Direct binding to RPA-coated ssDNA allows recruitment of the ATR activator TopBP1 to sites of DNA damage

Abstract

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation, however the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated ssDNA. We identify a point mutant that abrogates this interaction, and show that this mutant fails to accumulate at sites of DNA damage, and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

TopBP1 assembles nuclear condensates to switch on ATR signaling

“…ATR/Chk1 signalling ensures cell and organismal survival through coordination of DNA repair and DNA replication with physiological processes, including cell cycle progression and transcription (Saldivar et al, 2017). Studies using Xenopus egg extracts have largely contributed to defining the orchestrated set of events leading to ATR activation (Acevedo et al, 2016;Byun et al, 2005;Duursma et al, 2013;Kumagai et al, 2006; Van et al, 2010). ATR is recruited to DNA lesions and replication intermediates via the ATR interacting protein ATRIP, which binds RPA-covered single-stranded DNA (Zou and Elledge, 2003).…”