APE1 recruits ATRIP to ssDNA in an RPA-dependent and -independent manner to promote the ATR DNA damage response
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eLife assessment
In the DNA damage response (DDR) pathway, single-stranded DNAs (ssDNAs) activate a downstream kinase cascade with ATR/Chk1. Replication protein A (RPA) is believed to be essential for DDR activation by recruiting an ATR-partner protein, ATRIP, to RPA-coated ssDNAs through direct protein-protein interaction. This important paper provides convincing results, showing that an AP endonuclease, APE1 (APEX1), plays a role not only in RPA-dependent but also in RPA-independent recruitment of ATRIP on ssDNAs for DDR activation.
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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.
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Author Response
Reviewer #1 (Public Review):
By the in vitro DNA damage response (DDR) assay with a defined DNA substrate using Xenopus extracts and in vitro binding assays with purified proteins, the authors nicely showed the role of APE1 (APEX1) in ATRIP recruitment for DDR activation, particularly a non-enzymatic (structural) role of APE1 in the binding to both ssDNAs and ATRIP. The results described in the paper are very convincing to support the authors' claim. However, these studies lack the quantification with proper statistics (and/or mentioning the reproducibility of the results). And, given the important discovery of APE1 in the DDR activation in vitro, it would be nice to demonstrate the role of APE1(APEX1) in ATR activation in vivo using siRNA-mediated knockdown of mammalian cells or yeast cells.
Thanks for the …
Author Response
Reviewer #1 (Public Review):
By the in vitro DNA damage response (DDR) assay with a defined DNA substrate using Xenopus extracts and in vitro binding assays with purified proteins, the authors nicely showed the role of APE1 (APEX1) in ATRIP recruitment for DDR activation, particularly a non-enzymatic (structural) role of APE1 in the binding to both ssDNAs and ATRIP. The results described in the paper are very convincing to support the authors' claim. However, these studies lack the quantification with proper statistics (and/or mentioning the reproducibility of the results). And, given the important discovery of APE1 in the DDR activation in vitro, it would be nice to demonstrate the role of APE1(APEX1) in ATR activation in vivo using siRNA-mediated knockdown of mammalian cells or yeast cells.
Thanks for the suggestion. As shown in our response to the #2 Essential Revisions, we have addressed this question by additional experiment and added extra description in our revised manuscript showing that APE1 is important for the ATR DDR following oxidative stress in culture human cell U2OS cells (Figure 1-figure supplement 1B). In addition, we have performed at least three independent experiments and statistical analysis to support our claims.
Reviewer #2 (Public Review):
ATM and Rad3-related (ATR) interact with ATRIP and plays a central role in DNA damage response. Previous studies have established the idea that ATR is recruited to RPA-covered ssDNA via ATRIP-RPA interaction. In this paper, the authors propose a new RPA-independent mechanism for ATR recruitment.
Thanks for the nice summary of our major findings from the manuscript.
Reviewer #3 (Public Review):
In this manuscript, the authors explore the mechanism of ATRIP recruitment to single-stranded DNA (ssDNA), which is important for ATR activation and the subsequent control of DNA repair and cell cycle progression. Using Xenopus egg extracts, in vitro interaction assays, and ssDNA constructs, the authors found that AP endonuclease 1 (APE1) plays a role in the recruitment of ATRIP to ssDNA independently of RPA. Moreover, APE1 domains are characterized for ssDNA, ATRIP, and RPA interaction, determining that the nuclease activities of APE1 are not required for this new mode of ATRIP recruitment. Overall, the work presented makes a compelling case for a novel role for APE1 in ATRIP recruitment that seems crucial for ATR activation (at least in the Xenopus system). The results are likely to have an important impact on our understanding of the determinants for activation of ATR signaling and cellular responses to DNA damage and replication stress. It remains unclear whether the findings will be extended to other organisms and be relevant for different types of DNA lesions. Also, there are several points of concern in the manuscript that require further clarification, especially regarding some of the quantitative analyses presented and the claimed importance of the RPA-independent mode of ATRIP recruitment for ATR activation.
We thank the reviewer’s overall positive evaluation of our initial submission. We have included additional experimental data using mammalian cells showing the significance of APE1 in the ATR DDR, and also additional discussion of other studies in the literature. We also provided further clarifications or responses to the major/minor concerns (please see below detailed responses). In particular, we revised the proposed model of APE1 in ATRIP recruitment and ATR DDR (Please see revised Figure 5).
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eLife assessment
In the DNA damage response (DDR) pathway, single-stranded DNAs (ssDNAs) activate a downstream kinase cascade with ATR/Chk1. Replication protein A (RPA) is believed to be essential for DDR activation by recruiting an ATR-partner protein, ATRIP, to RPA-coated ssDNAs through direct protein-protein interaction. This important paper provides convincing results, showing that an AP endonuclease, APE1 (APEX1), plays a role not only in RPA-dependent but also in RPA-independent recruitment of ATRIP on ssDNAs for DDR activation.
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Reviewer #1 (Public Review):
By the in vitro DNA damage response (DDR) assay with a defined DNA substrate using Xenopus extracts and in vitro binding assays with purified proteins, the authors nicely showed the role of APE1 (APEX1) in ATRIP recruitment for DDR activation, particularly a non-enzymatic (structural) role of APE1 in the binding to both ssDNAs and ATRIP. The results described in the paper are very convincing to support the authors' claim. However, these studies lack the quantification with proper statistics (and/or mentioning the reproducibility of the results). And, given the important discovery of APE1 in the DDR activation in vitro, it would be nice to demonstrate the role of APE1(APEX1) in ATR activation in vivo using siRNA-mediated knockdown of mammalian cells or yeast cells.
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Reviewer #2 (Public Review):
ATM and Rad3-related (ATR) interact with ATRIP and plays a central role in DNA damage response. Previous studies have established the idea that ATR is recruited to RPA-covered ssDNA via ATRIP-RPA interaction. In this paper, the authors propose a new RPA-independent mechanism for ATR recruitment.
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Reviewer #3 (Public Review):
In this manuscript, the authors explore the mechanism of ATRIP recruitment to single-stranded DNA (ssDNA), which is important for ATR activation and the subsequent control of DNA repair and cell cycle progression. Using Xenopus egg extracts, in vitro interaction assays, and ssDNA constructs, the authors found that AP endonuclease 1 (APE1) plays a role in the recruitment of ATRIP to ssDNA independently of RPA. Moreover, APE1 domains are characterized for ssDNA, ATRIP, and RPA interaction, determining that the nuclease activities of APE1 are not required for this new mode of ATRIP recruitment. Overall, the work presented makes a compelling case for a novel role for APE1 in ATRIP recruitment that seems crucial for ATR activation (at least in the Xenopus system). The results are likely to have an important …
Reviewer #3 (Public Review):
In this manuscript, the authors explore the mechanism of ATRIP recruitment to single-stranded DNA (ssDNA), which is important for ATR activation and the subsequent control of DNA repair and cell cycle progression. Using Xenopus egg extracts, in vitro interaction assays, and ssDNA constructs, the authors found that AP endonuclease 1 (APE1) plays a role in the recruitment of ATRIP to ssDNA independently of RPA. Moreover, APE1 domains are characterized for ssDNA, ATRIP, and RPA interaction, determining that the nuclease activities of APE1 are not required for this new mode of ATRIP recruitment. Overall, the work presented makes a compelling case for a novel role for APE1 in ATRIP recruitment that seems crucial for ATR activation (at least in the Xenopus system). The results are likely to have an important impact on our understanding of the determinants for activation of ATR signaling and cellular responses to DNA damage and replication stress. It remains unclear whether the findings will be extended to other organisms and be relevant for different types of DNA lesions. Also, there are several points of concern in the manuscript that require further clarification, especially regarding some of the quantitative analyses presented and the claimed importance of the RPA-independent mode of ATRIP recruitment for ATR activation.
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