Abstract
The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hypersensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR-activating proteins.
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Acknowledgements
The research was supported primarily by R01GM116616 to D.C. with additional support from R01CA102729 and the Vanderbilt-Ingram Cancer Center. NMR experiments were supported by R01GM65484 and P01CA092584 to W.J.C. T.E.B. is supported by training grant T32CA009582-28. We thank R. Guo for performing the initial Flag-RPA1 immunopurifications and W. Hayes McDonald in the Vanderbilt Proteomics Laboratory for performing the mass spectrometry.
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T.E.B., J.W.L., G.K., C.C., H.D., G.G.G. and D.C. performed most of the experiments. M.D.F. and R.P. performed the NMR experiments with supervision from W.J.C. T.E.B. and D.C. conceived of the project and wrote the manuscript. D.C. supervised the project.
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Supplementary Figure 1 RPA recruits ETAA1 to damaged replication forks.
(a) Examples of GFP-Flag-ETAA1 localization in cells treated with HU or cisplatin for 3 h. (b) Schematic of ETAA1 fragments tested for their ability to bind RPA. (c–e) Nuclear extracts were prepared from HEK293T cells after transfection with the indicated GFP-Flag-NLS-ETAA1 expression vectors. Flag immunoprecipitates were separated by SDS-PAGE and examined by immunoblotting. Mock, mock-transfected. Cells were treated with 100 nM CPT for 3 h prior to lysis where indicated. The interaction between ETAA1 and RPA was not changed substantially in cells treated with CPT. Representative blots from one of two independent experiments are shown. (f) Quantitation of cells containing ETAA1 foci after transfection with the indicated ETAA1 expression vectors. Mean and SEM from n = 3 experiments is shown. (g) U2OS cells were transfected with an ETAA1 expression vector missing the C-terminal RPA32 interaction motif (ETAA1Δ32) in combination with non-targeting or RPA siRNA and imaged for ETAA1 and RPA localization after a challenge with 100 mM CPT. (h) ETAA1Δ cells were transduced with lentivirus to express empty vector (GFP-Flag), GFP-Flag-ETAA1 (WT), or GFP-Flag-ETAA1 with point mutations (residues 606-611 mutated from DVDDDL to NAAIRS) in the RPA70N motif and deletion of the ETAA1-RPA32C (deletion of residues 885–926) interaction motif (ETAA1ΔRPA). Cells were treated with 100 nM CPT for 3 h. RPA, and Flag-GPF-ETAA1 were visualized by immunofluorescence. Representative images from one of two independent experiments in g and h are shown. Scale bars are 5 μm. Unprocessed original scans of blots in c, d and e are shown in Supplementary Fig. 8, and source data for f is in Supplementary Table 1.
Supplementary Figure 2 ETAA1 knockdown causes hypersensitivity to replication stress.
(a–b) HeLa, H157, and BT549 cells transfected with non-targeting (NT) or ETAA1 siRNAs were left untreated or treated with 100 nM CPT for 3 h. Cells were fixed and RPA foci and γH2AX intensity were quantified by immunofluorescence imaging. The intensity of each nucleus, mean intensity, and number of nuclei imaged is depicted. (c) U2OS cells were transfected with non-targeting or ETAA1 siRNAs. All siRNAs target different regions of the ETAA1 coding sequence or 3′ UTR. siE1, siE3, and siE3 are different from the four ETAA1 siRNAs in the pool. Viability was measured 48 h after challenging the cells with 0.5 μM CPT for 24 h. Data show the mean of three independent experiments for siNT and siETAA1 pool and two for the other siRNAs. [OK?] (d–k) HeLa, HCT116, H157, and BT549 cells were transfected with non-targeting or ETAA1 siRNAs and viability was measured 72 h after challenge with CPT or HU. Data show the mean from three technical replicates and the experiment was performed once. (l) U2OS cells were transfected with non-targeting or ETAA1 siRNAs and exposed to 0, 3, or 5 Gy ionizing radiation. Cell viability was determined by clonogenic assay. (m–o) U2OS cells were transfected with non-targeting or ETAA1 siRNAs and viability was measured 72 h after challenge with cisplatin, olaparib, or BMN673. ATR siRNA was used as a positive control. In l–o the mean viability from from three replicates is shown. One representative experiment is shown and the experiments were completed twice independently. Source data for all panels is in Supplementary Table 1.
Supplementary Figure 3 ETAA1 interacts with multiple DNA damage response proteins.
(a) Flag-ETAA1 was immunoprecipitated from nuclear extracts of HEK293T cells, and immunoprecipitated proteins were identified by mass spectrometry. Extracts in the second replicate were treated with benzonase and RNAse prior to the immunopurification. Shown are peptide counts from two experiments including untransfected cell populations (Ctl). (b,c) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a Flag-GFP-ETAA1 expression vector (lanes 2 and 4) and ETAA1 was immunoprecipitated using Flag antibodies. Co-precipitating proteins were separated by SDS-PAGE and detected by immunoblotting. (d) HEK293T nuclear extracts were fractionated on a Superdex 200 column. Protein fractions were then separated using SDS-PAGE and immunoblotted with the indicated antibodies. To detect ETAA1, it was immunoprecipitated from the fractions prior to immunoblotting. The reason ETAA1 migrates as a doublet in some gel conditions is not known but may represent post-translational modifications. Unprocessed original scans of blots in b,c and d are shown in Supplementary Fig. 8. Blots in b and c are representative from one of two experiments. The experiment in d was completed once.
Supplementary Figure 4 ETAA1 is required for RPA phosphorylation in multiple cell types.
(a) U2OS, HeLa, HCT116, H157, BT549, and A549 cells transfected with non-targeting or ETAA1 siRNAs were left untreated or treated with 100 nM CPT for 8 h. Proteins were separated by SDS-PAGE and detected by immunoblotting. Unprocessed original scans of blots are shown in Supplementary Fig. 8. This experiment with all of the cell lines was completed once although the results in U2OS, HeLa, and HCT116 were confirmed in independent replicates.
Supplementary Figure 5 ETAA1 over-expression causes DNA damage signaling.
(a–h) U2OS cells were transfected with the indicated GFP-Flag-NLS-ETAA1 protein expression vectors, and GFP and γH2AX were visualized by immunofluorescence imaging. (a, b) Cell Profiler was used to quantitate γH2AX intensity in ETAA1 expressing cells. The intensity of each nucleus, mean intensity, and number of nuclei imaged is depicted. Data represent one out of three experiments in a and one out of two experiments in b. (c,d) The percentage of cells with γH2AX or ETAA1 foci were scored manually in blinded samples. Mean and SEM from n = 4 experiments is graphed. (e) Immunoblot of proteins to examine expression levels. (f) Immunoblot of untransfected (lane 1), transiently transfected (lane 3), and stable ETAA1 expressing cell lines after sorting (lane 2). (g,h) GFP-Flag-ETAA1-2-250 or 2-250-W107A intensity versus γH2AX intensity is plotted. Pearson correlation coefficient is shown. Data in e–h are representative of two experiments. Unprocessed original scans of blots in e are shown in Supplementary Fig. 8, and source data for a–d is in Supplementary Table 1.
Supplementary Figure 6 An interaction with RPA is needed for ETAA1 to maintain genome integrity.
(a,b) ETAA1Δ cells were infected with wild-type or RPA binding-deficient ETAA1 lentiviruses. After antibiotic selection, the cell populations were sorted to select for the 10% of cells expressing the lowest amount of ETAA1 protein. (b) Immunoblot shows that the complemented cells overexpress approximately equal amounts of wild-type (WT) and ETAA1ΔRPA proteins. (c) Cell populations were challenged with the indicated concentrations of CPT and cell viability measured after 72 h. The mean viability from three technical replicates from one of two independent experiments is shown. (d) The percentage of cells with micronuclei was scored in the indicated cell populations. Data from two independent complemented ETAA1Δ clones are shown. The mean percentage of cells with micronuclei and total number of nuclei scored from five technical replicates of one of two biological replicates is shown. Unprocessed original scans of blots in b are shown in Supplementary Fig. 8, and source data for c and d are in Supplementary Table 6.
Supplementary Figure 7 ETAA1- and TOPBP1-deficient cells maintain BLM expression.
WT and ETAA1Δ U2OS cells were transfected with non-targeting or two siRNAs targeting TOPBP1. Cell lysates were immunoblotted with the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 8. Representative blots from one out of two experiments is shown.
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Bass, T., Luzwick, J., Kavanaugh, G. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nat Cell Biol 18, 1185–1195 (2016). https://doi.org/10.1038/ncb3415
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DOI: https://doi.org/10.1038/ncb3415
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