Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes

TLDR

The ATR–ATRIP kinase complex is essential for detecting replication stress and DNA damage. Our work shows that RPA‑coated single‑stranded DNA is required to recruit ATR–ATRIP to damage sites, enabling ATR‑mediated Chk1 activation and phosphorylation of Rad17, and that this mechanism is conserved in yeast where the ATRIP homolog Ddc2 is similarly recruited in an RPA‑dependent manner, underscoring RPA‑ssDNA as the key structure that initiates checkpoint signaling.

Abstract

The function of the ATR (ataxia-telangiectasia mutated– and Rad3-related)–ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11 , is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.