The nuclease EXO1 promotes genomic instability by degrading nascent DNA in BRCA-proficient cells

Domain: chemistry
Published: 2026-02-25
Editorial outcome: major_revision

Abstract
DNA repair genes are generally considered tumor suppressors, as their inactivation is observed in tumors and is associated with carcinogenesis. Mutations in BRCA1 and BRCA2 genes are observed in breast, ovarian, and other cancers. This results in defective homologous recombination DNA repair, as well as degradation of nascent DNA during replication stress, catalyzed by exonucleases including EXO1 and MRE11. However, most tumors are BRCA pathway-proficient. Here, we show that EXO1 is overexpressed in a significant proportion of tumors. EXO1 overexpression causes the degradation of nascent DNA at both single stranded DNA (ssDNA) gaps and reversed replication forks. Importantly, this degradation occurs efficiently in BRCA-proficient cells, through cooperation with MRE11. This results in increased double strand break formation and hypersensitivity to genotoxic agents. We thus identify increased EXO1 activity as a mechanism of genomic instability similar to BRCA pathway inactivation, but occurring more frequently in tumors compared to BRCA inactivation.

Introduction
Genomic instability is an enabling characteristic of carcinogenesis, allowing cancer cells to acquire new phenotypes and evolve constantly1. To ensure continuous proliferation, cancer cells need to replicate their DNA at appropriate rates. This exposes them to increased replication stress, caused by the encounter of the DNA replication machinery with unrepaired DNA lesions or by reduced availability of nucleotides, resulting in slowing or arrest of replication forks2–4. Cancer cells harness cellular genome stability mechanisms to control replication stress and may even use them to drive genome instability5,6.

Stalled replication forks can be restarted through several mechanisms, primarily among those being fork repriming and fork reversal. Fork repriming entails the assembly of a new replication complex downstream of the stalled fork, initiated by the repriming activity of the PRIMPOL primase-polymerase7–18. This leaves behind a single stranded DNA (ssDNA) gap to be filled at a later time. In contrast, fork reversal involves the annealing of the nascent strands of the sister chromatids, catalyzed by the ZRANB3, HLTF, and SMARCAL1 translocases, generating a 4-way junction which stabilizes the fork and allows restart using the nascent strand of the sister chromatid as template6,8,19–22.

EXO1 is a 5′−3′ exonuclease which participates in multiple DNA repair processes involving exonucleolytic processing of DNA lesions23. EXO1 plays an important role in long-range DNA resection at double stranded DNA breaks (DSBs)24. This process generates long single stranded DNA (ssDNA) overhangs which are subsequently coated by RAD51 molecules loaded by the BRCA2 protein. EXO1 is thus an important player in BRCA-mediated homologous recombination (HR) repair of DSBs. EXO1 also participates in DNA mismatch repair (MMR), by degrading the nascent DNA strand containing the mismatch, to allow repair DNA synthesis from the parental strand25.

However, beyond these roles of EXO1 in promoting genomic stability, recent research has indicated that EXO1 activity can also promote genomic instability in certain genetic backgrounds. On reversed forks, BRCA-mediated loading of RAD51 is essential to protect the DSB end on the reversed arm formed by the nascent strand of the sister chromatids. In BRCA-mutant cells, this structure is degraded by nucleolytic activities, primarily those of MRE11 and EXO1. Indeed, it was proposed that one mechanism through which BRCA-deficient cells are sensitive to genotoxic cancer therapeutic drugs is the degradation of nascent DNA at reversed forks6,8,19–22. We recently showed that EXO1 engages the reversed arm to degrade the nascent DNA in the 5’−3’ direction, while MRE11 degrades the complementary strand in the 3’−5’ direction26.

Similarly, the filling of nascent strands ssDNA gaps is essential to suppress their nucleolytic expansion. Gap filling can be achieved by multiple mechanisms, including BRCA-mediated recombination using the nascent strand of the sister chromatid as a template, or by employing DNA polymerases able to bypass DNA lesions, a process termed translesion DNA synthesis (TLS). The inability to fill ssDNA gaps has been associated with genomic instability and chemosensitivity. Recent studies have proposed that accumulation of ssDNA gaps is one of the components causing increased sensitivity to cisplatin and PARP inhibitors in BRCA-deficient cells, since they get converted into DSBs, which are cytotoxic in these cells8,11,26–38. Moreover, concomitant inactivation of both the BRCA and TLS pathways synergize to provide increased chemosensitivity10,11, further highlighting the importance of ssDNA gap repair.

We recently showed that, in BRCA-deficient cells, cisplatin-induced ssDNA gaps are exonucleolytically expanded by MRE11 and EXO1 in opposing directions, in a process reminiscent of the degradation of reversed forks39,40. We also found that unfilled ssDNA gaps are processed into DSBs, and exonucleolytic gap expansion is a critical intermediate in this processing, since inactivation of EXO1 or of MRE11 suppressed ssDNA gap-induced DSB formation. Importantly, EXO1 depletion also suppressed the cisplatin sensitivity of BRCA2-deficient cells. This suggests that EXO1-mediated expansion of ssDNA gaps contributes to cisplatin toxicity in these cells.

The role of the BRCA pathway in protecting nascent DNA against nucleolytic degradation at ssDNA gaps and reversed replication forks is considered a critical component of its tumor suppression activity. While BRCA inactivation is found in a subset of cancers, most tumors are thought to be BRCA-proficient. Thus, other genetic changes to drive genome instability through these mechanisms may exist in cancer cells. Here, we show that EXO1 is overexpressed in a significant proportion of cancers, including breast, hepatocellular, skin, testicular, and cervical cancers and EXO1 expression is associated with increased genomic alterations. We moreover show that EXO1 overexpression causes nascent strand degradation, at both ssDNA gaps and reversed forks, through its exonuclease catalytic activity. EXO1-mediated nascent strand degradation occurs efficiently in BRCA-proficient cells, through its cooperation with MRE11. This results in increased DSB formation and hypersensitivity to genotoxic agents. We thus identify increased EXO1 activity as a mechanism of genomic instability, similar to BRCA inactivation but occurring in a larger proportion of tumors.

Results
EXO1 is overexpressed in tumors which is associated with increased genome instability
To assess the relevance of EXO1 in cancer, we queried the TCGA database41 for EXO1 alterations in tumor samples using the cBioPortal repository42. In line with previous studies43–47, we found that EXO1 is predominantly overexpressed, through both gene amplification and increased mRNA levels without gene amplification, in all tumor datasets analyzed (Supplementary Fig. 1a). In contrast, there were very few instances of mutations, deletions, or reduced mRNA levels. These findings indicate that, despite the known role of EXO1 in DNA repair, increased EXO1 expression, rather than loss of EXO1 function, is found in cancer samples.

In particular, we found that alterations in EXO1 are most predominant in breast cancer, with ~22% of all breast invasive carcinoma samples in the TCGA PanCancer Atlas dataset showing such alterations. Of those, the vast majority represents EXO1 overexpression (~21%) compared to EXO1 mutation/deletion (~1%). To validate these findings, we investigated EXO1 expression in breast cancer cell lines. Consistent with previous studies45, we found lower EXO1 expression in the MCF10A normal epithelial mammary gland cell line, compared to BRCA pathway-proficient breast cancer cell lines T47D, MDA-MB-231, MCF7 and MDA-MB-468 (with significant variation of EXO1 expression within the breast cancer cell lines) (Supplementary Fig. 1b).

When we compared breast cancer subtypes, analyses of TCGA PanCancer Atlas breast invasive carcinoma samples revealed an over-representation of basal-like breast cancer in EXO1-overexpressing samples, to about ~50% of all cases in the dataset, compared to control (non EXO1-overexpressing) samples, of which only about ~8% were basal-like (Supplementary Fig. 2a). Basal-like breast cancer is an aggressive subtype of breast cancer, characterized by lack of expression of estrogen, progesterone or HER-2/neu hormones receptors (triple negative). It is considered that basal-like breast cancer tumors have a different etiology than hormone receptor-positives breast tumors, with genomic instability rather than hormone-induced proliferation being the major determinant48–51. Indeed, basal-like breast cancer is sometimes associated with BRCA1 inactivation. However, in the TCGA PanCancer Atlas breast invasive carcinoma dataset analyzed, only 3% of samples had BRCA1 mutations. We thus sought to investigate if, similar to BRCA-inactivated tumors, EXO1-overexpressing tumors show increased genomic instability. We found that EXO1 expression in breast tumors in this dataset is not associated with increased point mutation rates (Supplementary Fig. 2b). However, we found that EXO1 expression positively correlates with genome alterations (amplifications, deletions, translocations) (Supplementary Fig. 2c), suggesting that EXO1 promotes chromosomal structural instability and formation of chromosome structural variants, which are known to result from incorrect repair of DSBs.

Besides breast invasive carcinoma, the TCGA PanCancer Atlas analyses showed that EXO1 alterations were also prevalent in hepatobiliary cancer (with ~20% of cases showing EXO1 alterations), melanoma and testicular cancers (~16% each), and cervical cancer (~14%) (Supplementary Fig. 1a). Increased EXO1 expression was vastly predominant in these cancers as well, at the expense of mutations or deletions/downregulation. To further evaluate the connection between EXO1 overexpression and genomic instability, we expanded the mutational analysis to these cancers. We combined the breast, hepatobiliary, melanoma, testicular, and cervical tumor TCGA PanCancer Atlas datasets and evaluated the pooled dataset for genomic instability. We were able to confirm a correlation between EXO1 expression and genome alterations (Supplementary Fig. 2d).

As controls, we investigated how expression of other DNA repair genes affected genome alterations. We previously showed that PARP10 is overexpressed in breast and ovarian cancers52. We also showed that PARP10 interacts with the ubiquitin ligase RAD18 to promote the ubiquitination of PCNA, an event which is critical for TLS-mediated lesion bypass53. TLS is catalyzed by mutagenic polymerases and causes point mutagenesis54,55. In contrast to EXO1, the expression of PARP10, RAD18, or of the TLS polymerase POLH were not correlated with increased genome alteration (Supplementary Fig. 2e–g). Overall, these findings argue that increased EXO1 levels may be associated with increased genomic instability in cancer samples.

EXO1 overexpression results in replication-associated DNA lesions upon replication stress
To directly test the impact of EXO1 expression levels on genomic instability, we stably overexpressed wild type EXO1 (EXO1OE) in BRCA pathway-proficient HeLa (cervical cancer) and U2OS (osteosarcoma) cells using a lentiviral expression system (Fig. 1a, b). As controls, we also obtained stable cell lines expressing the empty vector (EV) or overexpressing the EXO1 catalytic mutant D173A (EXO1-D173AOE), which inactivates its exonuclease activity56,57. Both variants were expressed at similar levels to each other. Moreover, the EXO1 overexpression achieved in this experimental system is comparable to EXO1 expression in breast cancer cell lines (Supplementary Fig. 1b).

We previously showed that, upon replication stress, EXO1 degrades nascent DNA in BRCA-deficient cells, both at reversed replication forks26, as well as upon fork restart by PRIMPOL and gap formation39. We thus first investigated if EXO1 overexpression causes replication-associated DNA lesions through its catalytic activity. We employed the alkaline comet assay, which can detect single strand DNA lesions, as well as double stranded DNA breaks. To only detect replication-associated lesions, we treated cells with the thymidine analog BrdU to label replicating cells, followed by 1 hr growth in BrdU-free media (“chase”) to allow the processing of normal replication intermediates such as DNA nicks on the lagging strand. During the BrdU labeling, we induced replication stress by treatment with a mild dose (0.4 mM for 30 mins) of hydroxyurea (HU), which depletes nucleotide pools, reducing replication fork progression, and thus mimicking nucleotide deprivation which is observed in hyper-replicating cells during cancer initiation58. Under these conditions, we observed an increase in the alkaline comet tail moment in BrdU-labeled EXO1-overexpressing cells, but not in cells overexpressing the catalytic mutant, in both HeLa and U2OS cells (Fig. 1c–e).

We also induced replication stress by acute treatment (150 μM for 30 mins) with cisplatin, a chemotherapeutic agent which causes guanine adducts on DNA and thus interferes with DNA replication59,60. Similar to the HU experiments, we observed an increase in the alkaline comet tail moment in BrdU-labeled EXO1-overexpressing cells, but not in cells overexpressing the catalytic mutant, in both HeLa and U2OS cells (Fig. 1f, g). As a positive control, we employed cells overexpressing PRIMPOL, which have increased ssDNA levels due to increased repriming downstream of DNA lesions8,11,40. As we previously showed40, PRIMPOL overexpression causes an increase in BrdU olive tail moment. These findings suggest that, upon replication stress, EXO1 overexpression causes replication-associated DNA lesions, and its catalytic activity is required for this.

EXO1 overexpression results in accumulation of ssDNA gaps in BRCA-proficient cells by promoting MRE11 recruitment to nascent DNA for gap expansion
We previously showed that EXO1 expands nascent strand ssDNA gaps formed upon replication stress in BRCA2-deficient cells39,40. Since the BrdU alkaline comet assay can detect ssDNA gaps, and the replication stress conditions we used for the experiments described above are known to cause ssDNA gaps38,40,61, we tested if the BrdU alkaline comet signal may derive from increased gap accumulation. To this end, we measured ssDNA gaps using the S1 nuclease DNA fiber combing assay62. We incubated cells consecutively with thymidine analogs IdU and CldU and treated them with 0.4 mM HU during the CldU incubation. Treatment of purified DNA fibers with the ssDNA endonuclease S1 results in cleavage of DNA regions exposing ssDNA.

Using this assay, we observed ssDNA gap accumulation in HU-treated EXO1-overexpressing HeLa and U2OS cells, as evidenced by a decrease in CldU/IdU ratios in S1-treated cells (Fig. 2a, b). In contrast, ssDNA gaps were not detected in EV control cells or in cells overexpressing the D173A catalytic mutant. Similar findings were observed upon treatment with 150 μM cisplatin, in both HeLa and U2OS cells (Fig. 2c, d). These findings suggest that increased EXO1 levels are enough to cause ssDNA gap accumulation, even in cells with functional BRCA pathway.

We previously showed that EXO1 interacts with MRE11 to bidirectionally expand ssDNA gaps in BRCA-deficient cells39,40. Since we observed ssDNA gap accumulation in EXO1-overexpressing cells, we employed the proximity ligation (PLA)-based SIRF (in situ quantification of proteins interactions at DNA replication forks) assay, which allows the quantification of specific protein recruitment to nascent DNA63, to investigate if increased EXO1 levels are enough to recruit MRE11 to nascent DNA under replication stress conditions. We employed a SIRF labeling scheme designed to detect MRE11 engagement on gaps considering its 3′ −5′ directionality of exonuclease activity: we first labeled cells with EdU for 30 min, then removed it and added 0.4 mM HU for 3 h in order to measure MRE11 engagement on the 3′ ends of the gaps (Fig. 3a). We found that MRE11 is recruited to nascent DNA in EXO1-overexpressing cells but not in cells overexpressing the D173A catalytic mutant, in both HeLa and U2OS cells (Fig. 3b–e). As a control, in the absence of HU treatment, MRE11 was not recruited to nascent DNA (Fig. 3d).

We next investigated the localization of EXO1 itself. We employed a SIRF labeling scheme designed to detect EXO1 engagement on gaps considering its 5′ −3′ exonuclease activity: we labeled cells with EdU for 30 min, with 0.4 mM HU added only during the first 20 min of EdU labeling, in order to measure EXO1 engagement on the 5′ ends of the gaps (Fig. 4a). In both HeLa and U2OS cells, we found increased EXO1 recruitment to nascent DNA in cells overexpressing wild type EXO1, but not in those overexpressing the D173A mutant (Fig. 4b–e).

Since we previously showed that MRE11 and EXO1 co-localize in BRCA-deficient cells39, we also tested if EXO1 overexpression causes an increase in the formation of EXO1-MRE11 complexes. Using PLA experiments we found that, in both HeLa and U2OS cells, MRE11 and EXO1 show increased co-localization upon treatment with 0.4 mM HU in EXO1-overexpressing cells, and this interaction is reduced in cells overexpressing the EXO1 D173A mutant (Fig. 4f, g). This suggests that increased EXO1 activity enhances the localization of MRE11 to nascent DNA in BRCA-proficient cells, and the co-localization of EXO1 and MRE11 at these structures.

EXO1 overexpression promotes the degradation of reversed replication forks in BRCA-proficient cells
Upon prolonged fork arrest, replication forks reverse into a 4-way structure which exposes a DSB end. The BRCA pathway stabilizes the reversed fork6,8,19–22. We previously showed that EXO1 collaborates with MRE11 to catalyze the degradation of both strands of the DSB end of reversed replication forks in BRCA-deficient cells26. We thus tested if EXO1 overexpression can promote the degradation of stalled replication forks, even in BRCA-proficient cells. To induce replication fork reversal, we treated cells with high-dose (4 mM) HU which causes acute fork arrest64. We used the DNA fiber combing assay using a labeling scheme designed to investigate nascent strand degradation, namely consecutive incubation with thymidine analogs IdU and CldU for 30 mins each, followed by treatment with 4 mM HU for 4 h64. Nascent strand degradation is detected as a reduction in CldU/IdU ratios. We found that EXO1 overexpression, but not overexpression of the D173A mutant, resulted in nascent strand degradation, in both HeLa and U2OS cells (Fig. 5a, b). We also employed this assay to examine stalled fork degradation in BRCA-proficient breast cancer cell lines. We found that T47D cells, which showed the highest level of EXO1 expression in our hands, have increased replication fork degradation compared to MDA-MB-468 and MCF7 cells, which showed comparatively less EXO1 expression (Supplementary Fig. 1b, 3a–e).

We next tested if, similar to ssDNA gap expansion, MRE11 activity is also involved in the degradation of reversed forks in EXO1-overexpressing cells. To this end, we first employed SIRF experiments to investigate the engagement of MRE11 to reversed replication forks21. We labeled cells with EdU for 20 min and then treated them with 4 mM HU for 4 h to induce fork arrest and reversal (Fig. 5c). Under these conditions, we found increased MRE11 engagement on nascent DNA in EXO1-overexpressing cells but not in cells overexpressing the D173A catalytic mutant, in both HeLa and U2OS cells (Fig. 5d–g). Confirming that the MRE11 SIRF signal in EXO1-overexpressing cells reflects MRE11 engagement on reversed replication forks, this signal was abolished upon knockdown of the fork reversal translocase SMARCAL1, which is required for fork reversal65 (Fig. 5h, Supplementary Fig. 4a).

The increased MRE11 binding to reversed forks in EXO1-overexpressing cells was surprising, considering that these cells are proficient for BRCA-mediated loading of RAD51 to these structures. Surprisingly, we found that EXO1-overexpressing cells show increased MRE11 SIRF signal compared to BRCA2-depleted cells (Fig. 5h), arguing that increased EXO1 activity promotes MRE11 engagement on reversed forks to a greater extent than loss of BRCA2-mediated RAD51 coating of reversed forks. Importantly, when we knocked down BRCA2 in EXO1-overexpressing cells, we found no additional increase in MRE11 loading, suggesting that EXO1 overexpression completely alleviates the fork protection activity of BRCA2.

In line with this, DNA fiber combing experiments showed that both inhibition of MRE11 exonuclease activity using the specific inhibitor mirin, as well as its siRNA-mediated depletion, suppress the fork degradation observed in EXO1-overexpressing HeLa and U2OS cells, indicating that MRE11 nuclease activity is critical for this degradation (Fig. 5i, Supplementary Fig. 4b–d). Confirming that this degradation occurs on reversed replication forks, knockdown of the fork reversal translocases ZRANB3 and SMARCAL165 also suppressed fork degradation in EXO1-overexpressing cells (Fig. 5i). Moreover, the levels of ZRANB3 and SMARCAL1, as well as those of PRIMPOL and MRE11 were not affected in EXO1-overexpressing cells compared to control cells (Supplementary Fig. 5a–d), ruling out an unspecific effect of EXO1 overexpression. These findings indicate that EXO1 overexpression causes degradation of reversed forks in BRCA-proficient cells, through engagement of MRE11.

EXO1 overexpression causes DSB accumulation and chemotherapy sensitization
We previously showed that, in both BRCA-deficient and PRIMPOL-overexpressing cells, ssDNA gap expansion by MRE11 and EXO1 results in DSB formation39,40. Moreover, degradation of reversed forks may also promote DSB formation36. We thus investigated if EXO1 overexpression is enough to cause DSB accumulation upon replication stress, even in BRCA-proficient cells. We quantified DSBs using the neutral comet assay. Treatment with either 0.4 mM HU or 150 μM cisplatin for 2 h resulted in DSB accumulation in EXO1-overexpresing cells but not in cells overexpressing the D173A catalytic mutant, in both HeLa and U2OS cells (Fig. 6a–d). Moreover, γH2AX foci were specifically increased upon treatment with 500 nM cisplatin for 20 h (Fig. 6e, f). These findings indicate that EXO1 overexpression causes DSB formation under replication stress conditions, even in BRCA-proficient cells.

Since the experiments presented above indicated that EXO1 overexpression causes DSB formation in response to cisplatin treatment, we measured if EXO1 overexpression increases the cellular sensitivity to cisplatin. We treated cells with 0.2 μM cisplatin for 10 days and observed that EXO1-overexpressing HeLa cells were more sensitive to the drug treatment compared to control cells (Fig. 7a). In contrast, overexpression of the D173A catalytic mutant did not affect cisplatin sensitivity compared to control cells. Increased sensitivity to DNA damaging agents, degradation of reversed replication forks, and accumulation of ssDNA gaps are hallmarks of BRCA pathway deficiency. BRCA-mutant cells are hypersensitive to PARP1 inhibitors. We thus tested the PARP1 inhibitor sensitivity of EXO1-overexpressing cells. We observed that HeLa cells overexpressing wild type EXO1, but not cells overexpressing the EXO1 catalytic mutant D173A, are also sensitive to the PARP1 inhibitor olaparib (Fig. 7b, c). The olaparib sensitivity of EXO1-overexpressing cells was similar to that of BRCA2-knockout cells (Fig. 7c). Overall, these findings show that increased EXO1 activity sensitizes BRCA-proficient cells to cisplatin and PARP1 inhibitors.

Methods
Cell culture and protein techniques
HeLa (ATCC CCL-2), U2OS (ATCC HTB-96), T47D (ATCC HTB-133), MDA-MB-231 (ATCC HTB-26), MCF7 (ATCC HTB-22), MDA-MB-468 (ATCC HTB-132), and MCF10A (ATCC CRL-10317) cells, obtained from ATCC, were grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 15% FBS and penicillin/streptomycin. For overexpression of wildtype or D173A EXO1 variants, pLV[Exp]-Puro-CMV>hEXO1 lentiviral constructs (VectorBuilder) were used. Infected cells were selected by puromycin. PRIMPOL-overexpressing HeLa and U2OS cells were generated in our laboratory and previously described40. BRCA2-knockout HeLa cells were generated in our laboratory and previously described71.

Gene knockdown was performed using Lipofectamine RNAiMAX. AllStars Negative Control siRNA (Qiagen 1027281) was used as control.

The following oligonucleotide sequences (Stealth or SilencerSelect siRNA, ThermoFisher) were used:
ZRANB3: UGGCAAUGUAGUCUCUGCACCUAUA;
SMARCAL1: CACCCTTTGCTAACCCAACTCATAA;
BRCA2: AUUAGGAGAAGACAUCAGAAGCUUG;
MRE11: AAUAACUCGAGGCAGGUAUGUAAUG.

Denatured whole cell extracts were prepared by boiling cells in 100 mM Tris, 4% SDS, 0.5 M β-mercaptoethanol. Antibodies used for Western blot, at 1:500 dilution, were:
EXO1: Novus NBP2-16391;
GAPDH: Santa Cruz Biotechnology sc-47724;
SMARCAL1: Invitrogen PA5-54181;
ZRANB3: Novus NBP2-93301;
MRE11: Santa Cruz Biotechnology sc-135992;
PRIMPOL: Proteintech 29824-1-AP.

Functional assays
Neutral and BrdU alkaline comet assays were performed as previously described38 using the Comet Assay Kit (Trevigen, 4250-050). For the BrdU alkaline comet assay, cells were incubated with 100 μM BrdU as indicated. Chemical compounds (HU, cisplatin) were added according to the labeling schemes presented. Slides were stained with anti-BrdU (BD347580) antibodies and secondary AF568-conjugated antibodies (Invitrogen A-11031) and imaged on a Nikon microscope operating the NIS Elements V1.10.00 software. Olive tail moment was analyzed using CometScore 2.0. Immunofluorescence was performed as previously described72 using a γH2AX antibody (Millipore Sigma JBW301). Slides were imaged on a confocal microscope (Leica SP5) and analyzed using ImageJ 1.53a software. For drug sensitivity assays, cells were grown for 10 days in the presence of 0.2 μM cisplatin, 0.2 μM olaparib, or 0.5 μM olaparib, with splitting every 2-3 days. Cellular viability was determined using a NanoEntek EVE Automated Cell Counter.

Proximity ligation-based assays
For PLA assays, cells were seeded into 8-chamber slides and 24 h later, treated as indicated in the respective labeling schemes. Cells were then permeabilized with 0.5% Triton for 10 min at 4°C, washed with PBS, fixed at room temperature with 3% paraformaldehyde in PBS for 10 min, washed again in PBS, and then blocked in Duolink blocking solution (Millipore Sigma DUO82007) for 1 hr at 37°C, and incubated overnight at 4°C with primary antibodies. Antibodies used were: MRE11 (Genetex GTX70212) and EXO1 (Novus NBP2-16391). Samples were then subjected to a proximity ligation reaction using the Duolink kit (Millipore Sigma DUO92008) according to the manufacturer’s instructions. Slides were imaged using a confocal microscope (Leica SP5) and images were analyzed using ImageJ 1.53a software.

For SIRF assays, cells were seeded into 8-chamber slides and 24 h later they were pulse-labeled with 50 µM EdU and treated with chemical compounds according to the labeling schemes presented. Cells were permeabilized with 0.5% Triton for 10 min at 4°C, washed with PBS, fixed at room temperature with 3% paraformaldehyde in PBS for 10 min, washed again in PBS, and then blocked in 3% BSA in PBS for 30 min. Cells were then subjected to Click-iT reaction with biotin-azide using the Click-iT Cell Reaction Buffer Kit (ThermoFisher C10269) for 30 min and incubated overnight at 4°C with primary antibodies diluted in PBS with 1% BSA. The primary antibodies used were: Biotin (mouse: Jackson ImmunoResearch 200-002-211; rabbit: Bethyl Laboratories A150-109A); MRE11 (GeneTex GTX70212); EXO1 (Santa Cruz Biotechnology sc-56092). Next, samples were subjected to a proximity ligation reaction using the Duolink kit (MilliporeSigma DUO92008) according to the manufacturer’s instructions. Slides were imaged using a confocal microscope (Leica SP5) and images were analyzed using ImageJ 1.53a software. To account for variation in EdU uptake between samples, for each sample, the number of protein-biotin foci were normalized to the average number of biotin-biotin foci for that respective sample. The scale bars for the SIRF micrographs shown represent 10 µm.

DNA fiber combing assays
Cells were incubated with 100 µM IdU and 100 µM CldU as indicated. Chemical compounds were added according to the labeling schemes presented. Next, cells were collected and processed using the FiberPrep kit (GenomicVision EXT-001) according to the manufacturer’s instructions. Samples were added to combing reservoirs containing MES solution (2-(N-morpholino)ethanesulfonic acid) and DNA molecules were stretched onto coverslips (GenomicVision COV-002-RUO) using the FiberComb Molecular Combing instrument (GenomicVision MCS-001). For S1 nuclease assays, MES solution was supplemented with 1 mM zinc acetate and either 40 U/mL S1 nuclease (ThermoFisher 18001016) or S1 nuclease dilution buffer as control, and incubated for 30 min at room temperature. Slides were then stained with antibodies detecting CldU (Abcam ab6326) and IdU (BD347580) and incubated with secondary AlexaFluor 488 (Abcam ab150117) and Cy5 (Abcam ab6565) conjugated antibodies. Finally, the cells were mounted onto coverslips and imaged using a confocal microscope (Leica SP5) and analyzed using LAS X 3.5.7.23225 software.

Statistics analyses
For immunofluorescence, SIRF, and PLA assays, as well as cellular viability assays the t test (two-tailed, unpaired) was used. For DNA fiber assays and comet assays, the Mann–Whitney statistical test (two-tailed) was performed. Western blot experiments were reproduced at least two times. Unless otherwise noted, schematic models were made with Microsoft PowerPoint v16.103. Statistical analyses were performed using GraphPad Prism 10 and Microsoft Excel v16.103. Statistical significance is indicated for each graph (ns = not significant, for p > 0.05; * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001, **** for p ≤ 0.0001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript examines the role of EXO1 overexpression in inducing genomic instability in BRCA positive breast cancers. The authors use the cBioPortal to interrogate TCGA datasets and find many cancers with amplifications and higher expression levels of EXO1, including around 20% of breast cancers. They then established Hela and U2OS based EXO1 over-expressing cell lines to examine the phenotypic effects. They find that EXO1 overexpression leads to nascent strand degradation at ssDNA gaps and reversed forks using S1 nuclease fork assays and BrdU comet assays. They show using SIRF/PLA that active EXO1 localizes to nascent DNA in proximity to MRE11 and find that EXO1 OE leads to nascent DNA degradation at forks in HU treated cells that can be prevented by MRE11 inhibition or depletion of fork reversal proteins like SMARCAL1 and ZRANB3. Finally, they demonstrate using neutral comet and IF that EXO1 OE leads to DSBs and higher sensitivity to cisplatin, that they conclude may explain the differences in survival seen in TCGA data. Overall, they find that elevating EXO1 expression leads to nascent strand degradation in BRCA proficient cancers, in addition to its previously known activity in BRCA deficient cancers.\n\nThe group has a strong track record performing the types of assays used in the paper and the experimental data is for the most part well-presented and convincing. With this, they show that EXO1 overexpression via lentivirus causes a number of phenotypes similar to its previously established role in BRCA deficient settings in multiple cell lines. My primary issue with the paper is the clinical data analysis and its interpretation and whether the OE system is truly modeling what they imply. The overexpression system is based on the finding that EXO1 is frequently amplified/upregulated in breast cancer. This occurs primarily in the context of a large 1q43 amplification that contains a large number of other genes. Whether EXO1 protein levels are in fact elevated and if so, to an extent that is similar to what they assay experimentally in their lentiviral system, remains an open question. I therefore have some doubts about the the relevance of the experimental system and I have very strong reservations about their bioinformatic analysis and its interpretation. I have separated my comments into those related to bioinformatics and experiments.\n\nMajor concerns\nBioinformatics\n1. The authors stated: These findings argue that, despite the known role of EXO1 in DNA repair, increased EXO1 activity, rather than loss of EXO1 function, is associated with carcinogenesis. And later they state: Overall, these findings argue that increased EXO1 activity, rather than loss of EXO1 function, is associated with carcinogenesis, through increased genomic instability. This is a massive overinterpretation of the data. First, there is no indication in any sample that EXO1 activity, let alone its protein levels, are elevated. This could be addressed in tissue arrays by IHC or other methods but is not. Thus, this is an assumption based on mRNA or copy number. While it is possible that high EXO1 is more oncogenic than its loss, this data allows only a speculative point, as there is no evidence that amplified/overexpressed EXO1 is associated with clinical outcome and no evidence that it is responsible for the elevated genome instability. I make this point not to discount the experimental data in the paper, only that I think that this clinical data, that is very superficially analyzed, is being overinterpreted to make general statements that are unsupported by the data they are describing. If one wants to make prognostic claims about clinical data, at the very least, multivariate analyses on annotated clinical datasets should be used to calculate hazard ratios. BRCA deficient samples should also be separated from those datasets and the correlations with numerous other genes in the proposed pathway could also be assessed.\n2. Related to this point, a major confounding factor in the types of univariate analyses produced by cBioPortal, and many other online platforms, is proliferation. EXO1 is an E2F target and its expression highly correlated with a number of proliferation related genes (CHEK1, FOXM1, CCNA2 etc). Proliferation status often correlates with clinical outcomes, thus whether EXO1 per se is driving any clinical outcomes remains unclear, and is in fact argued against with the data used in Figure 7 (see next point on this). This, and other clinical parameters, need to be considered in survival data analysis using multiple variables\n3. The authors stated: We found that breast cancer patients with altered EXO1 in tumors (which, as described above, represents amplification of EXO1 expression in the vast majority of cases) tend to have better survival than those with no EXO1 alterations (Fig. 7C,D). This is not what the data shows. First, this is a univariate analysis that is fine for hypothesis generation but not suited to interpret clinical data. Second, the p-values clearly indicate there is no significant difference in survival. The differences that the authors are referring to are a handful of the samples (in one case 4 patients of >1000). This in no way justifies a statement like the one made.\n4. The authors stated: This is in line with the increased sensitivity of EXO1-overexpressing cells to cisplatin and suggests that EXO1 overexpression in tumors sensitizes them to cisplatin chemotherapy. Aside from the fact that these graphs show no difference in survival, how many of these patients received cisplatin?\n5. The EXO1 amplifications that are apparent in the dataset used are also seen in other well curated cohorts like METABRIC. These do not represent EXO1 amplification in isolation but a very large duplication of 1q43 that contains a number of other genes that may also influence oncogenesis. If you run the same analyses with any of these genes (for example MROH9, SDHC, CHRM3, RGS7) you get precisely the same outcome- that there is no statistical difference in survival and there is a correlation with genome alterations. While the experimental data would lead to a reasonable hypothesis that EXO1 amp may play a role in genome alterations, it does not account for all of the other alterations happening with EXO1 amp in any way.\n6. The authors stated: EXO1 is overexpressed in a significant proportion of cancers, including in over 20% of breast tumors. First, the graph referred to in Figure 1A is based on a specific dataset and cannot be generalized to all breast cancer. One can look at this in cBioPortal across many different BC datasets and see variations, as each one has its own biases in tumor subtypes and other factors. Second, it is not actually an accurate description of the cohort shown, as amplification is only shown in just over 5% of the Breast Cancer cohort (the actual cohort name should be used for proper reference), while the rest is mRNA high.\n7. The phrase “all breast cancers” is used repeatedly in Figure 1 while describing data related to a single cohort. The proportion of basal cancers with elevated EXO1 is higher in many of the breast cohorts, however it is worth noting there are a lot more of other subtypes that have EXO1 high expression or amplifications. In fact, the p-value for survival is lower in some of these cohorts with fewer basal cancers (ex. Metabric), calling into question the relevance to TNBC. However, I still maintain that this type of 1:1 correlation is not a serious way to establish what the authors wish to conclude. If you use another platform that conducts multivariate analysis on the same dataset, the answer is in fact the opposite (ex MammoOnc-DB), where you see statistically worse survival in the high EXO1 cohort. I would highly recommend the authors consult a statistician to do this analysis in a more rigorous way across multiple datasets to make conclusions better supported by the clinical data. I would also point out previous data (I am not vouching for its accuracy, only its existence) that proposes that 1q amps that contain EXO1 are associated with poor prognosis in breast cancer (https://doi.org/10.1371/journal.pone.0077553).\n\nExperimental data\n1. For Figure 2, it would be nice to see some representative data in the figure rather than just the quantification.\n2. All the experiments have been performed with EXO1 over-expression (WT/catalytically dead) in Hela/U2OS cells. As EXO1 activity is shown to be dependent on MRE11, PRIMPOL, SMARCAL1 and ZRANB3, it would be reassuring to see that their relative levels are similar in the parental, WT and catalytically dead EXO1 lines.\n3. The authors stated: This suggests that increased EXO1 levels results in recruitment of MRE11 to nascent DNA under replication stress through MRE11-EXO1 complex formation. Prior literature showed that the D173A mutant of EXO1 is catalytically dead, however it is capable of binding DNA as well as the WT, as shown previously by multiple groups (PMIDs:11842105, 40319035, 23178594) and it retains the PCNA binding PIP box (23939618). Has the localization of the WT and dead mutant been examined? It would seem possible that it is not simply levels, but levels and its activity that are needed.\n4. Additionally, the authors argue that “increased EXO1 levels result in recruitment of MRE11 to nascent DNA under replication stress through MRE11-EXO1 complex formation”. The MRN complex can bind DNA lesions in the absence of EXO1, so what more specifically are the authors proposing as the mechanism here? In the case of gaps, why is EXO1 needed for MRE11 to act in the other direction? While I would expect the resulting ssDNA to be shorter, I do not understand why MRN would not be able to act on these substrates without EXO1.\n5. The authors stated: Thus, our study suggests that EXO1 overexpression causes a “BRCAness” phenotype, characterized by genomic instability and hypersensitivity to genotoxic chemotherapy. Are cells sensitive to PARP inhibitors?", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The study investigates the role of the nuclease EXO1 in promoting genomic instability by degrading nascent DNA in cells that are proficient in the BRCA pathway. The researchers found that EXO1 is overexpressed in various cancers, leading to increased degradation of nascent DNA and double-strand break (DSB) formation, even in the presence of functional BRCA genes. EXO1 is overexpressed (EXO1 OE) in a significant proportion of cancers, including breast, hepatocellular, skin, testicular, and cervical cancers. This overexpression is associated with increased genomic instability.\n\nEXO1 OE in BRCA-proficient cells leads to replication-associated DNA lesions upon replication stress. This is evidenced by increased single-strand DNA (ssDNA) gaps and reversed replication forks. EXO1 OE promotes the recruitment of MRE11 to nascent DNA, facilitating the degradation of ssDNA gaps and reversed forks. This interaction is critical for the observed genomic instability. The degradation of nascent DNA by EXO1 results in increased DSB formation. This leads to hypersensitivity to genotoxic agents like cisplatin. EXO1 OE sensitizes cells to chemotherapy, particularly in triple-negative breast cancer, which lacks hormone receptors and is typically treated with chemotherapy.\n\nOverall, the findings highlight the dual role of DNA repair enzymes like EXO1 in maintaining genomic stability under normal conditions and promoting genomic instability when overexpressed. This manuscript is a good contender for Nature Communications.\n\nFigure 2 : The alkaline comet assay also detects dsDNA breaks, wouldn’t their assay then also detect replication-associated dsDNA breaks ? (like fork collapse) or do they consider these breaks negligible, also because of the low dose of HU ?\nFigures 2C and 2D indicate that the EXO1 D173A mutant exhibits lower activity in U2OS cells compared to HeLa cells. This suggests that the role or recruitment/localization dynamics of EXO1 D173A may vary depending on the cellular context, highlighting potential cell line–specific differences in how the mutant functions. Has this been addressed experimentally ?\nThe authors state that “MRE11 is recruited to nascent DNA in cells overexpressing EXO1 but not in cells overexpressing the catalytic mutant D173A, in both HeLa and U2OS cells.” However, in Figure 4D, no significant difference in MRE11 recruitment is observed between EXO1 overexpression and EXO1 D173A overexpression in HeLa cells. Notably, there is also no significant difference between the empty vector (EV) and EXO1 D173A conditions, raising concerns about the robustness of this conclusion in the HeLa context.\n\nFigure 4D : The error bar in the EXO1 D173A condition is very high. Could the authors show individual N (like on Figures 7A & B) ? It appears that the EXO1 mutant is still able to recruit Mre11 to some extent. Could the authors comment on this?\nFigures 5C–G: How can the authors be certain that the SIRF signal they observe reflects degradation at inverted replication forks, especially without colocalization data using markers such as CtIP or MRE11?\n\nFigure 5H: To strengthen the conclusion that degradation occurs at inverted forks via MRE11 activity, the authors should consider including an siMRE11 condition and assess for potential epistasis by performing a double siRNA knockdown.\nThe authors should compare the levels of EXO1 Overexpression in a panel of cancer cell lines to those in their HeLa and U2OS cells: are they comparable?\nThe most impactful conclusion of the paper is that EXO1-overexpressing cells exhibit a BRCAness phenotype in terms of sensitivity to DNA damage agents; however, the authors don't thoroughly explore this aspect. Figures 7C and D only bring circumstantial evidence that EXO1-overexpressing cancers are more sensitive to cisplatin. I think Figure 7 (and the paper) would be greatly improved if the authors could examine cell survival following cisplatin treatment in a panel of cancer cell lines that overexpress or do not overexpress EXO1.\n\nAdditionally, a major point lacking in the discussion is that many publications have shown that EXO1 over-expression is associated with a poor prognosis and cancer progression, due to the increase in genome instability.", "reviewer_4": "EXO1 is an exonuclease with important roles in long-range resection at sites of replication stress or DNA double-strand breaks, generating single-stranded DNA that can then be engaged by homologous recombination repair or be subject to further nuclease attack, potentially leading to more damage.\n\nThe study starts off with showing that EXO1 mRNA levels are increased in cancer samples, such as from breast and cervical cancer. This finding is not new as it has been reported before by several groups (PMID: 24147022, PMID: 33623391, PMID: 39718579, PMID: 33569437). It is not investigated whether EXO1 protein levels are also increased in cancer, which has been attempted by others using breast cancer cell lines (PMID: 24147022).\n\nThe further premise of the study is that increased EXO1 expression in cancers could cause genome instability phenotypes that resemble those caused by loss of DNA repair factors such as BRCA1. The study goes on to show that ectopic EXO1 protein overexpression in model cancer cell lines causes phenotypes consistent with increased proximity of MRE11 nuclease to EXO1 and nascent DNA, increased resection at nascent DNA and increased DNA double strand break formation – if these cells are treated with reagents that induce replication stress. EXO1-overexpressing cells are potentially more sensitive to cisplatin, although the evidence for this is limited. The conclusion is that EXO1 overexpression might cause a BRCAness-like phenotype that could sensitise cancers to chemotherapy.\n\nThe experiments are of good quality and nicely controlled by using the catalytically inactive (nuclease-deficient) EXO1 D173A mutant. This shows that most phenotypes are caused by the EXO1 nuclease catalytic activity rather than overexpression per se. EXO1 D173A is still able to bind DNA and interact with other DNA repair factors, which helps explain why nuclease-independent effects are sometimes seen such as in Figure 3A or Figure 4D, F.\n\nThe mechanistic findings are generally convincing and useful for the field, but are limited in breadth, using a small number of assays that have not evolved much from the author’s previous publications on EXO1 and MRE11. The experiments show that increased EXO1 activity causes certain DNA replication and DNA damage phenotypes that would be expected if high EXO1 activity is detrimental, but do not provide much new insight into EXO1 function. The study does not directly measure effects on genomic instability or whether EXO1 might be an oncogene. The cancer data analysis is too rudimentary to add anything to the already existing literature.\n\nSpecific comments:\nTop of page 5: The results section starts with: “To investigate the relevance of EXO1-mediated genome instability in cancer, (…)”. This pre-supposes the findings of the study and does not explain why EXO1 expression levels in cancer were investigated to begin with. The authors could justify this much better. EXO1 is on located chromosome 1q, which is frequently amplified in breast cancer, and several aspects of high EXO1 expression in cancer have been characterised in previous studies (see above). This literature should be considered and cited.\nBottom of page 6: “However, we found that EXO1 expression positively correlates with genome alterations (Fig. 1D), suggesting that EXO1 promotes chromosomal structural instability (…)” Correlation is not causation. High EXO1 expression could be a consequence of chromosomal instability.\nBottom of page 7: “Overall, these findings argue that increased EXO1 activity, rather than loss of EXO1 function, is associated with carcinogenesis, through increased genomic instability.” See previous comment. It seems fair to say that increased EXO1 expression is associated with cancers with high genomic instability. It is not certain that EXO1 is highly expressed during cancer development or that it increases genomic instability.\nFigure 4: The authors should elaborate more on why they think EXO1 would recruit MRE11 to nascent DNA.\nThe labelling scheme in Figure 4A is not very convincing as a gap-specific assay. Data in Figure 3 support that gaps may be formed in the DNA that is synthesised during the treatment with 0.4 mM HU. But for the experiments in Figure 4A-E, nascent DNA is labelled in absence of HU and 0.4 mM HU is added afterwards, providing less opportunity for the MRE11 that co-localises with EdU to be at gaps. It would be helpful to show a SIRF experiment without any HU treatment to see the baseline in unchallenged cells and to support the conclusion that the effect in Figures 4D,E and 5F,G shows MRE11 recruitment specifically to gaps and arrested forks, respectively.\nFigure 7A, B: The link between the molecular findings in previous figures and cisplatin sensitivity would be more convincing if the sensitivity of EXO1 OE was normalised to the sensitivity of EXO1 D173A OE.\nMaterials and methods: Much more detail should be given on how the TCGA dataset analysis was performed and which datasets and search parameters used to generate Figure 1A and Figure 7C,D."}, "author_response": "Response to referees\nWe would like to thank the reviewers for their helpful and constructive comments, which led to a\nsignificantly improved manuscript. To address the reviewers’ concerns, we are submitting a\nsubstantially revised manuscript with 21 new figure panels.\nWe would also like to extend our gratitude for being allowed an extended revision timeline, as\nthe first author of this study, graduate student Alexandra Nusawardhana, was out of the\nlaboratory for an industry fellowship over the summer.\nReviewer #1\n[...]\nWe thank the reviewer for their helpful and encouraging comments.\nWe now show that EXO1 protein levels are increased in breast tumor tissues and cell lines\ncompared to normal samples, and the overexpression obtained in our system is comparable to\nthat found in breast cancer cell lines (new Supplementary Figures S1B,C). This argues that\nour model does measure the impact of EXO1 expression in tumors. To provide further evidence\nthat the mechanistic results obtained using this system are relevant, we now also show that\nbreast cancer cell lines with higher endogenous EXO1 expression have increased nascent\nstrand degradation compared to cell lines with lower endogenous EXO1 expression levels (new\nSupplementary Figures S1C, S3A, S3B) thus confirming the data obtained using the\ncontrolled overexpression system.\nAs detailed below, we use clinical data analysis to show that EXO1 expression correlates with\ngenomic alterations in clinical samples. Beyond this, clinical data analysis, particularly in terms\nof patient outcomes, is not a critical aspect of our manuscript and we removed them. Instead,\nthe main point of our manuscript is that EXO1 overexpression in tumors is associated with\ngenomic instability, and the mechanism of this genomic instability is nascent strand\ndegradation in BRCA-proficient cells. This is novel, as nascent strand degradation is not\ngenerally known to occur outside of deficiencies in the BRCA or TLS fork protection pathways.\nIn the revised manuscript, we now show that EXO1 overexpression results in comparatively\nmore MRE11 loading on reversed forks than BRCA2 knockdown, and BRCA2 depletion does\nnot further increase MRE11 recruitment to these structures in EXO1-overexpressing cells (new\nFigure 5H). This indicates that EXO1 overexpression completely alleviates the fork protection\nactivity of BRCA2, arguing that RAD51 loading on nascent DNA is not critical for fork protection,\nas previously thought, and instead suggesting that the BRCA proteins promote fork protection in\na RAD51-independent manner. In our opinion, this is novel and important.\nMajor concerns\nBioinformatics\n1. [...]\nWe agree with the reviewer that we have no indication that EXO1 activity is increased in patient\ntumors, and we removed the reference to activity from the manuscript. Studying the enzymatic\nactivity of EXO1 in patient tumors is unfortunately not feasible. However, in the revised\nmanuscript, we now investigate the EXO1 protein levels in breast cancer cell lines as well as in\nbreast cancer tissue microarrays, as indicated by the reviewer. Overall, we find increased EXO1\nprotein expression in breast cancer samples compared to normal breast tissue (new\nSupplementary Figures S1B,C). Moreover, we show that the expression of the EXO1 protein\nin our overexpression system is similar to that found in some of these breast cancer cell lines\n(new Supplementary Figures S1C).\nWe agree with the reviewer that there is no direct evidence that amplified EXO1 is associated with\nclinical outcomes, and this is only speculation at this time. We have reframed our revised\nmanuscript to clearly indicate this. We apologize if we were unclear and, potentially,\nunintentionally misleading when presenting our manuscript’s significance. We are not claiming\nto show that increased EXO1 causes clinical outcomes. While this may be a possibility, as\nperhaps suggested by the survival data in the original Figure 7C,D, the currently available\nTCGA data are not strong enough to assess this. As pointed out by the reviewer in their\ncomment #7, analyses of other databases or using other parameters can show the opposing\ntrend. We thus removed this figure from the manuscript, along with any claim that our data\nshows an impact of EXO1 on patient outcomes.\nRegarding the genomic instability aspect, on the other hand, we would like to respectfully argue\nthat our data, overall, does indicate that EXO1 overexpression promotes genomic instability. \nThere is a clear association between EXO1 levels and genomic instability in tumor datasets\n(Supplementary Figures S2B-G). This suggests that EXO1 overexpression may be\nresponsible for this genomic instability. Since we cannot study it directly in patient tumors, we\ncreated the overexpression system in cell lines and show that EXO1 overexpression causes\nnascent strand degradation and subsequently double stranded DNA breaks. Mechanistically,\nthis can account for the genome alterations observed in EXO1-overexpressing, BRCA-proficient\ntumors. This is the main point of our work, and we believe that the data presented support this\nmodel.\nWe agree that the statement that increased EXO1 activity, rather than loss of EXO1 function, is\nassociated with carcinogenesis is a speculation. We are not drawing this conclusion from our\nresults, but we believe that, generally speaking, our data support this possibility because we\nshow increased genomic instability in EXO1-overexpressing cells, and increased genomic\ninstability is known to cause carcinogenesis. Nevertheless, we agree that this is a speculation\nand have revised the manuscript accordingly (stating now that These findings argue indicate\nthat, despite the known role of EXO1 in DNA repair, increased EXO1 expression, rather than\nloss of EXO1 function, is found in cancer samples).\n2. [...]\nAs mentioned above, we agree that it is difficult to demonstrate that EXO1 overexpression\nimpacts clinical outcomes. While our data suggests that this may be the case, this is not the\npoint of our manuscript. We have thus removed the patient outcomes data from our revised\nmanuscript (Figures 7C,D in our original submission).\n3. [...]\nAs mentioned above, we agree that this point is difficult to make. This was a minor component\nof our original manuscript, and we now removed these analyses (Figures 7C,D in our original\nsubmission).\n4. [...]\nThe reviewer’s comment illustrates the difficulty of using these datasets to draw meaningful\nconclusions. We do not know how many of these patients received cisplatin. This is why, we\nagree with the reviewer’s comments on the lack of rigor of patient outcomes data and have\nremoved it (Figures 7C,D in our original submission).\n5. [...]\nWe thank the reviewer for pointing this out. We agree with the reviewer that EXO1 expression\nseems to be part of a very large duplication of 1q43, which includes many other genes, and we\nare not investigating the other alterations.\nOur aim is not to investigate the effect of this large scale amplification, but of EXO1 specifically.\nEven if the gene most relevant for carcinogenesis present on this amplification is not EXO1, and\nEXO1 amplification is simply a passenger event, this does not mean that its amplification does\nnot have a functional impact on tumor cells. Many passenger events have been shown to\ncontribute to cancer cell fitness, including in response to therapy (PMID: 28536279, PMID:\n29875280, PMID: 32084333, PMID: 20876136). Thus, we do not believe that our data are any\nless relevant because EXO1 is amplified as a part of a large chromosomal arm event rather\nthan as an individual amplification event. We are now discussing these issues in the revised\nmanuscript.\n6. [...]\nWe made the changes in the text to reflect these nuances. We thank the reviewer for pointing\nthem out.\n7. [...]\nWe thank the reviewer for this comment. We have updated the text referring to the original\nFigure 1 (Supplementary Figures S1 and S2 in the revised manuscript) to clearly indicate the\nsubset and database used. Regarding the patient prognosis, as mentioned above, we agree\nthat one cannot draw strong conclusions and we thus removed the patient outcomes data\n(Figures 7C,D in our original submission).\nExperimental data\n1. [...]\nWe now show representative figures for the BrdU alkaline comet assays (new Figure 1D).\n2. [...]\nWe now show that the levels of these proteins are similar in parental, wildtype-overexpressing,\nand catalytic mutant overexpressing cells (new Supplementary Figures S5A-D).\n3. [...]\nWe thank the reviewer for this suggestion. We investigated EXO1 recruitment to nascent DNA\nby SIRF experiments, under ssDNA gap-inducing conditions. We now show increased EXO1\nrecruitment to DNA in cells overexpressing wildtype EXO1, but not in those overexpressing the\nD173A catalytic mutant (new Figures 4A-E). While this mutant still binds DNA in vitro as pointed\nout by the reviewer, our results suggest that its retention on chromatin is impaired.\nThese experiments imply that, as indicated by the reviewer, increased EXO1 activity is driving\nMRE11-mediated nascent strand degradation by enhancing the localization of MRE11 to\nnascent DNA in BRCA proficient cells, and the co-localization between EXO1 and MRE11 at\nthese structures. We speculate that EXO1 initiates degradation at the 5’ end of the gap,\nexpanding the gap and thus allowing more opportunities for MRE11 to engage the 3’ end of the\ngap. This could drive gap expansion in BRCA-proficient cells, and subsequently genomic\ninstability.\n4. [...]\nWe agree with the reviewer that MRE11 can bind DNA lesions in the absence of EXO1, so it\nshould act on these structures without EXO1. Indeed, we and others previously showed that\nMRE11 does act on ssDNA gaps in BRCA-deficient cells. However, in BRCA-proficient cells, the\ngaps are quickly filled by recombination-mediated gap filling. We now speculate that increased\nEXO1 activity may interfere with this process, by expanding the gap and thus providing more\nopportunities for MRE11 to engage the other end of the gap and bidirectionally expand it. This\nultimately alleviates the ability of the BRCA pathway to fill the gap.\n5. [...]\nWe thank the reviewer for this suggestion. Indeed, in the revised manuscript, we now show that\nEXO1-overexpressing cells have increased sensitivity to PARP inhibitors (new Figure 7B).\nReviewer #2\n[...]\nWe thank the reviewer for participating in reviewing the manuscript and for their useful\ncomments.\nReviewer #3\n[...]\nWe thank the reviewer for their encouraging and useful remarks.\nFigure 2 : [...]\nThe reviewer is correct in pointing out that, technically, the alkaline comet assay can also detect\ndsDNA breaks. We have revised the text to reflect this possibility.\nFigures 2C and 2D indicate that the EXO1 D173A mutant exhibits lower activity in U2OS cells\ncompared to HeLa cells. This suggests that the role or recruitment/localization dynamics of\nEXO1 D173A may vary depending on the cellular context, highlighting potential cell line–specific\ndifferences in how the mutant functions. Has this been addressed experimentally ?\nWe have not consistently observed differences between the phenotypes of the EXO1 D173A\nmutant in HeLa compared to U2OS cells (see also our responses to the reviewer’s comments\nbelow). In the revised manuscript, we investigated the localization of EXO1 in both HeLa and\nU2OS cells overexpressing wildtype or D173A EXO1 mutant. In both cell lines, the wildtype\nprotein was found at nascent DNA gaps while the mutant was not (new Figures 4A-E), with no\napparent differences between the cell lines.\nThe authors state that “MRE11 is recruited to nascent DNA in cells overexpressing EXO1 but\nnot in cells overexpressing the catalytic mutant D173A, in both HeLa and U2OS cells.” However,\nin Figure 4D, no significant difference in MRE11 recruitment is observed between EXO1\noverexpression and EXO1 D173A overexpression in HeLa cells. Notably, there is also no\nsignificant difference between the empty vector (EV) and EXO1 D173A conditions, raising\nconcerns about the robustness of this conclusion in the HeLa context.\nThese differences between HeLa and U2OS cells are likely due to experimental variation. In the\nrevised manuscript, we provided a new experiment showing that the D173A mutant abolishes\nMRE11 recruitment to nascent DNA to the same extent in HeLa and U2OS cells (new Figure\n3D).\nFigure 4D : [...]\nAs mentioned above, we now provide a new experiment showing that the mutant is unable to\nrecruit MRE11 (new Figure 3D). Because of the large number of samples, it is impractical to\nshow the individual values in the graph, but those values are listed in the Source Data file.\nFigures 5C–G: [...]\nThe SIRF signal investigated in these figures is in fact the MRE11 signal. To demonstrate that\nthis MRE11 SIRF signal indeed occurs at reversed replication forks reflecting their degradation,\nwe now show that depletion of SMARCAL1, the translocase responsible for fork reversal,\nabolishes this MRE11 SIRF signal in EXO1-overexpressing cells (new Figure 5H).\nFigure 5H: [...]\nIn the revised manuscript, we show that depletion of MRE11 by siRNA, similar to its inhibition by\nmirin, fully suppresses the fork degradation observed in EXO1-overespressing cells in both\nHeLa and U2OS cells (new Supplementary Figure S4). Since individual depletions of\nSMARCAL1, ZRANB3 or MRE11 all restore fork protection to the same extent as in control cells\n(CldU/IdU ratio of 1), co-depletion of any of them (by double siRNA knockdown) would result in\nthe same phenotype (CldU/IdU ratio of 1). In other words, an additive phenotype would not be\ndetectable, since the CldU/IdU ratio cannot get bigger than 1. Because of this, an epistatic\nrelationship cannot be determined from such an experiment.\nThe authors should compare the levels of EXO1 Overexpression in a panel of cancer cell lines\nto those in their HeLa and U2OS cells: are they comparable?\nWe thank the reviewer for this comment. In the revised version, we now show that EXO1\nexpression is increased in breast cancer cell lines T47D, MDA-MB-231, MCF7 and MDA-MB-\n468 compared to MCF10A normal epithelial mammary gland cells. Moreover, we show that the\nEXO1 overexpression in our experimental system is comparable to that found in these breast\ncancer cell lines (new Supplementary Figures S1C).\nThe most impactful conclusion of the paper is that EXO1-overexpressing cells exhibit a\nBRCAness phenotype in terms of sensitivity to DNA damage agents; however, the authors don't\nthoroughly explore this aspect. Figures 7C and D only bring circumstantial evidence that EXO1-\noverexpressing cancers are more sensitive to cisplatin. I think Figure 7 (and the paper) would\nbe greatly improved if the authors could examine cell survival following cisplatin treatment in a\npanel of cancer cell lines that overexpress or do not overexpress EXO1.\nWe thank the reviewer for this thoughtful comment. First of all, to enhance the relevance of our\nfindings, we now show that EXO1-overexpressing cells are also sensitive to PARP1 inhibitors\n(new Figure 7B).\nWe appreciate the reviewer’s suggestion to use the breast cancer cell lines to corelate cisplatin\nsensitivity with EXO1 expression levels. Unfortunately, because of the big genetic variation\nbetween the different cell lines grown in vitro for many years, affecting many different\nmechanisms of drug resistance, any EXO1-dependent effects may be masked. In fact, this is\nexactly why we decided to create our exogenous EXO1 overexpression system to begin with,\ninstead of using breast cancer cell lines with different EXO1 expression.\nThis being said, we agree with the reviewer that it is important to show that EXO1 expression\nlevels correlates with specific genome instability phenotypes in breast cancer cell lines. In the\nrevised manuscript, using the DNA fiber combing assay we now show that T47D cells, which\nshow the highest level of EXO1 expression in our hands, have increased replication fork\ndegradation compared to MDA-MB-468 and MCF7 cells, which show comparatively lower EXO1\nexpression (new Supplementary Figure S3). (Unfortunately, because of varying replication\nrates and culturing conditions making them less amenable to the DNA fiber combing assay, we\nwere not able to include the other cell lines in this analysis.) These findings support our model\nthat EXO1 expression in tumors is associated with genomic instability through nascent strand\ndegradation.\nAdditionally, a major point lacking in the discussion is that many publications have shown that\nEXO1 over-expression is associated with a poor prognosis and cancer progression, due to the\nincrease in genome instability.\nWe thank the reviewer for this comment. As also explained in the answers to Reviewer #4, we\nacknowledge those studies. The issue of deriving patient prognosis conclusions from cancer\ndatasets is a complicated one, as this is affected by many factors which are not known or well\ndocumented. Critically among these factors is whether the patients received treatment with\ngenotoxic chemotherapeutic agents such as cisplatin. Not having that information to stratify the\nsamples results in confounding findings. As explained in our answer to Reviewer #1, this is why\nwe decided to remove the patient outcomes data (Figures 7C,D in our original submission).\nThe main point of our manuscript is that EXO1 overexpression in tumors is associated with\ngenomic instability, and the mechanism of this genomic instability is nascent strand degradation\nin BRCA-proficient cells. This is novel, as nascent strand degradation is not generally known to\noccur outside of deficiencies in the BRCA or TLS fork protection pathways. We do not believe\nthat any of the previous studies demonstrated that EXO1 overexpression causes genomic\ninstability, and identified the mechanism of this genomic instability, as we do here.\nReviewer #4\n[...]\nWe thank the reviewer for their very useful comments.\nIndeed, we are not directly measuring whether EXO1 may be an oncogene. EXO1-\noverexpressing animal models represent an important next step in studying the oncogenic\neffects of EXO1, but those costly, long-term studies are beyond the scope of the current\nmanuscript. We sincerely hope the reviewer agrees with us.\nRegarding genomic instability, we would like to respectfully argue that our manuscript does\nindicate that EXO1 overexpression promotes genomic instability: 1. We show that EXO1\noverexpression causes nascent strand degradation and double strand break formation. (In the\nrevised manuscript, we now provide additional evidence of this, by showing that breast cancer\ncell lines with increased EXO1 expression have increased nascent strand degradation\ncompared to cell lines with lower EXO1 expression levels – new Supplementary Figures S1C,\nS3A, S3B). 2. We show that EXO1 overexpressing cells are sensitive to cisplatin and PARP1\ninhibitors (the PARPi sensitivity is shown in the revised manuscript in the new Figure 7B); 3. We\nshow that EXO1 overexpression in patient tumor samples correlates with increased rates of\ngenome-wide alterations. While the increased expression of EXO1 in tumors was indeed shown\nbefore, to our knowledge the association with chromosome level genome alterations was not.\nIn addition, our work reveals critical novel insights into the mechanism of fork protection. In\nour opinion, nascent strand degradation is, in fact, not expected in BRCA pathway proficient\ncells, which are able to load RAD51 on reversed forks. For many years, this has been\nconsidered to represent the mechanism of fork protection, as RAD51 coating of reversed arms\nis supposed to block nuclease-mediated DNA resection. However, we find that EXO1\noverexpression is enough to induce MRE11-mediated fork degradation, regardless of the BRCA\nstatus. In the revised manuscript, we now show that EXO1 overexpressing cells have more\nMRE11 loading to reversed forks than BRCA2-depleted cells (new Figure 5H). This shows that\nincreased EXO1 activity promotes MRE11 engagement on reversed forks to a greater extent\nthan loss of BRCA2-mediated RAD51 coating of reversed forks. Importantly, when we knocked\ndown BRCA2 in EXO1-overexpressing cells, we found no additional increase in MRE11 loading.\nThis epistatic interaction indicates that EXO1 overexpression completely alleviates the fork\nprotection activity of BRCA2. These results argue that the presence of RAD51 on reversed forks\nis not enough to block MRE11-mediated fork degradation. Thus, our manuscript shows that\nRAD51 loading on nascent DNA is not critical for fork protection, as previously thought, and\ninstead suggest that the BRCA proteins can promote fork protection in a RAD51-independent\nmanner.\nSpecific comments:\nTop of page 5: [...]\nWe thank the reviewer for pointing out these studies, which we now cite, and we apologize for\nomitting to cite them in our original submission.\nBottom of page 6: [...]\nWe agree with the reviewer, which is why we used the term “correlates”. As explained above,\nour conclusion that EXO1 expression promotes genomic instability is based on the collective\ndata we present, not only this correlation. For example, we show that EXO1 overexpression\ncauses double stranded DNA breaks, a known intermediate through which chromosomal\ntranslocations occur.\nBottom of page 7: [...]\nWe agree with the reviewer, and we changed this sentence to: “Overall, these findings argue\nthat increased EXO1 levels may be associated with increased genomic instability in cancer\nsamples.”\nFigure 4: [...]\nIn the revised manuscript, we investigated EXO1 recruitment to nascent DNA by SIRF\nexperiments, under ssDNA gap-inducing conditions. We now show increased EXO1 recruitment\nto DNA in cells overexpressing wildtype EXO1, but not in those overexpressing the D173A\ncatalytic mutant (Figure 4A-E). These experiments imply that increased EXO1 activity is driving\nMRE11-mediated nascent strand degradation by enhancing the localization of MRE11 to\nnascent DNA in BRCA proficient cells, and the co-localization between EXO1 and MRE11 at\nthese structures. We speculate that EXO1 initiates degradation at the 5’ end of the gap,\nexpanding the gap and thus allowing more opportunities for MRE11 to engage the 3’ end of the\ngap. This could drive gap expansion in BRCA-proficient cells, and subsequently genomic\ninstability.\nThe labelling scheme in Figure 4A is not very convincing as a gap-specific assay. Data in Figure\n3 support that gaps may be formed in the DNA that is synthesised during the treatment with 0.4\nmM HU. But for the experiments in Figure 4A-E, nascent DNA is labelled in absence of HU and\n0.4 mM HU is added afterwards, providing less opportunity for the MRE11 that co-localises with\nEdU to be at gaps. It would be helpful to show a SIRF experiment without any HU treatment to\nsee the baseline in unchallenged cells and to support the conclusion that the effect in Figures\n4D,E and 5F,G shows MRE11 recruitment specifically to gaps and arrested forks, respectively.\nWe thank the reviewer for this useful comment. In the revised manuscript, we now show that in\nthe absence of HU treatment, there is no recruitment of MRE11 to nascent DNA (new Figure\n3D).\nFigure 7A, B: [...]\nWe thank the reviewer for this useful comment. In the revised manuscript, we show that the\noverexpression of the catalytic mutant does not cause cisplatin sensitivity (new Figure 7A).\nMoreover, we also show that the overexpression of wildtype, but not of catalytic mutant EXO1\ncauses PARP1 inhibitor sensitivity (new Figure 7B).\nMaterials and methods: [...]\nWe now provide the requested information (since these analyses were moved to the\nSupplementary Information, we provide this information in the Legends to Supplementary\nFigures S1 and S2)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed many of the reviewer comments and included new data that helps support their original model and add additional depth. This included demonstrating that EXO1 activity is required for its retention, that its OE is epistatic with BRCA2 loss, that MRE11 is binding reversed forks with new SIRF data and that OE is roughly similar to some BC cell lines that are widely used. The overinterpretation of superficially analyzed clinical data has been toned down, anda subset has been moved to the supplement. My biggest reservation is that experiments are carried out for the most part in a single cell line background that is not a BC and has its own host of other genetic abnormalities, making some of the more generalized interpretations proposed difficult to support. While some data from cell lines expressing different levels of EXO1 is presented as further proof, this data is not particularly compelling.\n\nSpecific comments-\n1. Referring to this comment in the rebuttal- “by showing that breast cancer cell lines with increased EXO1 expression have increased nascent strand degradation compared to cell lines with lower EXO1 expression levels – new Supplementary Figures S1C, S3A, S3B).” The authors argue that they have demonstrated that nascent strand degradation is elevated in T47D that are high EXO1 expressors of the lines examined. The data in S3A and S3B does not appear to support this. In S3A there is a minor difference, but not in S3B, and no statistics employed anywhere here. MDA-MB-468 cells that potentially have more than two-fold OE of EXO1 and no effect on nascent strand degradation, making this data unsupportive of authors claims.\n2. The authors report a tissue microarray using EXO1 IF to determine levels. No primary data is provided to evaluate and no indication of what controls were carried out to ensure that the EXO1 antibody was on target are provided. The methods provided state that 2 different primary EXO1 antibodies were used. Please clarify these points.\n3. In referring to Figure S2D as measuring genomic instability, the authors should clarify exactly what types of lesions are measured in this assessment, as it is relevant to the potential mechanism. Do these types of aberrations make sense with the proposed elevation in nascent strand resection based on other examples?\n4. In S2E-G, to me the most outstanding confounders are proliferation and the other genes amplified on 1q with EXO1. Do all 1q genes correlate with higher genome instability by this measure? Does proliferation? Looking quickly at CHK1 or KI67, it would appear so. The same applies to S2A, is this at all specific to EXO1 as is implied?\n5. In the first sentence of the discussion, and several other places, it is stated that EXO1 OE causes nascent strand degradation. However, in the absence of RS, there is no indication of elevated breaks or damage, so it would be worth clarifying these statements to some extent in that they are dependent on fork stalling, as nicely shown by a number of data points. I am not asserting that the authors are trying to misrepresent anything, I just think it is not always clearly stated. Particularly, considering that all the experiments are done biologically unrelated cell types and the BC cell lines seem to have no significant effect of EXO1 OE.\n6. I think it is a big leap to go from a HeLa cell model to proposing EXO1 as a biomarker generally for chemotoxic therapy. Is there any indication that it would outperform a proliferative marker for example? You would expect the same effects with a wide range of DNA damage types? This could be stated more specifically to align with the data.\n7. The authors stated: Since increased EXO1 expression is found in a higher proportion of cancers than BRCA inactivation, this has important implications for both cancer etiology as a prevalent mechanism of genomic instability, as well as cancer treatment, suggesting that EXO1 could be a biomarker for the response to genotoxic chemotherapy. Relevant to this, the authors do show new data indicating that there is PARPi sensitivity in the OE HeLa cells. However, this data is somewhat weak as a single dose is used and not compared to BRCA mutations (including epistasis) in this background.\n8. The EXO1 SIRF experiments are performed with treatment of HU for just 20 mins (Fig 4 A-E) while the EXO1-MRE11 PLA has been done after 4hrs of HU. Did the authors have any rational for selecting the two different time points, did authors test earlier time points for EXO1-MRE11 PLA? If so, mentioning that would be useful to further substantiate that EXO1 activity triggers MRE11 recruitment.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have satisfactorily addressed all my comments, and the manuscript is now acceptable for publication.", "reviewer_4": "The authors have addressed my comments to my satisfaction."}, "author_response": "Response to referees\nWe would like to thank the reviewers for their helpful and constructive comments.\nReviewer #1\nThe authors have addressed many of the reviewer comments and included new data that helps support their original model and add additional depth. This included demonstrating that EXO1 activity is required for its retention, that its OE is epistatic with BRCA2 loss, that MRE11 is binding reversed forks with new SIRF data and that OE is roughly similar to some BC cell lines that are widely used. The overinterpretation of superficially analyzed clinical data has been toned down, anda subset has been moved to the supplement. My biggest reservation is that experiments are carried out for the most part in a single cell line background that is not a BC and has its own host of other genetic abnormalities, making some of the more generalized interpretations proposed difficult to support. While some data from cell lines expressing different levels of EXO1 is presented as further proof, this data is not particularly compelling.\n\nWe thank the reviewer for their comments. We would like to respectfully point out that our experiments are done in two different cell lines, from different backgrounds -namely U2OS osteosarcoma cells and HeLa cervical cancer cells. The large number of genetic variations present in cancer cell lines is exactly the reason why we chose to employ an isogenic system, by overexpressing EXO1 (or the catalytic mutant and the empty vector, as controls) in these cells. This system allows us to specifically investigate the impact of EXO1 overexpression, eliminating confounding effects caused by other genetic changes.\n\nIn addition, we provide correlative data using breast cancer cell lines with varying EXO1 expression, which supports our proposed model. In the revised manuscript, we provide additional replicate experiments using these breast cancer cell lines, as requested by the reviewer (see comment #1 below).\n\nSpecific comments-\n1. Referring to this comment in the rebuttal- “by showing that breast cancer cell lines with increased EXO1 expression have increased nascent strand degradation compared to cell lines with lower EXO1 expression levels – new Supplementary Figures S1C, S3A, S3B).” The authors argue that they have demonstrated that nascent strand degradation is elevated in T47D that are high EXO1 expressors of the lines examined. The data in S3A and S3B does not appear to support this. In S3A there is a minor difference, but not in S3B, and no statistics employed anywhere here. MDA-MB-468 cells that potentially have more than two-fold OE of EXO1 and no effect on nascent strand degradation, making this data unsupportive of authors claims.\n\nIn the revised manuscript, we included two new, independent replicates of this nascent strand degradation experiment (new Supplementary Figures S3C, S3D). Moreover, we plotted the median values of the CldU/IdU ratios from each of the four experiments and performed statistical analyses. We show that nascent strand degradation is statistically significantly increased in T47D cells, which show the highest level of EXO1 expression, compared to MDA-MB-468 and MCF7 cells, which show comparatively lower EXO1 expression (new Supplementary Figures S3E). We would like to point out that MDA-MB-468 and MCF7 cells do show nascent strand degradation (as shown by the fact the CldU/IdU ratio is less than 1), but not to the same extent as T47D cells.\n\n2. The authors report a tissue microarray using EXO1 IF to determine levels. No primary data is provided to evaluate and no indication of what controls were carried out to ensure that the EXO1 antibody was on target are provided. The methods provided state that 2 different primary EXO1 antibodies were used. Please clarify these points.\n\nWe apologize but we do not understand why the reviewer states that “2 different primary EXO1 antibodies were used”. We clearly state that the EXO1 primary antibody Santa Cruz Biotechnology sc-56092 was used.\n\nThe experiment presented was performed on a commercially available tissue microarray slide (Novus NBP2-30212). We were not able to perform control experiments (eg. no primary antibody) since all samples were on the same slide, so they could not be differentially treated. We did perform control single antibody experiments but those were performed on a different tissue sample that was available to us. We observed minimal background signal in those control experiments. We are presenting below a representative micrograph showing this. We prefer to not include this in the manuscript, as the sample used for the test is different than the breast cancer microarray samples quantified in the figure. In addition, to this control, we would like to point out that the observed difference in EXO1 expression between breast cancer and normal tissue we observed, clearly indicates the specificity of the antibody.\n\n[Figure for Reviewer #1. Control tissue immunofluorescence experiment showing minimal background staining when the primary antibody was not included (blue - Hoechst staining; red - AF568 fluorescence).]\n\n3. In referring to Figure S2D as measuring genomic instability, the authors should clarify exactly what types of lesions are measured in this assessment, as it is relevant to the potential mechanism. Do these types of aberrations make sense with the proposed elevation in nascent strand resection based on other examples?\n\nWe thank the reviewer for this comment. The TCGA cBioPortal analysis defines Genome Alterations as large-scale copy number changes (deletions, amplifications, translocations). These are events initiated upon double strand break formation. Since we show that nascent strand degradation correlates with double strand break formation (Figure 6), these findings align with our proposed model. We now discuss this in the revised manuscript.\n\n4. In S2E-G, to me the most outstanding confounders are proliferation and the other genes amplified on 1q with EXO1. Do all 1q genes correlate with higher genome instability by this measure? Does proliferation? Looking quickly at CHK1 or KI67, it would appear so. The same applies to S2A, is this at all specific to EXO1 as is implied?\n\nWe have not analyzed all 1q genes for correlation with genome stability. As mentioned in the Discussion section, even if EXO1 was a “passenger” event, its overexpression may still have a functional impact. In other words, if expression of other genes on 1q correlate with genomic instability, this could still be caused by EXO1 overexpression. This is why, we do not think investigating all 1q genes for genomic instability in cancer samples would be that informative. Our experiments clearly indicate that EXO1 overexpression in cells causes genomic instability through nascent strand degradation, which is the main point of our manuscript.\n(Regarding proliferation, as we discussed in the previous revision, we removed any data related to patient outcomes and are not considering proliferation in our analysis.)\n\n5. In the first sentence of the discussion, and several other places, it is stated that EXO1 OE causes nascent strand degradation. However, in the absence of RS, there is no indication of elevated breaks or damage, so it would be worth clarifying these statements to some extent in that they are dependent on fork stalling, as nicely shown by a number of data points. I am not asserting that the authors are trying to misrepresent anything, I just think it is not always clearly stated. Particularly, considering that all the experiments are done biologically unrelated cell types and the BC cell lines seem to have no significant effect of EXO1 OE.\n\nWe thank the reviewer for this comment. We updated these statements to clearly indicate that the degradation is in response to replication stress (or at stalled/reversed/gapped forks).\n\n6. I think it is a big leap to go from a HeLa cell model to proposing EXO1 as a biomarker generally for chemotoxic therapy. Is there any indication that it would outperform a proliferative marker for example? You would expect the same effects with a wide range of DNA damage types? This could be stated more specifically to align with the data.\n\nWe thank the reviewer for this comment. While this is ultimately just a speculation, we now clarify that EXO1 could be a biomarker for “replication stress-inducing cancer therapy”.\n\n7. The authors stated: Since increased EXO1 expression is found in a higher proportion of cancers than BRCA inactivation, this has important implications for both cancer etiology as a prevalent mechanism of genomic instability, as well as cancer treatment, suggesting that EXO1 could be a biomarker for the response to genotoxic chemotherapy. Relevant to this, the authors do show new data indicating that there is PARPi sensitivity in the OE HeLa cells. However, this data is somewhat weak as a single dose is used and not compared to BRCA mutations (including epistasis) in this background.\n\nIn the revised manuscript, we added a new figure showing olaparib sensitivity with an additional dose (new Figure 7C). Moreover, we also included BRCA2-knockout cells in these new experiments. The olaparib sensitivity of EXO1-overexpressing cells was comparable to that of BRCA2-knockout cells. We were not able to perform epistasis experiments since it was not feasible to generate cell lines with concomitant EXO1 overexpression and BRCA2 knockout during this second round of manuscript revision, and we could not use BRCA2 downregulation by siRNA for this experiment, as cells are treated with olaparib for 10 days and siRNA-mediated downregulation is transient.\n\n8. The EXO1 SIRF experiments are performed with treatment of HU for just 20 mins (Fig 4 A-E) while the EXO1-MRE11 PLA has been done after 4hrs of HU. Did the authors have any rational for selecting the two different time points, did authors test earlier time points for EXO1-MRE11 PLA? If so, mentioning that would be useful to further substantiate that EXO1 activity triggers MRE11 recruitment.\n\nThe EXO1-MRE11 PLA experiments were performed using 3hr HU treatment, similar to the MRE11 SIRF, for which conditions were previously published (PMID: 29053959, PMID: 37805499). The EXO1 SIRF was performed using conditions our laboratory recently published (PMID: 38180818). Because of the 5’-3’ directionality of EXO1 (as opposed to the 3’-5’ directionality of MRE11), we think that longer HU treatment conditions may result in removal of EXO1 from DNA was the 3’ end is reached. We agree with the reviewer that additional time points for the EXO1-MRE11 PLA experiments would be useful, but these experiments are complicated and time consuming, and in any case the HU treatment conditions may not translate similarly between the PLA and SIRF assay (which also require EdU incorporation).\n\nReviewer #2\nWe thank the reviewer for participating in reviewing the manuscript and for their useful comments.\n\nReviewer #3\nWe thank the reviewer for their comments and for their helpful suggestions during the manuscript revision.\n\nReviewer #4\nWe thank the reviewer for their comments and for their helpful suggestions during the manuscript revision."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed the majority of my concerns and the expanded data in Figure 7 and Figure S3 add more confidence to the conclusions. I remain concerned about the data in S1B due and have to reiterate one of my primary concerns that was ignored in the response- no primary data is provided to evaluate.\n\n2. The authors report a tissue microarray using EXO1 IF to determine levels. No primary data is provided to evaluate and no indication of what controls were carried out to ensure that the EXO1 antibody was on target are provided. The methods provided state that 2 different primary EXO1 antibodies were used. Please clarify these points.\n\nWe apologize but we do not understand why the reviewer states that “2 different primary EXO1 antibodies were used”. We clearly state that the EXO1 primary antibody Santa Cruz Biotechnology sc-56092 was used.\n\nI think due to the wording, I thought the Novus product was also an antibody, thanks for clarifying that.\n\nThe experiment presented was performed on a commercially available tissue microarray slide (Novus NBP2-30212). We were not able to perform control experiments (eg. no primary antibody) since all samples were on the same slide, so they could not be differentially treated. We did perform control single antibody experiments but those were performed on a different tissue sample that was available to us. We observed minimal background signal in those control experiments. We are presenting below a representative micrograph showing this. We prefer to not include this in the manuscript, as the sample used for the test is different than the breast cancer microarray samples quantified in the figure. In addition, to this control, we would like to point out that the observed difference in EXO1 expression between breast cancer and normal tissue we observed, clearly indicates the specificity of the antibody.\n\nThe SC antibody listed seems to be discontinued, but archived data sheets do not mention IHC as a tested application or show examples of IF or tissue staining. If a tissue microarray was stained and scored, that “clearly indicates the specificity of the antibody”, I think providing some example images of what was actually scored is a very reasonable request that was ignored in the author response. I also think that the case made for the control data is reasonable and do not understand why it would not be shown, absent any other more rigorous controls (we typically prepare FFPE blocks using KO cell line pellets for example).\n\nThere is currently a lot of clinical interest in EXO1, with several new inhibitors recently published. The confident visualization of the endogenous protein in IF, and certainly in IHC, has been historically difficult. If the authors are confident that this antibody is specifically recognizing EXO1 in cancer tissues based on their data, than showing the data would be beneficial to the larger community as a step towards proving this is a potential biomarker that can be assessed with existing reagents. However, the fact that it is a monoclonal, that should not be quantity limited, and has been apparently discontinued, makes me somewhat concerned as to why it is no longer available. If the authors are not confident enough to show primary data examples or controls using an antibody that is no longer available, then I think they should consider removing this data. While this data would provide evidence that EXO1 protein levels are indeed higher in actual cancer tissues, the paper as a whole does not hinge on this data that, as presented, raises numerous concerns.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to referees\nWe would like to thank the reviewers for their helpful and constructive comments.\nReviewer #1\nThe authors have addressed the majority of my concerns and the expanded data in Figure 7 and Figure S3 add more confidence to the conclusions. I remain concerned about the data in S1B due and have to reiterate one of my primary concerns that was ignored in the response- no primary data is provided to evaluate.\n\n2. The authors report a tissue microarray using EXO1 IF to determine levels. No primary data is provided to evaluate and no indication of what controls were carried out to ensure that the EXO1 antibody was on target are provided. The methods provided state that 2 different primary EXO1 antibodies were used. Please clarify these points.\n\nWe apologize but we do not understand why the reviewer states that “2 different primary EXO1 antibodies were used”. We clearly state that the EXO1 primary antibody Santa Cruz Biotechnology sc-56092 was used.\n\nI think due to the wording, I thought the Novus product was also an antibody, thanks for clarifying that.\n\nThe experiment presented was performed on a commercially available tissue microarray slide (Novus NBP2-30212). We were not able to perform control experiments (eg. no primary antibody) since all samples were on the same slide, so they could not be differentially treated. We did perform control single antibody experiments but those were performed on a different tissue sample that was available to us. We observed minimal background signal in those control experiments. We are presenting below a representative micrograph showing this. We prefer to not include this in the manuscript, as the sample used for the test is different than the breast cancer microarray samples quantified in the figure. In addition, to this control, we would like to point out that the observed difference in EXO1 expression between breast cancer and normal tissue we observed, clearly indicates the specificity of the antibody.\n\nThe SC antibody listed seems to be discontinued, but archived data sheets do not mention IHC as a tested application or show examples of IF or tissue staining. If a tissue microarray was stained and scored, that “clearly indicates the specificity of the antibody”, I think providing some example images of what was actually scored is a very reasonable request that was ignored in the author response. I also think that the case made for the control data is reasonable and do not understand why it would not be shown, absent any other more rigorous controls (we typically prepare FFPE blocks using KO cell line pellets for example).\n\nThere is currently a lot of clinical interest in EXO1, with several new inhibitors recently published. The confident visualization of the endogenous protein in IF, and certainly in IHC, has been historically difficult. If the authors are confident that this antibody is specifically recognizing EXO1 in cancer tissues based on their data, than showing the data would be beneficial to the larger community as a step towards proving this is a potential biomarker that can be assessed with existing reagents. However, the fact that it is a monoclonal, that should not be quantity limited, and has been apparently discontinued, makes me somewhat concerned as to why it is no longer available. If the authors are not confident enough to show primary data examples or controls using an antibody that is no longer available, then I think they should consider removing this data. While this data would provide evidence that EXO1 protein levels are indeed higher in actual cancer tissues, the paper as a whole does not hinge on this data that, as presented, raises numerous concerns.\n\nWe thank the reviewer for the supportive comments. We agree that the paper as a whole does not hinge on the data presented in Supplementary Figure S1B. This data (breast tissue staining) agrees with the bioinformatic analysis (Supplementary Figure S1A) and the cell lines western blots (Supplementary Figure S1C). Nevertheless, considering the reviewer’s comments, we are now removing Supplementary Figure S1B from the manuscript, as suggested by the reviewer.\n\nReviewer #2\nWe thank the reviewer for participating in reviewing the manuscript and for their useful comments."}]

Decision letter