Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination

Abstract

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

Keywords: ATM; BRCA1-A; Camptothecin; NDRG3; NHEJ; PLK1; UBAP2; homologous recombination; nascent chromatin capture; replication stress.

Graphical abstract

Figure 1

Protein composition of broken replication forks (A) SILAC-NCC-MS strategy for proteomics analysis of broken replication forks. Cells were released from a single thymidine block into mid-S phase and labeled with biotin-dUTP (b-dUTP) in the absence (untreated [Unt]) or presence of CPT (1 μM) prior to NCC purification. (B) Left: NCC pull-downs analyzed by western blotting. Unt samples were harvested immediately after b-dUTP labeling (0 h) or 2 h. Right: b-dUTP-labeled S-phase cells show CPT-induced DNA damage (γH2AX and pRPAS33). An arrowhead indicates b-dUTP-negative cells (outside of S phase). Scale bar, 15 μm. (C) GO analysis of proteins recruited (top 10% based on H/L ratio) or depleted (bottom 10% based on H/L ratio) at CPT-damaged forks. All identified proteins were used as background. p vvalue is shown with fold enrichment. (D) Illustration of dormant origin firing in response to CPT and suppression of new origin firing by roscovitine (rosco). (E) Experimental design for NCC-SILAC-MS using roscovitine to block CPT-induced dormant origin firing. (F) GO analysis as in (C). (G) Heatmap of replication and DDR factor enrichment, indicating the mean of three independent experiments. Not detected is indicated by gray coloring. See also Figure S1.

Figure 2

Protein dynamics in response to fork breakage and fork stalling (A) Left: heatmap of replication and DDR factor enrichment at broken (CPT+Rosco/Rosco) and stalled (HU/Unt) replication forks. To normalize for fork numbers, CPT+Rosco/Rosco data were used. The mean of three independent experiments is shown. Right: enrichment of DDR proteins across CPT and HU NCC-SILAC-MS datasets. Broken forks, CPT+Rosco/Rosco; stalled fork, HU/Unt. (B) Overview of DDR factor recruitment to broken forks (blue bars, CPT+Rosco/Rosco) and stalled forks (yellow bars, HU/Unt). The box gradient fill color indicates enrichment at undamaged forks (red) over mature chromatin (blue), as illustrated on the right (Alabert et al., 2014). Well-established protein complexes (red lines), functional groups (dotted line), and interactions (gray lines) are shown. Not detected (ND) is indicated by gray coloring. See also Figure S2.

Figure 3

Distinct chromatin environment at broken and stalled replication forks (A and B) Heatmap showing enrichment of inner nuclear membrane (INM) proteins (A), nuclear pore complex (NPC; A), and chromatin regulators (B) at broken (CPT+Rosco/Rosco) and stalled (HU/Unt) replication forks. The mean of three independent experiments is shown. (C) Enrichment of canonical histones across all NCC-SILAC-MS data (see Figures S7A and S7B for details). Mean is shown with SEM; n = 3. CHX, cycloheximide. (D) Left: the ratio of newly synthesized (new) and old recycled (old) histone H4 at replication forks isolated from CPT- and CHX-treated cells. New and old histones were analyzed by pulse-SILAC labeling followed by NCC-MS (Figure S3A). The CHX dataset is from Alabert et al. (2015). Mean is shown with SEM; n = 3 (CHX), 2 (CPT). Right: illustration of new and old histones in nascent chromatin. See also Figure S3.

Figure 4

ATM inhibition rewires the broken replication fork proteome toward a stalled fork response (A) Experimental design of NCC-SILAC-MS analysis of ATM function at CPT-damaged forks (CPT+ATMi/CPT). The ATMi AZD0156 (250 nM) was added 5 min before CPT (1 μM) treatment. (B) Enrichment of DDR (left) and chromatin (right) proteins, shown as log₂ SILAC ratios of CPT+ATMi (heavy) over CPT (light) (CPT+ATMi/CPT). The mean of six independent experiments is shown. NE, nuclear envelope. (C) Enrichments of DDR proteins from (B), grouped according to function. Symbol size indicates the number of replicates in which a protein was identified. ^∗, RPA binding proteins. (D and E) High-content microscopy of U-2-OS cells exposed to CPT or HU for 1 h with or without ATMi added 5 min before. Pre-extracted cells were stained for γH2AX and BRCA1 (D) or RPA (E). (D) BRCA1 foci in γH2AX positive cells are shown relative to cells treated with CPT alone. Error bars indicate SEM; n = 3. Individual measurements are indicated by dots and correspond to the mean of more than 1,092 cells. ^∗p = 0.0303; NS, not significant by ratio-paired two-sided t test. See the gating strategy for γH2AX positive cells in Figure S4I. (E) RPA intensity shown as mean (+), with whiskers indicating 10^th–90^th percentiles; from left, n = 3,858, 3,402, 3,398, 3,249, 8,946, 9,509, 8,525, and 8,068 cells. See the gating strategy for RPA-positive cells in Figure S4J. (F) Correlation plot showing DDR proteins identified in NCC-SILAC-MS as indicated. Pearson correlations (r) are shown. See also Figure S4.

Figure 5

ATM and PLK1 suppress RNF168 recruitment and downstream accumulation of RAP80 (A) DNA fiber analysis of replication fork restart. Top: experimental design. Fork restart, fork arrest, and new origin firing were scored as shown in Figure S5A. Bottom: percentage of labeled DNA tracks is shown as the mean, with error bars indicating SEM. Individual measurements are indicated by dots (CPT, n = 3; HU, n = 2). From the left, ^∗∗p = 0.0046, 0.0028, and 0.2658; NS, not significant by ratio-paired two-sided t test. (B–D) High-content microscopy of RNF168 and RAP80 recruitment to CPT-induced DNA repair foci. U-2-OS cells were exposed to CPT for 1 h in the presence or absence of ATMi (AZD0156) and PLK1 inhibitor (volasertib). In (D), cells were treated with control or RNF168 siRNAs for 48 h prior to drug treatment. Pre-extracted cells were stained for γH2AX and RNF168 (B) or RAP80 (C and D). RNF168 or RAP80 foci in γH2AX-positive cells are shown relative to cells treated with CPT alone. Error bars indicate SEM; n = 4 (B), n = 5 (C), n = 3 (D). Individual measurements are indicated by dots and correspond to the mean of more than 894 cells. A representative experiment is shown in Figures S5B and S5C. ^∗p < 0.05, ^∗∗p < 0.01 by ratio-paired two-sided t test. (E) Model illustrating suppression of H2A ubiquitination by ATM and PLK1 kinase activity at broken forks. See also Figure S5.

Figure 6

NDRG3 and UBAP2 are new HR factors required for CPT resistance (A) Overview of putative novel DDR factors identified by NCC-SILAC-MS. Recruitment to broken forks (blue, CPT+Rosco/Rosco) and stalled forks (yellow, HU/Unt) is indicated as in Figure 2B. A red dotted line encircles ATM/ATR substrates (Boeing et al., 2016; Matsuoka et al., 2007; Stokes et al., 2007). Factors were selected based on the enrichment of a higher than 0.3 log2 SILAC ratio and being putative ATM/ATR targets (Table S4). (B) Colony formation analysis. Left: siRNA depletion of NDRG3 in U-2-OS cells treated with CPT (50 nM) for 24 h. Mean with SEM, n = 3. Right: CRISPRi depletion of UBAP2 in U-2-OS cells treated with CPT (50 nM) for 24 h. Mean with SEM, n = 3. ^∗p < 0.05, ^∗∗p < 0.01 by ratio-paired two-sided t test. (C) High-content microscopy of γH2AX in siRNA-transfected U-2-OS cells exposed to CPT for 1 h. Mean intensity relative to control siRNA (siControl) is shown for γH2AX-positive cells, gated as shown in Figure S6F. Error bars indicate SEM, n = 4. Individual data points are indicated by dots and correspond to the means of more than 1,871 cells. (D) HR efficiency measured in DR-GFP U-2-OS reporter cells. The HR efficiency is shown as percent of control siRNA. Mean is shown with SEM, n = 3. ^∗p < 0.05, ^∗∗p < 0.01 by ratio-paired two-sided t test. (E) High-content microscopy of RPA in siRNA-transfected U-2-OS cells exposed to CPT for 1 h. Mean intensity relative to control siRNA (siControl) is shown for γH2AX-positive cells, gated as shown in Figure S6C. Error bars indicate SEM, n = 3. Individual data points are indicated by dots and correspond to the means of more than 1,871 cells. (F) High-content microscopy of RAD51 foci. Left: representative images of RAD51 and γH2AX. Scale bar, 10 μm. Right: bar diagram showing RAD51 foci in γH2AX-positive cells relative to control siRNA treatment. Mean with error bars showing SEM, n = 3. Data points are indicated by dots and correspond to the mean of more 449 cells. A representative experiment is shown in Figure S6H. siRNA-transfected U-2-OS cells were exposed to CPT for 3 h. See also Figure S6.

Figure 7

Model of proteome dynamics at broken replication forks Detailed model illustrating ATM function in protein recruitment at broken forks (this work) integrated with repair functions established in the literature (see text for references). ATM is recruited to broken forks, where it recruits PLK1 and promotes CtIP-dependent DNA end resection to facilitate HR repair. In parallel, ATM and PLK1 suppress H2A monoubiquitination and K63-linked poly-ubiquitination and, thereby, recruitment of BRCA1-A. ATM also promotes accumulation of POH1, a K63-specific deubiquitinase, and restricts accumulation of NHEJ factors such as SMCHD1, RIF1, and REV7.