Lin Y , Raj J , Li J , Ha A , Hossain MA , Richardson C , Mukherjee P , Yan S .

R01 CA225637 NCI NIH HHS , R15 GM101571 NIGMS NIH HHS , R15 GM114713 NIGMS NIH HHS , S10 OD026970 NIH HHS

	Figure 1. APE1 is required for SSB-induced ATR–Chk1 DDR pathway activation and APE2 recruitment to SSB sites, but not vice versa. (A) CTL or SSB plasmid was added to mock- or APE1-depleted HSS. After incubation for 30 min, the DNA-bound fractions and total egg extract were examined via immunoblotting analysis as indicated. (B) CTL or SSB plasmid was added to HSS supplemented with APE1i III or AR03 at a final concentration of 1 mM. (C–D) CTL or SSB plasmid was added to mock-, APE1- or APE2-depleted HSS. The DNA-bound fractions and total egg extract at the indicated timepoints were examined via immunoblotting analysis.
	Figure 2. APE1 exonuclease activity is important for APE2 recruitment to SSB sites and ATR–Chk1 DDR pathway activation. (A) Characterization of the exonuclease activities of WT and mutant GST–APE1 (4 μM) after different incubation times in in vitro exonuclease activity assays. WT, wild type; ED, E95Q–D209N; DA, D306A; CA, C92A–C98A. (B) Characterization of the endonuclease activities of WT and mutant GST–APE1 (0.08 μM) via in vitro endonuclease activity assays. * nonspecific dye band from sample buffer. (C) An amino acid alignment highlights the conserved D306 residue in Xenopus, humans, and mouse APE1. (D) WT or DA Myc-APE1 was added back to APE1-depleted HSS supplemented with CTL or SSB plasmid. After incubation for 30 min, the DNA-bound fractions and total egg extract were examined via immunoblotting. (E) CTL or SSB plasmid was added to APE1-depleted HSS that was mixed with WT or DA Myc-APE1. After different incubation times (5 and 10 min), the DNA-bound fractions and total egg extract were examined via immunoblotting.
	Figure 3. APE1 recognizes and binds to SSB structures in vitro. (A) EMSAs show the interactions between GST–APE1 and various DNA structures: dsDNA, dsDNA–SSB and dsDNA–gap. (B) Interactions between GST–APE1 and various DNA structures: dsDNA–AP, dsDNA–AP–SSB and dsDNA–AP–gap. (C) An EMSA showing the interaction between WT, DA and CA GST–APE1 and the dsDNA–SSB structure. (D) Interactions between WT, DA, and CA GST–APE1 and the dsDNA–AP–SSB structure via an EMSA. *nonspecific dye band from sample buffer in panels (A–D). (E) Interactions of GST-tagged WT and DA APE1 as well as GST to the defined Cy5–SSB structure via MST (Microscale Thermophoresis) assays.
	Figure 4. APE1 is critical for SSB end resection and SSB repair in the HSS system. (A) The dsDNA–SSB structure was added to mock- or APE1-depleted HSS supplemented with WT or DA Myc-APE1. After different incubation times, samples were examined via denaturing urea PAGE electrophoresis and visualized. (B) The dsDNA–AP–SSB structure was added to mock- or APE1-depleted HSS, and samples were analyzed the same as in Panel (A). (C) The SSB plasmid was added to mock- or APE1-depleted HSS supplemented with WT or DA Myc-APE1. After different incubation times, the DNA repair products were isolated and analyzed on an agarose gel. (D) The SSB plasmid was added to HSS supplemented with the different APE1-specific inhibitors (E3330, CRT0044876 (CRT), APE1i III and AR03) at a final concentration of 1 mM. After different incubation times, the DNA repair products were isolated and analyzed on an agarose gel.
	Figure 5. APE1 interacts with APE2. (A) GST or GST-APE2 was examined for interaction with Myc-APE1 in an interaction buffer. (B) GST, WT or DA GST-APE1 was examined for interaction with Myc-APE2 in an interaction buffer. (C) GST or GST-tagged different fragments (i.e. AA 1–316, AA 35–316 and AA 101–316) of APE1 was examined for interaction with Myc-APE2 in an interaction buffer. (D) GST or GST-tagged different fragments (i.e. AA 101–316, AA 101–200 and AA 201–316) of APE1 was examined for interaction with Myc-APE2 in an interaction buffer. (E) GST or GST-APE1 was examined for interaction with FL Myc-APE2 or ΔZF Myc-APE2 (i.e. Zf-GRF motif deletion mutant in APE2) in an interaction buffer. (F) GST, and WT/C470A/R502E GST-APE2-ZF were analyzed for interaction with Myc-APE1 in an interaction buffer. WT GST-APE2-ZF is GST-tagged Zf-GRF motif of APE2 (i.e. AA 456–517 in APE2). (A–F) Input and pulldown samples were analyzed via immunoblotting analysis.
	Figure 6. Characterization of patient-derived APE1 variants in SSB repair and signaling. (A) CBiolPortal analysis of human APE1 mutations from cancer patients. The F266L and F266Y mutations in human APE1 were identified in data from a cancer patient. A comparison of the sequences of the peptide surrounding the F266 residue in human (Hs), frog (Xl) and mouse (Mm) APE1 is shown under the schematic diagram. (B) The dsDNA–SSB structure was used to characterize the exonuclease activities of WT, F264L, and F264Y APE1 at different concentrations. (C) The endonuclease activities of WT, F264L and F264Y APE1 protein (0.08 μM) were examined in vitro using the dsDNA–AP as a substrate. (D) An EMSA shows the interaction between WT, F264L and F264Y APE1 and the dsDNA–AP–SSB structure in vitro. * nonspecific dye band from sample buffer in panels C and D. (E) The SSB plasmid was added to mock- or APE1-depleted HSS supplemented with WT or F264Y Myc-APE1. After different incubation times, the DNA repair products were isolated and analyzed on an agarose gel. (F) WT or F264Y Myc-APE1 was added back to APE1-depleted HSS supplemented with CTL or SSB plasmid. After incubation for 30 min, total egg extract were examined via immunoblotting analysis as indicated.
	Figure 7. A two-step mechanism for SSB end resection in SSB repair and signaling.Step 1: An SSB is recognized by APE1, which then initiates SSB end resection in the 3′–5′ direction to generate a small gap (this work). Step 2: APE2 is recruited to the small gap likely via PCNA, and its exonuclease activity is activated and enhanced via several distinct regulatory mechanisms (28–30). After this two-step SSB end resection, the ATR–Chk1 DDR pathway is activated and the SSB is eventually repaired. See the text for more details.

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