Lin Y et al. (2018), APE2 promotes DNA damage response pathway from ...

XB-ART-54574

Nucleic Acids Res 2018 Mar 16;465:2479-2494. doi: 10.1093/nar/gky020.

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APE2 promotes DNA damage response pathway from a single-strand break.

Lin Y , Bai L , Cupello S , Hossain MA , Deem B , McLeod M , Raj J , Yan S .

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As the most common type of DNA damage, DNA single-strand breaks (SSBs) are primarily repaired by the SSB repair mechanism. If not repaired properly or promptly, unrepaired SSBs lead to genome stability and have been implicated in cancer and neurodegenerative diseases. However, it remains unknown how unrepaired SSBs are recognized by DNA damage response (DDR) pathway, largely because of the lack of a feasible experimental system. Here, we demonstrate evidence showing that an ATR-dependent checkpoint signaling is activated by a defined plasmid-based site-specific SSB structure in Xenopus HSS (high-speed supernatant) system. Notably, the distinct SSB signaling requires APE2 and canonical checkpoint proteins, including ATR, ATRIP, TopBP1, Rad9 and Claspin. Importantly, the SSB-induced ATR DDR is essential for SSB repair. We and others show that APE2 interacts with PCNA via its PIP box and preferentially interacts with ssDNA via its C-terminus Zf-GRF domain, a conserved motif found in >100 proteins involved in DNA/RNA metabolism. Here, we identify a novel mode of APE2-PCNA interaction via APE2 Zf-GRF and PCNA C-terminus. Mechanistically, the APE2 Zf-GRF-PCNA interaction facilitates 3'-5' SSB end resection, checkpoint protein complex assembly, and SSB-induced DDR pathway. Together, we propose that APE2 promotes ATR-Chk1 DDR pathway from a single-strand break.

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Species referenced: Xenopus laevis
Genes referenced: apex2 atm atr atrip chek1 clspn ghrh gmnn myc parp1 pcna rpa1 ssb topbp1 usp9x xrcc1

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	Figure 1. A defined site-specific SSB structure can be repaired in the Xenopus HSS system. (A) Schematic diagram of generating SSB and DSB plasmid structures. (B) The defined SSB structure is gradually repaired in HSS system. SSB repair intermediate products from HSS system were isolated at different time points and treated by SbfI, followed by examination via agarose gel electrophoresis. (C) (Top) CTL or SSB plasmid was added to HSS, which was supplemented with [32P-α]-dATP, for a 30-min incubation. Then NPE was added for continuous incubation for different time as indicated and samples were examined on agarose gel. (Bottom) Quantification of DNA synthesis of CTL or SSB plasmid in the HSS/NPE system shown in the top.
	Figure 2. An ATR–Chk1 DNA damage response pathway is induced by the defined SSB structure in the HSS system. (A) CTL or SSB plasmid was added to HSS at different concentrations as indicated, and incubated for 30 min. Extracts were examined via immunoblotting analysis for Chk1 phosphorylation (i.e., Chk1 P-Ser344) and total Chk1. (B) CTL or SSB plasmid was added to HSS at a final concentration of 75 ng/μl. After different time of incubation at room temperature, the extracts were examined via immunoblotting analysis. (C–E) ATR inhibitor VE-822, ATM inhibitor KU55933, DNA-PK inhibitor NU7441, recombinant geminin, or DNA polymerase inhibitor Aphidicolin was added to HSS supplemented with CTL or SSB plasmid at a final concentration of 75 ng/μl for 30 min. Extracts were examined via immunoblotting analysis as indicated. (F) Geminin or roscovitine was added to HSS supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, extracts were examined via immunoblotting analysis as indicated. (G) CTL, SSB or DSB plasmid was added to HSS at different concentrations as indicated. Samples were examined via immunoblotting analysis.
	Figure 3. APE2 is required for checkpoint signaling from the defined SSB structure in the HSS system. (A–E) CTL or SSB plasmid was added to mock-, ATRIP-, TopBP1-, Rad9-, Claspin-, or XRCC1-depleted HSS, respectively, at a concentration of 75 ng/μL for 30 min. Extracts were examined via immunoblotting analysis, as indicated. (F) PARP1 specific inhibitor (4-amino-1,8-naphthalimide, 0.1 mM) was added to HSS supplemented with CTL or SSB plasmid. Extracts were examined via immunoblotting analysis. (G) Recombinant Myc-APE2 was added to APE2-depleted HSS supplemented with CTL or SSB plasmid. Extracts were examined via immunoblotting analysis. ‘Endo. APE2’ represents endogenous APE2.
	Figure 4. The SSB-induced ATR DDR pathway is essential for SSB repair. (A) SSB plasmid was added to HSS with or without VE-822 for different time as indicated. SSB repair products were isolated and examined on agarose gel (Ethidium bromide staining). (B) Quantification of SSB repair capacity (circular/(circular + nicked) × 100) with or without VE-822 treatment in the HSS system from (A). (C) SSB plasmid was added to mock- or ATRIP-depleted HSS for different time as indicated. SSB repair products were isolated and examined on agarose (Ethidium bromide staining). (D) Quantification of SSB repair capacity in mock- or ATRIP-depleted HSS system from (C) using a similar method described in (B).
	Figure 5. APE2 Zf–GRF interacts with PCNA as the second mode of APE2–PCNA interaction. (A) Schematic diagram of APE2 Zf–GRF region and the IDCL and CTM regions of PCNA. (B) GST pulldown assays with GST, GST-APE2 and GST-APE2-ZF from HSS. The input and pulldown samples were examined via immunoblotting analysis. (C) GST pulldown assays with GST or GST-APE2-ZF as well as WT/mutant PCNA (i.e., LI PCNA, PK PCNA, or LIPK PCNA) in an interaction buffer. The input and pulldown samples were examined via immunoblotting analysis. (D) GST pulldown assays with GST or WT/mutant GST-APE2-ZF (i.e., G483A-R484A, F486A-Y487A, or C470A) as well as WT His-tagged PCNA in an interaction buffer. The input and pulldown samples were examined via immunoblotting analysis. (E) Biotin-coupled ssDNA (80nt) was coupled to streptavidin dynabeads and utilized for protein–DNA interaction assays with GST or WT/mutant GST-APE2-ZF (i.e., G483A-R484A, F486A-Y487A or C470A) in an interaction buffer. The input and bead-bound fractions were analyzed via immunoblotting analysis.
	Figure 6. APE2 Zf–GRF–PCNA interaction promotes SSB end resection, the assembly of a checkpoint protein complex onto SSB plasmid, and Chk1 phosphorylation in the HSS system. (A) CTL or SSB plasmid was added to mock- or APE2-depleted HSS, which was supplemented with WT or C470A Myc-APE2. DNA-bound fractions and total extract samples were examined via immunoblotting analysis as indicated. ‘Endo. APE2’ represents endogenous APE2. (B) FAM-labeled dsDNA with a site specific SSB (designed as FAM-SSB) was added to HSS for different time as indicated. Then samples were examined via TBE-Urea gel and visualized via Typhoon imager. ‘Marker’ represents four FAM-labeled different-length ssDNA. (C) The length dependence of ssDNA for the recruitment of ATR-ATRIP complex and RPA to ssDNA in the HSS. Streptavidin Dynabeads coupled with different length of Biotin-coupled ssDNA (i.e., 0, 10, 20, 40, 60 or 80nt) were added to HSS. After incubation, the Biotin-ssDNA bead-bound fractions were isolated from HSS. The Input and bead-bound fractions were examined via immunoblotting analysis. (D) The FAM-SSB substrate was added to mock- or APE2-depleted HSS, which was supplemented with WT or C470A Myc-APE2. DNA structures were examined via TBE-Urea gel and visualized via Typhoon imager (top). Samples were also analyzed via immunoblotting analysis as indicated. ‘Endo. APE2’ represents endogenous APE2 (bottom).
	Figure 7. Exonuclease activities of APE2 with purified proteins in vitro. (A) In vitro analysis of exonuclease activity of GST-APE2 with the presence or absence of WT or mutant His-tagged PCNA using the FAM-labeled gapped dsDNA structure. (B) In vitro analysis of exonuclease activity of WT and mutant GST-APE2 with the presence or absence of WT His-tagged PCNA using the FAM-labeled gapped dsDNA structure.
	Figure 8. A working model of the molecular mechanism of APE2-mediated ATR–Chk1 DDR pathway from a defined SSB structure. Please see text for more details.

References [+] :

Acevedo, Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage. 2016, Pubmed, Xenbase