The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling

dc.contributor.authorJordan, Matthew R.
dc.contributor.authorOakley, Greg G.
dc.contributor.authorMayo, Lindsey D.
dc.contributor.authorBalakrishnan, Lata
dc.contributor.authorTurchi, John J.
dc.contributor.departmentMedicine, School of Medicine
dc.date.accessioned2024-10-09T06:36:00Z
dc.date.available2024-10-09T06:36:00Z
dc.date.issued2024-08-26
dc.description.abstractThe ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein–protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.
dc.eprint.versionFinal published version
dc.identifier.citationJordan MR, Oakley GG, Mayo LD, Balakrishnan L, Turchi JJ. The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling. Sci Rep. 2024;14(1):19791. Published 2024 Aug 26. doi:10.1038/s41598-024-70589-y
dc.identifier.urihttps://hdl.handle.net/1805/43816
dc.language.isoen_US
dc.publisherSpringer Nature
dc.relation.isversionof10.1038/s41598-024-70589-y
dc.relation.journalScientific Reports
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 Internationalen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0
dc.sourcePMC
dc.subjectChemical biology
dc.subjectSmall molecules
dc.subjectMolecular biology
dc.subjectDNA damage and repair
dc.subjectDNA damage response
dc.titleThe effect of replication protein A inhibition and post-translational modification on ATR kinase signaling
dc.typeArticle
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