# The Effect of Replication Protein A Inhibition and Post-Translational Modification on ATR Kinase Signaling

## TL;DR

This study explores how inhibiting RPA and its modifications affects ATR kinase signaling, a key pathway in DNA repair and cancer therapy.

## Contribution

The study reveals the mechanism of RPA inhibitors in blocking ATR signaling and shows how RPA post-translational modifications influence this pathway.

## Key findings

- RPA inhibitors block ATR-dependent phosphorylation of downstream target proteins.
- Phosphorylation of RPA32 and TopBP1 stimulates ATR kinase activity.
- RPA70 acetylation does not affect ATR phosphorylation of target proteins.

## Abstract

The 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 towards ensuring 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, termed RPA-DBi, and RPA protein-protein interactions, termed RPA-PPIi; both activities are required for ATR activation. Here, we employ a biochemically reconstituted ATR kinase signaling pathway and demonstrate that both RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of downstream target proteins. 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 has no effect on 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.