Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 8;75(3):605-619.e6.
doi: 10.1016/j.molcel.2019.05.026. Epub 2019 Jun 26.

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2

Affiliations
Free PMC article

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2

Jadwiga Nieminuszczy et al. Mol Cell. .
Free PMC article

Abstract

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.

Keywords: BRCA1; BRCA2; DNA replication; EXD2; EXDL2; fork regression.

Conflict of interest statement

W.N. is an inventor on a filed patent application covering targeting of EXD2 for cancer therapy.

Figures

None
Figure 1
Figure 1
EXD2 Is Recruited to Stressed Replication Forks (A) Western blot of iPOND samples. Thymidine chase analysis illustrates that EXD2 specifically associates with the replisome. PCNA acts as a control. (B) Schematic of the proximity ligation assay (PLA) employed to detect colocalization of target proteins with nascent DNA. (C) Percentage of cells with MRE11/biotin PLA foci (mean ± SEM, n = 3 independent experiments, t test). Right: representative images of PLA foci (red), DAPI acts as a nuclear counterstain. Scale bar, 10 μm. (D) Percentage of cells with GFP/biotin PLA foci (mean ± SEM, n = 3 independent experiments, t test) in U2OS control cells and U2OS cells expressing GFP-EXD2. Right: representative images of PLA foci (red), DAPI acts as a nuclear counterstain. Scale bar, 10 μm. (E) Laser microirradiation induces rapid redistribution of GFP-EXD2 to damaged chromatin; representative images showing GFP-EXD2 accumulation at laser-generated DNA lesions. GFP-CtIP was used as a positive control. Scale bar, 10 μm. (F) Quantification of GFP-EXD2 (left panel) and GFP-CtIP (right panel) recruitment kinetics (intensity versus time) to laser-generated DNA lesions (mean ± SE, n ≥ 10 cells from 2 independent experiments).
Figure 2
Figure 2
EXD2 Promotes Global Replication Fork Dynamics in Response to Replicative Stress (A) Survival of HeLa control and HeLa EXD2−/− cells treated with the indicated doses of cisplatin, gemcitabine, mitomycin C, or hydroxyurea (mean ± SEM, n = 3 independent experiments). (B) Boxplot of CldU tract length ratios of associated sister forks from HeLa WT and EXD2−/− cells. (5–95 percentile, n ≥ 60 sister fork pairs pooled from 3 independent experiments, Mann-Whitney). (C) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells treated with 1 mM HU (5–95 percentile, n ≥ 300 tracts pooled from 3 independent experiments, Mann-Whitney). (D) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells (left panel) and quantification of the percentage of stalled forks (red only tracts) in HeLa WT and EXD2−/− cells (right panel) (5–95 percentile, n ≥ 300 tracts pooled from 3 independent experiments Mann-Whitney [left panel]; n = 3 independent experiments, t test [right panel]).
Figure 3
Figure 3
EXD2’s Nuclease Activity Is Required to Suppress Replication Fork Collapse (A) Quantification of the frequency of 53BP1 foci in HeLa WT and EXD2−/− S/G2 cells and representative images. Cyclin A (green) acts as a marker for S/G2 cells, DAPI acts as a nuclear stain (mean ± SEM, n = 3 independent experiments, Mann-Whitney). Scale bar, 10 μm. (B) Quantification of the frequency of chromosomal aberrations from mitotic spreads from HeLa WT and EXD2−/− cells (mean ± SEM, n = 75 metaphase spreads pooled from 3 independent experiments, t test). (C) Representative images of metaphase spreads from B). Arrows indicate chromatid breaks. Scale bar, 6.5 μm. (D) Boxplot of CldU tract length ratios of associated sister forks from HeLa WT, EXD2−/−, and EXD2−/− cells complemented with either Flag-EXD2 WT or Flag-EXD2 nuclease dead (ND) mutant protein (5–95 percentile, n ≥ 60 sister fork pairs pooled from 3 independent experiments, Mann-Whitney).
Figure 4
Figure 4
Loss of EXD2 Leads to Mitotic Abnormalities Associated with Under-Replicated DNA (A) Quantification of the HeLa WT and EXD2−/− anaphase or telophase cells showing DAPI-positive bridges (mean ± SEM, n = 3 independent experiments, chi-square). Scale bar, 10 μm. (B) Quantification of HeLa WT and EXD2−/− G1 cells with 53BP1 OPT domains in G1 cells (left panel). Quantification of the number of 53BP1 OPT domains per positive cell in HeLa WT and EXD2−/− cells (right panel) and representative images (mean ± SEM, n = 3 independent experiments, chi-square). Scale bar, 20 μm. (C) Quantification of HeLa WT and EXD2−/− cells showing MN and representative images. Phalloidin acts as a cytosolic marker (mean ± SEM, n = 3 independent experiments, chi-square). Scale bar, 20 μm. (D) Quantification of HeLa WT, EXD2−/−, and EXD2−/− cells complemented with either Flag-EXD2 WT or Flag-EXD2 nuclease dead (ND) mutant protein for anaphase or telophase cells showing DAPI-positive bridges (mean ± SEM, n = 3 independent experiments, chi-square). (E) Quantification of HeLa WT, EXD2−/−, and EXD2−/− cells complemented with either Flag-EXD2 WT or Flag-EXD2 nuclease dead (ND) mutant protein for G1 cells with 53BP1 OPT domains (mean ± SEM, n = 3 independent experiments, chi-square). (F) Quantification of HeLa WT, EXD2−/−, and EXD2−/− cells complemented with either Flag-EXD2 WT or Flag-EXD2 nuclease dead (ND) mutant protein for cells showing MN (mean ± SEM, n = 3 independent experiments, chi-square).
Figure 5
Figure 5
EXD2 Protects Replication Forks against Uncontrolled Degradation of Nascent DNA (A) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells (left panel) and U2OS WT and EXD2−/− cells (right panel) (5–95 percentile, n ≥ 300 tracts pooled from 3 independent experiments, Mann-Whitney). (B) Quantification of U2OS cells or U2OS cells stably expressing GFP-EXD2 for GFP/BRCA1 PLA foci (mean ± SEM from 3 independent experiments, Mann-Whitney) and representative images. Scale bar, 10 μm. (C) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells untreated or pre-treated with MRE11 inhibitor Mirin (50 μM) (5–95 percentile, n ≥ 300 tracts pooled from 3 independent experiments, Mann-Whitney). (D) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells upon MRE11 knock-down (5–95 percentile, n ≥ 300 tracts pooled from 3 independent experiments, Mann-Whitney).
Figure 6
Figure 6
EXD2 Acts to Counteracts Replication Fork Regression (A) Schematic of the process of replication fork reversal and RECQ1-mediated restart of regressed forks (upper panel); upon loss of EXD2 reversed forks undergo pathological degradation. Knockdown of SMARCAL1 suppresses fork reversal and promotes fork restart (bottom left panel); RECQ1 is inhibited by parylation thus, PARP inhibition de-represses RECQ1 and increases its activity counteracting fork regression (bottom right panel). (B) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells upon SMARCAL1 knockdown (5–95 percentile, n ≥ 200 tracts pooled from 3 independent experiments, Mann-Whitney). (C) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells untreated or treated with 10 μM olaparib (5–95 percentile, n ≥ 200 tracts pooled from 2 independent experiments, Mann-Whitney). (D) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells upon SMARCAL1 knockdown (5–95 percentile, n ≥ 180 tracts pooled from 3 independent experiments, Mann-Whitney). (E) Boxplot of CldU/IdU tract ratios of HeLa WT and EXD2−/− cells untreated or treated with 10 μM olaparib (5–95 percentile, n ≥ 160 tracts pooled from 3 independent experiments, Mann-Whitney). (F) Quantification of the average number of PLA foci per focus positive cell in HeLa and EXD2−/− cells upon SMARCAL1 knockdown and representative images (mean ± SEM, n = 4 independent experiments, Mann-Whitney). Scale bar, 10 μm. (G) Phosphor imaging of 5′ radiolabeled indicated DNA substrates (labeled strand shown in red, length of the regressed arm indicated) incubated for indicated amounts of time with EXD2 WT protein.
Figure 7
Figure 7
EXD2 Is Required for Survival of BRCA1/2-Deficient Cells (A) Proliferation of HeLa WT and EXD2−/− cells upon BRCA1 knockdown (mean ± SEM, n = 3 independent experiments). (B) Proliferation of HeLa WT and EXD2−/− cells upon BRCA2 knockdown (mean ± SEM, n = 3 independent experiments). (C) Proliferation of U2OS WT and EXD2−/− cells upon BRCA1 knockdown (mean ± SEM, n = 4 independent experiments). (D) Proliferation of DLD1 WT and BRCA2−/− cells upon EXD2 knockdown (mean ± SEM, n = 3 independent experiments). (E) Proliferation of SUM149 and SUM149 revertant cells upon EXD2 knockdown (mean ± SEM, n = 3 independent experiments). (F) Proliferation of RPE1 WT, EXD2−/−, and EXD2ND/ND cells upon BRCA1 knockdown (mean ± SEM, n = 3 independent experiments). (G) Quantification of the HeLa WT and EXD2−/− cells upon BRCA2 knockdown showing MN and representative images (mean ± SEM, n = 3 independent experiments). Scale bar, 20 μm. (H) Quantification of the relative alt-EJ efficiency upon EXD2, MRE11 or BRCA2 knockdown as indicated (mean ± SEM, n = 5 independent experiments, t test). (I) Proposed model for EXD2 function during genome duplication. Replicative stress leads to fork stalling. Stressed forks are protected by EXD2 activity in a pathway cooperating with RECQ1, allowing for efficient fork restart and timely accomplishment of DNA replication. Loss of EXD2 leads to extensive degradation of nascent DNA at stalled forks, compromising fork restart and ultimately adversely impacting genome stability. Combined deficiency in EXD2 and BRCA1/2 results in loss of fork protection and in the absence of functional HR also compromises cells’ ability to rescue collapsed forks by the backup Alt-EJ pathway.

Similar articles

See all similar articles

References

    1. Berti M., Vindigni A. Replication stress: getting back on track. Nat. Struct. Mol. Biol. 2016;23:103–109. - PMC - PubMed
    1. Berti M., Ray Chaudhuri A., Thangavel S., Gomathinayagam S., Kenig S., Vujanovic M., Odreman F., Glatter T., Graziano S., Mendoza-Maldonado R. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 2013;20:347–354. - PMC - PubMed
    1. Bhowmick R., Hickson I.D. The “enemies within”: regions of the genome that are inherently difficult to replicate. F1000Res. 2017;6:666. - PMC - PubMed
    1. Biehs R., Steinlage M., Barton O., Juhász S., Künzel J., Spies J., Shibata A., Jeggo P.A., Löbrich M. DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination. Mol. Cell. 2017;65:671–684. - PMC - PubMed
    1. Broderick R., Nieminuszczy J., Blackford A.N., Winczura A., Niedzwiedz W. TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution. Nat. Commun. 2015;6:6572. - PMC - PubMed

Publication types

Feedback