Author response: APE1 recruits ATRIP to ssDNA in an RPA-dependent and -independent manner to promote the ATR DNA damage response

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-stranded DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence suggests that APE1 recruits ATRIP onto ssDNA in an RPA-dependent and -independent manner in the ATR DDR pathway. Editor's evaluation This important paper provides new insight into the mechanism of the activation of DNA damage checkpoint (DDR) in response to the single-stranded DNAs (ssDNAs). The authors used Xenopus egg extracts and a reconstitution reaction with purified proteins and presented convincing results to support the authors' claims on a non-catalytic role of APE1 endonuclease to recruit DDR activator, ATRIP, to the ssDNA for DDR activation. The work would be of interest to researchers who work on the cell cycle and DNA damage responses as well as DNA repair. https://doi.org/10.7554/eLife.82324.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The DNA damage response (DDR) signaling pathways such as ATR-Chk1 and ATM-Chk2 are activated by DNA replication stress or different DNA damage to coordinate DNA repair with cell cycle as well as apoptosis and senescence (Bartek et al., 2004; Branzei and Foiani, 2010; Ciccia and Elledge, 2010; Cimprich and Cortez, 2008; Harper and Elledge, 2007; Harrison and Haber, 2006; Su, 2006). In response to stalled DNA replication forks and different DNA lesions including DNA double-strand breaks (DSBs) and single-strand breaks (SSBs), ATR DDR can be recruited to and activated by RPA-coated single-stranded DNA (ssDNA) derived from functional uncoupling of MCM helicase and DNA polymerase activities, DSB end resection in the 5'–3' direction, or SSB end resection in the 3'–5' direction (Ciccia and Elledge, 2010; Cimprich and Cortez, 2008; Lin et al., 2018; Maréchal and Zou, 2015; Shiotani and Zou, 2009). ATR activation also requires several mediator proteins such as ATRIP, TopBP1, and the Rad9-Rad1-Hus1 (9-1-1) complex (Delacroix et al., 2007; Kumagai et al., 2006; Yan and Michael, 2009; Zou and Elledge, 2003). Activated ATR kinase then phosphorylates a variety of substrates such as Chk1, among others, and phosphorylated Chk1 is the activated version of Chk1 kinase to regulate cell cycle progression and often serves as an indicator of ATR DDR activation (Chen and Sanchez, 2004; Guo et al., 2000; Matsuoka et al., 2007). Since the discovery of ATRIP in ATR DDR pathway about 20 years ago, it has been an active subject of studies regarding how the ATR-ATRIP complex is recruited to ssDNA and activated by ATR activator/mediator proteins to maintain genome integrity (Cortez et al., 2001; Zou and Elledge, 2003). Earlier studies from several groups using human and yeast cells as well as Xenopus egg extracts have revealed independently that ATR and ATRIP associate with each other into a tight complex and that the direct ATRIP recognition and interaction with RPA-ssDNA is essential for the recruitment of ATR to ssDNA regions at sites of DNA damage for ATR activation (Lee et al., 2003; Unsal-Kaçmaz and Sancar, 2004; Zou and Elledge, 2003). However, the RPA requirement of ATRIP recruitment to ssDNA for ATR DDR activation is sort of questioned by several follow-up reports demonstrating that ATR-ATRIP complexes can bind to ssDNA in an RPA-independent manner in vitro, and that the low affinity RPA-independent recruitment of ATRIP to ssDNA is mediated by an unknown protein in mammalian cell nuclear extracts (Bomgarden et al., 2004; Kim et al., 2005). Although the exact molecular determinants of ATRIP (such as the N-terminal 1–108 amino acid fragment of human ATRIP) interaction with RPA-ssDNA remain to be determined and reconciled (Ball et al., 2005; Namiki and Zou, 2006), additional lines of investigations have demonstrated that TopBP1 can directly activate the ATR-ATRIP complex in Xenopus egg extracts and reconstituted human proteins in an RPA-dependent and RPA-independent manner (Choi et al., 2010; Choi et al., 2007; Kumagai et al., 2006). Whereas a good progress has been made regarding the implication of post-translational modifications such as sumoylation and phosphorylation in ATRIP regulation in ATR DDR (Memisoglu et al., 2019; Wagner et al., 2019; Wu et al., 2014), it remains an outstanding question in the field of genome integrity of how exactly ATRIP is recruited to ssDNA in an RPA-dependent and/or -independent fashion for ATR DDR activation. As the major AP endonuclease, AP endonuclease 1 (APE1) has fast AP endonuclease activity but slow 3'–5' exonuclease and 3'-phosphodiesterase activities as well as 3'–5' RNA phosphatase and exoribonuclease activities (Boiteux and Guillet, 2004; Burkovics et al., 2006; Chohan et al., 2015; Hadi et al., 2002; Tell et al., 2009; Wilson and Barsky, 2001). In addition to its role in redox regulation for transcription, APE1 plays essential roles in various DNA repair pathways (Li and Wilson, 2014; Tell et al., 2009). Whereas APE1-knockout mice are embryonic lethal, the underlying mechanism of APE1 in cell viability remains unclear (Fung and Demple, 2005; Masani et al., 2013; Xanthoudakis et al., 1996). Human APE1 is genetically altered and aberrantly expressed and localized in cancer patients and has become an emerging therapeutic target for various cancer therapy (Abbotts and Madhusudan, 2010; Al-Safi et al., 2012; Fishel and Kelley, 2007; Koukourakis et al., 2001; Sengupta et al., 2016; Thakur et al., 2014; Yoo et al., 2008). Recent pre-clinical and clinical studies have shown encouraging finding of APE1 inhibitor APX3330 in anti-cancer therapy in solid tumors (Caston et al., 2021; Shahda et al., 2019). Our recent studies have demonstrated that the ATR-Chk1 DDR pathway is activated by hydrogen peroxide-induced oxidative DNA damage and defined plasmid-based SSB structures in Xenopus high-speed supernatant (HSS) system (Lin et al., 2018; Wallace et al., 2017; Willis et al., 2013). To promote the ATR DDR activation, APE1 initiates the 3'–5' end resection at SSB sites to generate a short ~1–3 nt-ssDNA gap via its exonuclease activity, followed by PCNA-mediated APE2-dependent SSB end resection continuation (Lin et al., 2018; Lin et al., 2020). This APE1/2-mediated two-step processing of SSBs generates a longer stretch of ssDNA (~18–26 nt) coated by RPA, leading to the assembly of the ATR DDR complex (ATR-ATRIP, TopBP1, and 9-1-1 complex), subsequent ATR DDR activation, and eventual SSB repair (Hossain et al., 2018; Lin et al., 2018; Lin et al., 2020). However, it remains unknown whether APE1 plays a direct role in the ATRIP recruitment to ssDNA via a non-catalytic function in the presence and/or absence of RPA for the ATR DDR pathway. Here, we provide direct evidence that in addition to its exonuclease-mediated function, APE1 plays a direct role in the recruitment of ATRIP to ssDNA in Xenopus egg extracts and in in vitro reconstitution system with purified proteins. The N-terminal motif of APE1 is required for its direct association with ssDNA in vitro, and such APE1-ssDNA interaction can be enhanced by RPA. Importantly, APE1 directly interacts with ATRIP and recruits ATRIP to ssDNA with the absence of RPA in vitro. A mutant APE1 deficient for ATRIP interaction but proficient for ssDNA association could not recruit ATRIP onto ssDNA in the presence of endogenous RPA in APE1-depleted Xenopus egg extracts. Similar to wild type APE1, a nuclease mutant APE1 still recruits ATRIP onto ssDNA in APE1-depleted HSS, suggesting that APE1’s role in ATRIP ssDNA recruitment is not dependent on its nuclease activity. APE1 directly associates with RPA in vitro via two distinct motifs within APE1. Notably, the RPA-interaction-deficient APE1 had no effect on the ATRIP recruitment onto ssDNA in Xenopus egg extracts. Overall, the data in this study demonstrating that APE1 is required for ATRIP recruitment to RPA-coated ssDNA for ATR DDR activation in Xenopus egg extracts, and that APE1 directly associates with and recruits ATRIP to ssDNA in the absence of RPA in vitro. These findings thus support a previously uncharacterized critical non-catalytic function of APE1 for direct ATRIP recruitment to ssDNA independent of RPA for the ATR DDR pathway. Results APE1 is required for the recruitment of ATRIP onto ssDNA in the ATR-Chk1 DDR pathway activation in Xenopus egg extracts Our recent studies have revealed that APE1 plays an important role in the defined SSB structure-induced ATR-Chk1 DDR pathway via its 3'–5' exonuclease activity in Xenopus HSS system (Lin et al., 2018; Lin et al., 2020). This APE1-mediated initiation of 3'–5' SSB end resection (~1–3 nt-ssDNA gap) will be followed by APE2 recruitment and activation to continue SSB end resection, generating a longer stretch of ssDNA (~18–26 nt-ssDNA) coated by RPA for subsequent assembly of an ATR checkpoint protein complex including ATR-ATRIP, TopBP1, and 9-1-1 complex to activate ATR DDR (Lin et al., 2018; Wallace et al., 2017; Willis et al., 2013). In addition to the 3'–5' SSB end resection initiation, we are interested in whether APE1 plays other roles such as non-catalytic function in the ATR DDR pathway. To test this question directly, we chose to utilize a defined plasmid DNA with a 1–3 nt small ssDNA gap structure (designated as Gap plasmid) in vitro as previously described (Lin et al., 2018), and tested whether APE1 is still required for the ATR DDR in response to this Gap plasmid in HSS (top of the panel, Figure 1A). As expected, the defined Gap plasmid (‘Gap’), but not control plasmid (‘CTL’) triggered Chk1 phosphorylation, suggesting the activation of the ATR-Chk1 DDR pathway by the defined Gap plasmid in the Xenopus HSS (Figure 1A). Notably, the Gap plasmid-induced Chk1 phosphorylation was still impaired in APE1-depleted HSS system (‘extract’, Figure 1A), suggesting that APE1 may play an additional non-catalytic function in the ATR DDR pathway. To further define the additional role of APE1 in ATR DDR activation, we isolated the DNA-bound fractions from the HSS and examined the abundance of checkpoint proteins via immunoblotting analysis. Although the recruitment of RPA70 and RPA32 to the Gap plasmid was slightly reduced when APE1 was depleted from the HSS (‘DNA-bound’, Figure 1A), the presence of RPA70 and RPA32 on Gap-plasmid DNA suggests that the gap structure can be further processed by APE2 in the APE1-depleted HSS, consistent with our previous studies (Lin et al., 2018; Lin et al., 2020). Notably, the recruitment of ATRIP onto Gap plasmid was significantly compromised in APE1-depleted HSS, suggesting that APE1 plays a more direct role in ATRIP recruitment to ssDNA regions in the defined Gap plasmid (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset AP endonuclease 1 (APE1) is required for the recruitment of ATRIP to RPA-coated single-stranded DNA (ssDNA) in Xenopus egg extracts. (A) CTL (control) or Gap plasmid was added to Mock- or APE1-depleted high-speed supernatant (HSS) and incubated for 30 min. The DNA-bound fractions and total egg extract were examined via immunoblotting analysis as indicated. (B) Streptavidin beads coupled with equal moles of biotin-labeled double-stranded DNA (dsDNA) with ssDNA gap structures (30 nt or 80 nt) were added to Mock- or APE1-depleted HSS. After incubation for 30 min at room temperature, the DNA-bound fractions and total egg extract were examined via immunoblotting analysis as indicated. (C) Streptavidin beads coupled with equal moles of biotin-labeled dsDNA with ssDNA gap structures (30 nt or 80 nt) were added to an interaction buffer containing purified His-ATRIP protein with/without His-RPA protein. After incubation for 30 min at room temperature, the DNA-bound fractions and the input were examined via immunoblotting analysis. (D) Streptavidin beads coupled with equal moles of biotin-labeled dsDNA with ssDNA gap structures (30 nt or 80 nt) were added to an interaction buffer containing His-ATRIP and His-RPA, which was supplemented with GST or GST-APE1. After incubation for 30 min at room temperature, the DNA-bound fractions and the input were examined via immunoblotting analysis. (A, B, D) ATRIP intensity was quantified, and the ratio of ATRIP from DNA-bound vs extract/input was examined. a.u., arbitrary unit. Mean ± SD, n=3. Figure 1—source data 1 Raw images of immunoblotting analysis referenced in Figure 1A. https://cdn.elifesciences.org/articles/82324/elife-82324-fig1-data1-v1.zip Download elife-82324-fig1-data1-v1.zip Figure 1—source data 2 Raw images of immunoblotting analysis referenced in Figure 1B. https://cdn.elifesciences.org/articles/82324/elife-82324-fig1-data2-v1.zip Download elife-82324-fig1-data2-v1.zip Figure 1—source data 3 Raw images of immunoblotting analysis referenced in Figure 1C. https://cdn.elifesciences.org/articles/82324/elife-82324-fig1-data3-v1.zip Download elife-82324-fig1-data3-v1.zip Figure 1—source data 4 Raw images of immunoblotting analysis referenced in Figure 1D. https://cdn.elifesciences.org/articles/82324/elife-82324-fig1-data4-v1.zip Download elife-82324-fig1-data4-v1.zip In light of different lengths of ssDNA in vitro reconstitution systems by previous studies (such as 75 nt or 80 nt) (Bomgarden et al., 2004; Choi et al., 2010; Zou and Elledge, 2003), we intended to study the recruitment of ATRIP onto defined double-stranded DNA (dsDNA) structures with different length of ssDNA gaps. We chose to test two 100 bp-dsDNA structures with either 30 nt- or 80 nt-ssDNA gap covalently linked with 5'-biotin on top strand (designated as ‘30 nt gap’ and ‘80 nt gap’) for subsequent streptavidin magnetic bead-bound isolation and analysis from Xenopus egg extracts (top of the panel of Figure 1B). When equal moles of dsDNA with 30 nt or 80 nt-ssDNA gap were added to HSS, more RPA70 and RPA32 as well as APE1 and ATRIP are recruited to the 80 nt-ssDNA gap and Chk1 phosphorylation was also enhanced (Figure 1B). This enhanced Chk1 phosphorylation is likely due to increased RPA complex recruitment onto the 80 nt-ssDNA gap (Figure 1B). Whereas APE1 depletion led to compromised Chk1 phosphorylation, the recruitment of ATRIP but not RPA70 nor RPA32 was compromised in APE1-depleted HSS (Figure 1B). Our observations so far suggest that APE1 is important for the recruitment of ATRIP onto RPA-coated ssDNA and Chk1 phosphorylation in Xenopus HSS. To re-evaluate the role of RPA in the recruitment of ATRIP onto ssDNA gaps, we performed RPA depletion experiment in HSS and characterized the phenotype of RPA depletion in ATRIP recruitment and ATR-Chk1 DDR pathway. With anti-RPA antibodies, majority of the endogenous RPA protein complex, if not all, was immunodepleted from HSS (extract panel, Figure 1—figure supplement 1A), and such RPA depletion led to almost no binding of RPA protein complex (RPA70 and RPA32) onto the defined ssDNA gaps and impairment of the ssDNA-induced Chk1 phosphorylation (Figure 1—figure supplement 1A). Whereas RPA deletion did not decrease the recruitment of APE1 onto ssDNA gaps, the recruitment of endogenous ATRIP protein onto ssDNA gaps (30 nt and 80 nt) was impaired in RPA-depleted HSS (bead-bound panel and quantification panel, Figure 1—figure supplement 1A). We noted that anti-RPA antibodies co-depleted some endogenous ATRIP protein from HSS, but had almost no co-depletion of endogenous APE1 protein in HSS (Lanes 1–3 vs Lanes 4–6 in extract panel, Figure 1—figure supplement 1A). This co-depletion of endogenous ATRIP protein is similar to a previous observation by the Dunphy group (Kim et al., 2005), and may suggest a tight complex formation between endogenous ATRIP and RPA protein complex in Xenopus egg extracts. Our quantifications of ssDNA-bound ATRIP normalized to available ATRIP protein in HSS showed that RPA depletion almost had no effect on ATRIP’s binding to the 30 nt-ssDNA gap, and only mildly decreased the binding of ATRIP (~20%) to 80 nt-ssDNA gap (quantification panel, Figure 1—figure supplement 1A). These observations suggest that the recruitment of ATRIP onto ssDNA gaps is RPA-dependent and RPA-independent in the HSS system. Because ssDNA gaps have been widely accepted as a central platform for protein recruitment and activation of the ATR/Chk1 DDR pathway (Cimprich and Cortez, 2008; Maréchal and Zou, 2015), we are prompted to evaluate the role of APE1 in the ATR/Chk1 DDR pathway in cultured cells. Hydrogen peroxide-induced oxidative DNA damage triggered Chk1 phosphorylation at Ser345 and Ser317 in human osteosarcoma U2OS cells, suggesting the activation of ATR DDR (Figure 1—figure supplement 1B). Notably, siRNA-mediated APE1 knockdown of human APE1 in U2OS cells compromised the hydrogen peroxide-induced Chk1 phosphorylation, suggesting that APE1 is important for the ATR/Chk1 DDR in cultured human cells (Figure 1—figure supplement 1B). Our observation here is consistent with two prior studies showing that APE1 is important for the ATR DDR pathway activation in response to oxidative DNA damage in human cancer cells MDA-MB-231 and PANC1 cells, and ultraviolet damage in non-dividing nucleotide excision repair-deficient (i.e. XPC-/-) cells (Li et al., 2022; Vrouwe et al., 2011). Next, to determine whether APE1 plays any direct role in the RPA-dependent ATRIP recruitment onto ssDNA gaps, we tested whether His-tagged ATRIP recombinant protein can be recruited to 30 nt- or 80 nt-ssDNA gap in vitro in the absence or presence of equal moles of recombinant RPA complex. Consistent with previously reported RPA-dependent ATRIP recruitment to ssDNA (Zou and Elledge, 2003), 30/80 nt-ssDNA coated with RPA70 and RPA32 significantly enhanced the recruitment of His-ATRIP in vitro, although almost no binding of ATRIP onto ssDNA (30 nt and 80 nt) was observed in the absence of recombinant RPA complex (Figure 1C). We noticed more binding of His-ATRIP onto 80 nt-ssDNA gap compared with 30 nt-ssDNA gap (Figure 1C). As expected, the recruitment of His-ATRIP onto 30 nt- and 80 nt-ssDNA was similar to each when same amount of ssDNA gap structures was coupled to beads (Figure 1—figure supplement 1C). Furthermore, the addition of GST-APE1 but not GST protein increased the recruitment of His-ATRIP onto ssDNA with the presence of His-RPA complex in vitro (Figure 1D). It is worth noting that the presence of GST-APE1 had almost no noticeable effect on the recruitment of His-RPA70 and His-RPA32 to ssDNA gap structures (Figure 1D). Similarly, the presence of GST-APE1 but not GST increased the recruitment of endogenous ATRIP but not endogenous RPA70/RPA32 to ssDNA gap structures in the Xenopus HSS (Figure 1—figure supplement 1D). Whereas RPA itself is sufficient for ATRIP recruitment onto ssDNA in vitro, our observations here suggest that APE1 may stimulate the RPA-dependent ATRIP recruitment onto ssDNA in vitro. Alternatively, it is possible that APE1 may play an additional but direct role in the recruitment of ATRIP onto ssDNA in vitro that is independent of RPA. APE1 recognizes and binds with ssDNA directly in a length-dependent manner in vitro Although APE1 is known as a DNA repair protein to specifically recognize and process AP site, it remains unclear whether and how APE1 interacts with ssDNA. To identify the possible direct role of APE1 in ATRIP recruitment onto ssDNA, we first performed systematic analysis of APE1 association with ssDNA. Our bead-bound experiments showed that GST-APE1 but not GST was recruited onto 30 nt- and 80 nt-ssDNA gap structures in vitro (Figure 2A–B). We also determined that GST-APE1 but not GST was recruited onto beads coupled with 70 nt-ssDNA in vitro (Figure 2—figure supplement 1). Furthermore, we demonstrated that GST-APE1 but not GST was recruited to beads coupled with 40 nt-, 60 nt-, and 80 nt-ssDNA, but not 10 nt- nor 20 nt-ssDNA (Figure 2C). Furthermore, the longer ssDNA is, the more GST-APE1 is recruited (Figure 2C). Collectively, these observations suggest an APE1-ssDNA interaction in a length-dependent manner in vitro (30–80 nt) regardless the ssDNA is alone or in gapped structures. Figure 2 with 2 supplements see all Download asset Open asset AP endonuclease 1 (APE1) recognizes and binds with single-stranded DNA (ssDNA) in a length-dependent fashion in vitro. (A) Schematic diagram of APE1 functional domains and a summary of its interactions with ssDNA, ATRIP, and RPA from this study. Various symbols indicate estimates of APE1 interactions: ‘+++’, indicates the strongest interaction; ‘+’ indicates moderate interaction; ‘+/-’ indicates minimal to no interaction; ‘-’ indicates almost no interaction; ‘nd’, not determined. (B) Streptavidin beads coupled with biotin-labeled double-stranded DNA (dsDNA) with ssDNA gap structures (30 nt or 80 nt) were added to an interaction buffer containing GST or GST-APE1. After incubation for 30 min at room temperature, the DNA-bound fractions and the input were examined via immunoblotting analysis as indicated. (C) Streptavidin beads coupled with biotin-labeled ssDNA with different lengths (10 nt, 20 nt, 40 nt, 60 nt, or 80 nt) were added to an interaction buffer containing GST or GST-APE1. After incubation for 30 min at room temperature, the DNA-bound fractions and the input were examined via immunoblotting analysis. (D) Streptavidin beads coupled with biotin-labeled ssDNA (70 nt) were added to an interaction buffer containing (70 nt) GST or WT or fragment of GST-APE1. After incubation for 30 min at room temperature, the DNA-bound fractions and the input were examined via immunoblotting analysis. (E) An electrophoretic mobility shift assay (EMSA) shows the interaction between WT, AA35-316 and AA1-34 GST-APE1, and the 70 nt-ssDNA structure in vitro. Figure 2—source data 1 Raw images of immunoblotting analysis referenced in Figure 2B. https://cdn.elifesciences.org/articles/82324/elife-82324-fig2-data1-v1.zip Download elife-82324-fig2-data1-v1.zip Figure 2—source data 2 Raw images of immunoblotting analysis referenced in Figure 2C. https://cdn.elifesciences.org/articles/82324/elife-82324-fig2-data2-v1.zip Download elife-82324-fig2-data2-v1.zip Figure 2—source data 3 Raw images of immunoblotting analysis referenced in Figure 2D. https://cdn.elifesciences.org/articles/82324/elife-82324-fig2-data3-v1.zip Download elife-82324-fig2-data3-v1.zip To further dissect domain requirements within APE1 for ssDNA association, we generated a series of deletion GST-tagged APE1 and found that WT GST-APE1 and AA101-316 GST-APE1 but not any other deletion GST-APE1 tested (i.e. AA35-316, AA1-100, AA1-34, AA35-100, AA101-200) associated with beads coupled with 70 nt-ssDNA in vitro (Figure 2A and D). Intriguingly, AA101-316 but not AA35-316 GST-APE1 associated with ssDNA (Figure 2D). We speculate that the fragment of AA35-100 within APE1 may somehow inhibit the APE1-ssDNA association due to a currently unknown mechanism. In addition, our electrophoretic mobility shift assays (EMSA) revealed that WT GST-APE1 but not GST formed protein-ssDNA complex in vitro (Figure 2E). Notably, neither AA35-316 nor AA1-34 GST-APE1 formed protein-ssDNA complex in EMSA (Figure 2E). These observations suggest that AA1-34 within APE1 is required but seems not sufficient for ssDNA association at least under our tested conditions, and that APE1 AA35-316 is deficient for ssDNA association while APE1 AA101-316 is proficient in ssDNA interaction (Figure 2). What are the effects of N-terminal motif of APE1 for its 3'–5' exonuclease and AP endonuclease activities? Similar to our previous report (Lin et al., 2020), WT GST-APE1 but neither ED (E95Q-D306A) GST-APE1 nor GST displayed 3'–5' exonuclease and AP endonuclease activities (Figure 2—figure supplement 2A–B). Notably, AA101-316 GST-APE1 is defective for 3'–5' exonuclease and AP endonuclease activities (Figure 2—figure supplement 2A–B); however, AA35-316 GST-APE1 is proficient in AP endonuclease activity but deficient for 3'–5' exonuclease activity (Figure 2—figure supplement 2C–D). These observations suggest the importance of the AA1-34 motif of APE1 for its 3'–5' exonuclease activity and the AA35-100 motif within APE1 for its AP endonuclease activity. APE1 interacts and recruits ATRIP onto ssDNA in an RPA-independent manner in vitro and promotes the ATR DDR pathway in Xenopus egg extracts using a non-catalytic mechanism We next tested whether and how APE1 might interact with ATRIP directly by protein-protein interaction assays. GST pulldown assays showed that GST-APE1 but not GST directly interacted with His-ATRIP in vitro (Figure 2A and Figure 3A). Domain dissection experiments revealed that both AA35-316 GST-APE1 and AA1-100 GST-APE1 associated with His-ATRIP to the similar capacity as WT GST-APE1 (Figure 3). However, AA101-316 GST-APE1 and other fragments of APE1 tested (i.e. AA1-34, AA35-100, and AA101-200) were deficient for interaction with His-ATRIP (Figure 3A). In addition, neither of the point mutants within GST-APE1’s active sites (i.e. ED, D306A, and C92A-C98A) affected the APE1-ATRIP interaction (Figure 3B), although they are deficient for 3'–5' exonuclease as shown previously (Lin et al., 2020). Thus, our findings indicate that AA35-100 within APE1 is required but not sufficient for ATRIP interaction and AA1-100 is the minimum fragment within APE1 sufficient for ATRIP association in vitro (Figures 2A and 3A). Figure 3 with 1 supplement see all Download asset Open asset AP endonuclease 1 (APE1) interacts and recruits ATRIP onto single-stranded DNA (ssDNA) in an RPA-independent manner in vitro and promotes the ATR DNA damage response (DDR) pathway in Xenopus egg extracts using a non-catalytic function mechanism. (A–B) GST pulldown assays with GST, WT, or fragment/mutant GST-APE1 as well as His-ATRIP in an interaction buffer. The input and pulldown samples were examined via immunoblotting analysis. (C) Streptavidin beads coupled with biotin-labeled double-stranded DNA (dsDNA) with ssDNA gap structures (30 nt or 80 nt) were added to an interaction buffer containing His-ATRIP and GST/GST-tagged proteins (WT, AA101-316, or AA35-316 GST-APE1) as indicated. DNA-bound fractions and input samples were examined via immunoblotting analysis as indicated. (D) Streptavidin beads coupled with biotin-labeled dsDNA with ssDNA gap structures (30 nt or 80 nt) were added to APE1-depleted high-speed supernatant (HSS), which was supplemented with GST or GST-tagged proteins (WT, AA101-316, or ED GST-APE1) as indicated. DNA-bound fractions and total extract samples were examined via immunoblotting analysis as indicated. Figure 3—source data 1 Raw images of immunoblotting analysis referenced in Figure 3A. https://cdn.elifesciences.org/articles/82324/elife-82324-fig3-data1-v1.zip Download elife-82324-fig3-data1-v1.zip Figure 3—source data 2 Raw images of immunoblotting analysis referenced in Figure 3B. https://cdn.elifesciences.org/articles/82324/elife-82324-fig3-data2-v1.zip Download elife-82324-fig3-data2-v1.zip Figure 3—source data 3 Raw images of immunoblotting analysis referenced in Figure 3C. https://cdn.elifesciences.org/articles/82324/elife-82324-fig3-data3-v1.zip Download elife-82324-fig3-data3-v1.zip Figure 3—source data 4 Raw images of immunoblotting analysis referenced in Figure 3D. https://cdn.elifesciences.org/articles/82324/elife-82324-fig3-data4-v1.zip Download elife-82324-fig3-data4-v1.zip Based on the observation of direct APE1-ATRIP interaction (Figure 3A), we intended to test whether APE1 could recruit ATRIP onto ssDNA directly in the absence of RPA in vitro. We found that His-ATRIP protein was recruited onto 30 nt- and 80 nt-ssDNA gap structures in the presence of WT GST-APE1 but not GST (compare Lanes 4–6 and Lane 1–3 in ‘bead-bound’, Figure 3C). Due to its deficiency in ssDNA interaction (Figures 2D and 3A), AA35-316 GST-APE1 was not recruited to 30 nt- and 80 nt-ssDNA gap structures, which led to the insufficient recruitment of His-ATRIP onto ssDNA (Lanes 10–12 in ‘bead-bound’, Figure 3C). Notably, AA101-316 GST-APE1 was recruited to 30 nt- and 80 nt-ssDNA gap structures but could not recruit ATRIP to ssDNA, due to deficiency in ATRIP association (Lanes 7–9 in ‘bead-bound’, Figure 3C). These observations strongly support that APE1 interacts with ssDNA via its AA1-34 fragment and recruits ATRIP onto ssDNA via its AA1-100 in in vitro reconstitution systems, and that such APE1-mediated ATRIP recruitment onto ssDNA is independent of RPA. To test the biological significance of APE1-mediated ATRIP onto ssDNA, we performed rescue experiments in APE1-depleted HSS. WT GST-APE1 but not AA101-316 GST-APE1 rescued the recruitment of endogenous ATRIP onto 30 nt- and 80 nt-ssDNA gap structures and subsequent Chk1 phosphorylation, although endogenous RPA70 and RPA32 as well as WT/AA101-316 GST-APE1 associated with ssDNA gap structures in APE1-depleted HSS (compare Lanes