa cells. The dose of H2O2 is displayed on the x-axis on a linear scale, while the percentage of cell survival is displayed on the y-axis on a logarithmic scale. (D) U2OS cells deficient in nucleotide excision repair, xeroderma pigmentosum group A- (XPA-) expressing Neurospora crassa UV-damage endonuclease (UVDE) were exposed to 100 J/m2 UV through micro pores in membrane filters. DNA single-strand breaks were produced at UV-irradiated region, where GFP-XRCC1 accumulated independently of ALC1 expression. Representative image of XPA-UVDE cells displaying GFP-XRCC1 and UV damage (CPD) signals. (E) GFP-XRCC1 and GFP-Polβ accumulate immediately at DNA damage sites after exposure to a 405 nm pulse laser in U2OS cells, which have been treated with siRNA against ALC1 or control siRNA for 48 h as previously described [41]. GFP-signal intensity for GFP-XRCC1 and GFP-Polβ is displayed in the histogram. Error bars represent standard deviations from three independent experiments. p-value was calculated by a student’s t-test: p (*) <0.05, and n.s. (not significant).
Fig 5.
ALC1 facilitates DNA-damage-induced chromatin relaxation.
(A) DT40 cells of the indicated genotypes were treated with H2O2, and cells were harvested at the indicated times after H2O2 treatment. The chromatin fraction was digested with 10 and 20 U/ml MNase. Digested genomic DNA products were analyzed by gel electrophoresis. The relative intensity of the band corresponding to the mono-nucleosome (146 bp, indicated by arrow) was quantified. (B) Histone H3 present in the nuclear soluble fraction was detected by western blot. The blot was probed with anti-Topoisomerase I (TopI) antibody as a loading control. The relative intensity of the band corresponding to histone H3 was quantified and normalized for the level of TopI. p-value was calculated by a student’s t-test: p (*) <0.05, and n.s. (not significant).
Fig 6.
Overexpression of ALC1 in a wide variety of cancer cell lines.
(A) Transcriptional characteristics of ALC1 were surveyed in 1000 cancer-cell lines using the Cancer Cell Line Encyclopedia [44]. The relative expression level of ALC1 in the cancer-cell lines is displayed on the x-axis on a logarithmic scale, while the relative copy number of the ALC1 gene is displayed on the y-axis on a linear scale. (B) Relationship between ALC1 and PARP1 expression levels in 1000 cancer cell lines. The relative expression of ALC1 is displayed on the x-axis, while the relative expression of PARP1 is displayed on the y-axis on a logarithmic scale. R-value represents correlation coefficients. p-value was calculated by t-test.