Abstract
ATR is a master regulator of cell response to replication stress. Adequate activation of ATR is essential for preventing genome aberrance induced by replication defect. However, the mechanism underlying ATR activation is not fully understood. Here, we identify that RBMX is an ssDNA binding protein that orchestrates a novel pathway to activate ATR. Using super-resolution STORM, we observe that RBMX and RPA bind to adjacent but nonoverlapping sites on ssDNA in response to replication stress. RBMX then binds to and facilitates positioning of TopBP1, which activates nearby ATR associated with RPA. In addition, ATR activation by ssDNA-RBMX-TopBP1 is independent of ssDNA–dsDNA junction and 9-1-1 complex. ChIP-seq analysis reveals that RBMX/RPA are highly enriched on repetitive DNAs, which are considered as fragile sites with high replication stress. RBMX depletion leads to defective localization of TopBP1 to replication stressed sites and inadequate activation of ATR. Furthermore, cells with deficient RBMX demonstrate replication defect, leading to formation of micronuclei and a high rate of sister-chromatin exchange, indicative of genome instability. Together, the results identify a new ssDNA-RBMX-TopBP1 pathway that is specifically required for activation of ATR on repetitive DNAs. Therefore, RBMX is a key factor to ensure genome stability during replication.
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Data availability
The raw ChIP-Seq data of RBMX and RPA1 have been submitted to GEO database. The GEO accession number is GSE134980.
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Acknowledgements
We thank all the members in Zhao’s laboratory for insightful scientific discussion. We thank Dr. Zhou Songyang at the School of Life Sciences, Sun Yat-sen University for providing plasmids of Bac-to-Bac system and Dr. Wenqing Zhang for providing Sf9 cells.
Funding
This work was supported by National Natural Science Foundation of China Grants [31970683, 81771506]; National Key R&D Program of China [2018YFA0107000]; Guangzhou Municipal People’s Livelihood Science and Technology Plan [201803010108].
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TZ performed most of the experiments. HZ performed protein purification and EMSA experiments. XL performed the STORM experiments. YY, DP, YZ, and JR analyzed the ChIP-seq data. TZ and YZ wrote the paper. TZ, HL, and YZ designed the experiments. YZ supervised the project.
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Zheng, T., Zhou, H., Li, X. et al. RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability. Cell Death Differ 27, 3162–3176 (2020). https://doi.org/10.1038/s41418-020-0570-8
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DOI: https://doi.org/10.1038/s41418-020-0570-8
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