ATRIP Binding to Replication Protein A-Single-stranded DNA Promotes ATR–ATRIP Localization but Is Dispensable for Chk1 Phosphorylation

Abstract

ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…Recent findings alluded to above would seem to support this idea, however, alternative pathways may exist. Mutant human and Xenopus ATRIP proteins lacking an N-terminal region required for interaction with RPA and stable DNA binding nevertheless support ATR-mediated phosphorylation of Chk1 in response to DNA damage (Ball et al, 2005;Kim et al, 2005), and TopBP1 has been postulated to be a direct activator of ATR via specific protein-protein interactions (Kumagai et al, 2006). Understanding why O 6 -meG poses a block to gap repair in the MMR pathway is an important unanswered question as it does not appear to pose a block to replication per se (Mojas et al, 2007).…”

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

“…For instance, RPA was reported to play a necessary role for chromatin association with ATR following DNA strand breaks (You et al, 2002). Intriguingly, RPA was also found to act as a sensor for ATR recruitment at DNA damage sites and further trigger downstream Chk1 activation (Ball et al, 2005;Zou and Elledge, 2003). In addition, RPA has been reported to be associated with cancers.…”

The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

“…The single-stranded DNA is then coated by replication protein A (RPA) (Walter and Newport 2000;Byun et al 2005), which signals the independent recruitment of two checkpoint complexes: the ataxia-telangiectasia mutated (ATM)-Rad3-related kinase-ATR-interacting protein (ATR-ATRIP) complex and the Rad9-Hus1-Rad1 (9-1-1) complex. The ATRIP-ATR complex is bound to DNA by a direct interaction between ATRIP and RPA Ball et al 2005Ball et al , 2007Kim et al 2005;Namiki and Zou 2006). In contrast, loading of the 9-1-1 complex requires several steps.…”