Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease

Abstract

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

Keywords: APE2; APEX2; BRCA1; BRCA2; RNASEH2; TDP1; TOP1; ribonucleotide; synthetic lethality.

Figure 1.. Characterization of the APEX2-BRCA1/2 synthetic lethality.

Clonogenic survival of RPE1 (A) and DLD1 cells (B) of the indicated genotypes expressing wild-type APE2 or the APE2 E48Q/D197N mutant (sgRNA-resistant) upon transduction with a sgRNA targeting APEX2. Shown are results of a two-way ANOVA with Dunnet post-test that compared the mock and APE2 E48Q/D197N mutant to the WT APE2 condition in the BRCA1^−/− (A) or BRCA2^−/− (B) cells (*** P ≤ 0.001). (C) Clonogenic survival of the indicated cell lines upon transduction with vectors expressing Cas9 and an sgRNA targeting LacZ (control), PARP1, or APEX2. APEX2 editing efficiency. % indel by TIDE is indicated. Also shown are results of a t-test that compared sgRNA vs LacZ sgRNA control in BRCA1 mutated cell lines (* FDR ≤ 0.05; ** FDR ≤ 0.01). (D) Clonogenic survival of RPE1-hTERT p53^−/− Cas9 cells of the indicated genotypes upon transduction with virus expressing an sgRNA targeting LacZ or APEX2. (E) Affinity purifications from 293T cell lysates transiently expressing C-terminal SBP/HA-tagged APE2 wild-type, Y396A/F397A or ΔPIP mutants. Bound proteins and whole-cell extracts (WCE) were immunoblotted with the indicated antibodies. (F) Quantitation of clonogenic survival of RPE1 wild-type or BRCA1^−/− cells expressing APE2 or the indicated mutants transduced with an sgRNA targeting endogenous APEX2. Also shown are the results of a two-way ANOVA with Dunnet post-test (APE2 mutants vs WT APE2) (* P ≤ 0.05). Data in all panels are shown as mean ± standard deviation (n ≥ 3 biologically independent experiments), circles. WT, wild-type. See Figure S1 for controls and additional data. See Table S6 for details on statistical tests.

Figure 2.. APEX2, APEX1 and TDP1 genetic interaction landscape.

(A) Genetic interaction with with APEX2, APEX1 or TDP1 (source nodes) determined by CRISPR screens (see Table S2). Thickness of the edges are scaled on ΔBF values. Source nodes are coloured in blue and DNA repair-coding genes in red. See Figures S1C and S2 for cell line validation and additional analysis of the screens. (B) Representative image of RPE1-hTERT Cas9 cells clonogenic survival assays of the indicated genotypes transduced with sgRNA targeting AAVS1 or APEX2. Data presented as mean ± standard deviation. The results of a t-test that compares sgAPEX2 to the sgAAVS1 condition (*** FDR ≤ 0.001) are shown. (C) Competitive growth assays in wild-type or BRCA1^−/− RPE1-hTERT p53^−/− Cas9 cells transduced with virus expressing the indicated sgRNAs. Data presented as mean ± standard deviation (n=3 biologically independent experiments). The results of a one-way ANOVA test that compared sgRNA targeting each sgRNA condition in BRCA1^−/− to their WT counterpart (* P ≤ 0.05, *** P ≤ 0.001) are shown. (D) Quantitation of sister chromatid exchanges (SCEs) on metaphase spreads of RPE1-hTERT p53^−/− Cas9 wild-type or two independent APEXZ^−/− cell lines. Black lines indicate the means (n=2 biologically independent experiments). The results of a t-test comparing APEX2 clones to WT is shown (FDR *** ≤ 0.001). (E) Quantitation of chromatid breaks (left) and radial chromosomes (right) in metaphase spreads from RPE1-hTERT p53^−/− Cas9 cells with indicated genotypes upon transduction with sgRNA targeting APEX2 (10 metaphases scored from at least 2 biologically independent experiments). The results of a pair-wise Wilcox (Mann Whitney) test comparing sgAPEX2 to sgAAVS1 conditions are shown (* FDR < 0.05; ** FDR < 0.01). (F) Representative image of a metaphase from a BRCA1^−/− cell sgAPEX2 showing multiple broken or aberrant chromosomes (arrowheads). See Table S6 for details on statistical tests.

Figure 3.. APE2 reverses 3′ blocked DNA lesions

(A) Competitive growth assays in RPE1-hTERT p53^−/− Cas9 cells of the indicated genotypes transduced with virus expressing an sgRNA targeting RNASEH2A. Results of a two-way ANOVA with Dunnet post-test comparing APEX2^−/− to WT is shown (P*** < 0.001). (B) As in A, but using an sgRNA targeting APEX2. Statistical test compares each knockout cell lines to WT (* P ≤ 0.05; ** P≤ 0.01; *** P< 0.001). (C) Competitive growth assays in cells transduced with an sgRNA targeting BRCA2 in RPE1 hTERT p53^−/− Cas9 wild-type or RNASEH2A^−/− cells overexpressing either RNASEH2A, RNASEH2A-P40D/Y210A (ribonucleotide excision repair deficient; RED), or RNASEH2A-D34A/D169A (nuclease-dead; ND). (D) Competitive growth assays in wild-type or BRCA1^−/− RPE1-hTERT p53^−/− Cas9 cells expressing APE2 or the E48Q/D197N mutant transduced with the indicated sgRNAs. (E) Clonogenic survival of RPE1 hTERT p53^−/− Cas9 wild-type or APEX2^−/− cells transfected with the indicated siRNA and exposed to camptothecin (CPT). Immunoblot of the depletion of TDP1 by siRNA is also shown. NT, non-targeting control siRNA. Results of a Fit of exponential decay (quadratic survival) with extra-sum-of-squares F test comparing APEX2^−/− siTDP1 condition to WT siNT, WT siTDP1 and APEX2^−/− siNT is shown (*** P ≤ 0.001) (F) Clonogenic survival of RPE1-hTERT p53^−/− Cas9 cells of the indicated genotypes exposed to KBrO₃. Results of a Fit of exponential decay (quadratic survival) with extra-sum-of-squares F test comparing APEX1^−/− to WT, APEX2^−/− or TDPT^−/− are shown (*** P ≤ 0.001). Data in panels A-F are represented as the mean ± standard deviation (n =3 biologically independent experiments). See Figure S3 for controls and additional data. See Table S6 for details on statistical tests.

Figure 4.. APE2 nucleolytic activity facilitates resolution of blocked 3′ ends.

(A) Denaturing gel electrophoresis of dsDNA oligonucleotide substrates carrying a 3′ hydroxyl (lanes 1–10), 3′ phosphotyrosine (lanes 11–20), terminal 2′,3′-cyclic phosphate (lanes 21–30) or internal model abasic site (THF; tetrahydrofuran, lanes 31–40) incubated with recombinant X. laevis APE2, human APE1, or with MBP as controls. Asterisk, position of FAM label. Numbers, oligonucleotide lengths. (B) Scheme of TOP1 suicide substrate containing either a deoxythymidine (dT) or ribouridine (rU) at the TOP1 incision hot spot, two nucleotides from the 3′ end of a 5′ Cy5-labeled scissile strand. (c) Scheme of TOP1cc processing assay performed in D and F. (D) Denaturing gel electrophoresis of products obtained from dT-containing TOP1 suicide substrate incubated with the indicated recombinant proteins. Low and high molecular weight products are shown cropped from the same gel. Blue numbers in circles annotate the reaction products summarized in panel (E). (F) Same as in (D), but rU-containing TOP1 suicide substrate was used. Gray numbers in circles annotate the reaction products corresponding to the schematic in panel (G). Asterisk annotates a background band. See Figures S4 and S5 for additional data and controls.

Figure 5.. APE2 X-ray structure.

(A) Domain architecture of APE2. (B) Magnesium ion coordination site with electron density maps overlaid (2F_o-F_c, 1.5 o at 1.54Å). (C) Crystal structure of the N-terminal EEP domain of X. laevis APE2. A single active site-bound magnesium ion is indicated in green. (D) An orthogonal view of the catalytic domain structure. (E) Structural overlay of APE2 (blue) with the APE1-THF-DNA complex (grey). AP site recognition residues (in grey surfaces) are also divergent in APE2. (F) Closeup view of the APE1-APE2 comparison. Critical APE1 AP site recognition residues R177, M270 and W280 interrogate the DNA base stack and the DNA lesion binding pocket, which is capped by a closed L5 loop. In APE2, these residues are not conserved, and are replaced by smaller side chain P148, T270 and A260. By comparison, L5 in APE1 is closed and secures the AP recognition pocket. (G) A surface representation of the APE1-DNA complex compared to APE2 in (H). See Figure S6 for additional data and Table S4 for crystallographic data collection and refinement statistics.

Figure 6.. SLX4-XPF-ERCC1 may process 3′ ends in APE2-deficient cells.

(A) Competitive growth assays in RPEl-hTERT p53^−/− Cas9 cells of the indicated genotypes transduced with virus expressing sgRNAs targeting SLX4 or AA VS1. Data presented as the mean ± standard deviation (n=3 biologically independent experiments). Results of a two-way ANOVA with Dunnet post-test comparing APEX2^−/− to WT are shown (*** P ≤ 0.001). (B) As in A but cells were transduced with virus expressing an sgRNA targeting APEX2. Results of a two-way ANOVA with Dunnet post-test comparing knock-out cell lines to WT are shown (*** P≤ 0.001). (C) Clonogenic survival of RPE1 hTERT p53^−/− Cas9 wild-type or SLX4^−/− cells with transgenes expressing either wild-type SLX4 or its L530A/W531A mutant. These cells were transduced with an sgRNA targeting either AAVS1 or APEX2. Data are presented as the mean ± standard deviation (n=4 biologically independent experiments). Results of a t-test comparing GFP-SLX4 to GFP-only and GFP-SLX4-L530A/W531A are shown (*** FDR ≤ 0.001). (D) Quantitation of chromatid breaks (left) and radial chromosomes (right) in metaphase spreads from RPE1-hTERT p53^−/− Cas9 cells with indicated genotypes upon transduction with an sgRNA targeting SLX4 (10 metaphases scored from at least 2 biologically independent experiments). The results of a Pair-wise Wilcox (Mann Whitney) comparing sgSLX4 to sgAAVSl conditions are shown (* FDR ≤ 0.05). See Figure S7 for controls and additional data. See Table S6 for details on statistical tests.

Figure 7.. DNA 3′ block metabolism.

DNA 3′ blocks are managed by a two-tier system. In Tier 1, RNase H2, TDP1 and APE1 promote 3′ block avoidance. Tier 2 consists of APE2. BRCA1/2-dependent recombination preserves cell viability and genomic integrity when cells undergo replication in the presence of 3ʼblocks.