Article Figures & Data
Figures
- Figure S1. Mitogen-deprived TBP MEFs rely on the intra–S phase checkpoint.
(A) Crystal violet assay of TBP MEFs cultured for 5 d with or without FCS in the absence or presence of 2 μM VE821 (ATR inhibitor), 2.5 mM Caffeine (ATM/ATR inhibitor), 10 μM KU55933 (ATM inhibitor), 100 nM AZD7762 (Chk1/Chk2 inhibitor), and 5 μM NU7441 (DNA-Pk inhibitor). (B, C, D, E, F) IncuCyte growth curves of TBP MEFs with FCS (red), with FCS and small-molecule inhibitor (black), without FCS (green) and without FCS and small-molecule inhibitor (blue). Small-molecule inhibitors used are 2 μM VE821 (ATR inhibitor) (B), 50 nM UCN01 (Chk1 inhibitor) (C), 10 μM KU55933 (ATM inhibitor) (D), 2.5 mM Caffeine (ATM/ATR inhibitor) (E), and 5 μM NU7441 (DNA-Pk inhibitor) (F). (G, H) Relative apoptosis of TBP MEFs with FCS (red), with FCS and small-molecule inhibitor (black), without FCS (green) and without FCS and small-molecule inhibitor (blue). Small-molecule inhibitors used are 2 μM VE821 (ATR inhibitor) (G) and 50 nM UCN01 (Chk1 inhibitor) (H). Apoptosis was measured by fluorescent signal upon caspase 3 cleavage and normalized to cell confluency. For (B, C, D, E, F, G, H), error bars show SDs.
- Figure S2. Cell cycle profiling of TBP MEFs in the presence of ATR/CHK1 inhibitors.
(A, B, C, D) FUCCI constructs (mKO-hCdt1 and mAG-hGEM) were introduced in TBP MEFs to assess the duration of cell cycle phases in individual cells in the presence or absence of FCS. TBP MEFs were treated with 2 μM VE821 (ATR inhibitor) (A, B) or 50 nM UCN01 (Chk1 inhibitor) (C, D). G1 phase: only mKO-hCdt1 expression. Early S phase: both mKO-hCdt1 and mAG-hGEM expression. S/G2 phase: only mAG-hGEM expression. M phase: both markers absent. Y-aches represent individual cells. (E) Duration of cell cycle phases in TBP MEFS with and without FCS treated with 2 mM VE821 or 50 nM UCN01. Data from untreated TBP MEFS were taken from Benedict et al (2020). Error bars show SDs.
- Figure 1. shRNA-based screen to identify genes essential for proliferation in replication stress conditions.
(A) Schematic outline of the shRNA screen. TBP MEFs were infected with the lentiviral DNA damage response shRNA library and cultured in the absence or presence of FCS. ShRNA inserts were subsequently recovered by PCR and analyzed by next generation sequencing. ShRNAs that were depleted in the abscence of serum likely target genes essential for proliferation in replication stress conditions. (B) The shRNA screen identified expression of CHK1 and RECQL essential for proliferation in replication stress conditions. The x-axis shows the average number of sequencing reads at the start point. The y-axis depicts the fold change in abundance of shRNAs in the cells cultured without FCS versus with FCS. (C) CHK1 expression levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (D) CHK1 protein levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs. Anti-actin was used as loading control. (E) Growth curve of TBP MEFs with FCS (black) and without FCS (green), TBP-Chk1KD#1 MEFs with FCS (grey), and without FCS (blue) measured using the IncuCyte. Error bars show SD.
- Figure S3. shRNA screen to identify replication-rescue genes.
(A) Experimental setup of the screen. TBP MEFs were infected with the DNA Damage Response shRNA library at a MOI of 1–2. 3 d later, all cells were mixed, genomic DNA at T = 0 was isolated and cells were plated with and without FCS. Cells were cultured for ∼10–12 cell divisions. For the with FCS condition, cells were replated every 2 d to a new dish and this step was repeated four times. For the without FCS condition, cells were replated to a new dish every 4 or 5 d and this step was repeated four times. After the final culture period, genomic DNA (gDNA) was isolated. (B) Correlation plots of the T = 0, with FCS and without FSC replicates used in the screen. (C) Cluster analysis of the T = 0, with FCS and without FCS replicates used in the screen. (D) Crystal violet assay of TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs cultured for 5 d with or without FCS.
- Figure 2. RECQL is essential for proliferation in replication stress conditions.
(A) RECQL expression levels in TBP and TBP-RecqlKD MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (B) RECQL protein levels in TBP and TBP-RecqlKD MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP (black) and TBP-RecqlKD (grey) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Relative apoptosis of TBP (black) and TBP-RecqlKD (grey) MEFs cultured without FCS. Apoptosis was measured by fluorescent signal upon caspase 3 cleavage and normalized to cell confluency. For (C, D, E), error bars show SD. (F) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs cultured with FCS or for 7 d without FCS. For each condition, more than 50 cells were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test). (G) Immunofluorescence images of γH2AX and CldU foci in TBP-shRNA control and TBP-RecqlKD MEFs after 2 d of serum starvation. DNA was labelled with Topro3. In the merged picture, DNA is blue, γH2AX is green and CldU is red. Colocalization of yH2AX and CldU is seen as yellow foci. Scale bar = 12 μm. (H) Quantification of the number of γH2AX foci per nucleus in CldU-negative TBP-shRNA control and TBP-RecqlKD MEFs in the presence and absence of serum. Mean is indicated in red. Results of three independent experiments are pooled and significance is indicated (t test).
- Figure S4. Two independently generated TBP-RecqlKD MEF cell lines and cell cycle analysis of mitogen-deprived TBP-RecqlKD MEFs.
(A) RECQL expression levels in TBP and TBP-RecqlKD MEFs generated by two newly designed shRNAs (TBP-RecqlKD#2 and TBP-RecqlKD#3) measured by qRT-PCR. Error bars show SD. (B) RECQL protein levels in TBP, TBP-RecqlKD#2 and TBP-RecqlKD#3 MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP-shRNA control (black), TBP-RecqlKD#2 (green) and TBP-RecqlKD#3 (blue) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Duration of G1 and G2 cell cycle phases of TBP and TBP-RecqlKD MEFs expressing FUCCI constructs (mKO-hCdt1 and mAG-hGEM) in the presence or absence of FCS. G1 phase: only mKO-hCdt1 expression. Early S phase: both mKO-hCdt1 and mAG-hGEM expression. S/G2 phase: only mAG-hGEM expression. Data from TBP MEFs were taken from Benedict et al (2020). Error bars show SDs.
- Figure S5. TBP-RecqlKD MEFs show an increase in DNA double-strand breaks after multiple days without serum.
(A) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs cultured with or without FCS for 1 or 2 d. (B) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs cultured with or without FCS for 4 d. (C) Relative apoptosis of serum starved TBP-RecqlKD in the absence (black) or presence (red) of 10 μM of the pan-caspase inhibitor QvD. Apoptosis was measured by fluorescent signal upon caspase 3 cleavage and normalized to cell confluency. Error bars show SD. (D) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs with or without FCS for 4 d in the absence or presence of 10 μM pan-nuclease inhibitor QvD. (E) Tail moments of TBP-shRNA control, TBP-RecqlKD#2, and TBP-RecqlKD#3 MEFs with or without FCS for 3 d. For (A, B, D, E), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure 3. RECQL protects stalled replication forks against MRE11-dependent DNA double-strand break formation.
(A) Timing of the neutral comet assay procedure after 1 h with 0, 300 μM, and 2 mM HU. (B) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs without HU treatment or immediately after 1 h 300 μM or 2 mM HU treatment. (C) Timing of the neutral comet assay procedure after 1 h with 300 μM or 2 mM HU or 30 min after washing away HU. (D) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs immediately after 1 h 300 μM HU treatment or 30 min after washing away HU. (E) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs immediately after 1 h 2 mM HU treatment or 30 min after washing away HU. (F) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs after 1 h 300 μM HU treatment in the presence or absence of 50 μM Mirin. We previously showed that Mirin was active in MEFs and reduced repair of double strand breaks induced by irradiation (Benedict et al, 2020). (G) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs after 7 d without FCS in the presence or absence of 12.5 μM Mirin. (H) Growth curve of TBP MEFs (black), TBP MEFs with 12.5 μM Mirin (grey), TBP-RecqlKD MEFs (red), and TBP-RecqlKD MEFs with 12.5 μM Mirin (green). Error bars show SDs. For (B, D, E, F, G), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure S6. DNA damage in HU-treated TBP-RecqlKD MEFs.
(A) Tail moments of TBP-shRNA control, TBP-RecqlKD#2 and TBP-RecqlKD#3 MEFs untreated and treated with 300 μM HU for 1 h. (B) Tail moments of TBP and TBP-RecqlKD MEFs cultured without HU or with 300 μM HU for 30 or 120 min. (C) Tail moments of TBP and TBP-RecqlKD MEFs cultured without HU or with 300 μM HU for 3 or 5 h. For (A, B, C), more than 50 cells were analyzed per condition using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure S7. RAD51 dependence of double-strand break repair.
(A, B) Tail moments of TBP and TBP-RecqlKD MEFs cultured in the presence of 20 μM of the RAD51 inhibitor B02 (A) or 5 μM of the DNA-PK inhibitor NU7441 (B) immediately after 1 h 2 mM HU treatment or 30 min after washing away HU. For each condition, more than 50 cells were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure S8. MRE11 dependence of double-strand break induction.
(A, B) MRE11 expression levels in TBP (A) and TBP-RecqlKD (B) MEFs transfected with control (black) and MRE11 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (C) Tail moments of TBP and TBP-RecqlKD MEFs transfected with control (black) and MRE11 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (D) Tail moments of TBP MEFs untreated, immediately after 1 h 2 mM HU treatment or 30 min after washing away HU either in the absence or presence of 50 μM Mirin. (E, F) DNA2 expression levels in TBP-shRNA control (E) and TBP-RecqlKD (F) MEFs transfected with control (black) or DNA2 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (G) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and DNA2 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (H, I) MUS81 expression levels in TBP-shRNA control (H) and TBP-Recql KD (I) MEFs transfected with control (black) and MUS81 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (J) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and MUS81 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. For (C, D, G, J), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure 4. RECQL is involved in fork restart at broken, but not stalled replication forks.
(A) Schematic overview of the DNA fiber assay procedure to measure replication fork restart. Cells were labelled with CldU, treated with HU for 1 h, washed and labelled with IdU as indicated. (B) Quantification of fork stalling in TBP (black) and TBP-RecqlKD MEFs (grey) after 300 μM HU treatment. (C) Quantification of fork stalling in TBP (black) and TBP-RecqlKD MEFs (grey) after 2 mM HU treatment. (D) Quantification of fork stalling in TBP (black) and TBP-RecqlKD MEFs (grey) after 300 μM HU treatment in the presence of 50 μM Mirin. For (B, C, D), at least 300 different fiber types were counted. Error bars show SDs. Significant differences between three independent experiments are indicated (t test).
- Figure 5. RECQL prevents MRE11-dependent double strand break formation upon c-MYC–induced replication stress.
(A) C-MYC protein levels in RPE1 cells with either an empty or c-MYC-inducible construct without doxycycline treatment or with 3 d of doxycycline treatment. Anti-actin was used as loading control. (B) Growth curves of RPE1 cells with empty and c-MYC-inducible construct cultured in the presence of doxycycline (black and grey, respectively) and absence of doxycycline (dark blue and light blue, respectively) measured with the IncuCyte. Error bars show SDs. (C) Replication fork speed of RPE1 cells with empty or c-MYC–inducible construct cultured in the presence or absence of doxycycline. Track lengths of at least 75 ongoing forks were measured with ImageJ. SD is plotted and red bars denote the mean. Significance is indicated (nonparametric Kruskal–Wallis test). (D) Quantification of origin firing in RPE1s with empty or c-MYC–inducible construct cultured in the presence or absence of doxycycline. First label and second label origins are shown as percentage of all labelled tracks. Error bars show SDs. Significant differences between three independent experiments are indicated (t test). (E) RECQL expression levels in empty, c-MYC, empty-RecqlKD, and c-Myc–RecqlKD RPE1s measured by qRT-PCR. Error bars show SDs of two independent experiments. (F) RECQL protein levels in empty construct and c-MYC doxycycline inducible RPE1s. Anti-actin was used as loading control. (G) Growth curves of c-MYC and c-MYC-RecqlKD RPE1s cultured in the absence (grey and green, respectively) and presence of doxycycline (blue and red, respectively) measured with the IncuCyte. Error bars show SDs. (H) Tail moments of c-MYC and c-MYC-RecqlKD RPE1s without doxycycline or with 3 d doxycycline treatment. (I) Tail moments of c-MYC-RecqlKD RPE1s in the presence (3 d) or absence of doxycycline in combination with or without 12.5 μM Mirin. For (H, I), more than 50 cells for each condition were analyzed using CASP software. SD and means are indicated in black. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
- Figure 6. RECQL prevents DNA double-strand break formation in human cancer cells.
(A) RECQL expression levels of different cancer cell lines transfected with control and RECQL siRNAs measured by qRT-PCR. Error bars denote the SD of two independent experiments. (B) Tail moments of different cancer cell lines transfected with control and RECQL siRNAs. Mean of each condition is indicated by the red line and number. P-values are indicated above each cell line. (C, D) Tail moments of VU120T (C) and OCUB-M (D) cancer cells transfected with control and RECQL siRNAs and treated with and without 12.5 μM (C) and 6.25 μM (D) Mirin, respectively. SDs are plotted in black and red bars denote the mean. For (B, C, D), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
Supplementary Materials
Table S1 Overview of shRNA vectors.
Table S2 Overview of sequencing primers.
