Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes

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.

Interplay between ATM and ATR in the regulation of common fragile site stability

“…RPA is a protein complex that associates with single-stranded DNA (ssDNA) and is required for the recruitment of ATR-ATRIP complex to DNA damage. Moreover, the presence of RPA on ssDNA is sufficient to recruit ATR-ATRIP complex to DNA (Zou and Elledge, 2003). Thus, RPA was used as an indication for the induction of the ATR pathway.…”

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

“…4 The potential interaction between ATM and RPA phosphorylation In this study, it has been found that ATM acts an early regulator rather than ATR-Chk1 in response to thymidine treatment-induced DNA replication stress in hTERT-NHU cells. Although RPA has already been reported to coat ssDNA and recruit ATR to trigger ATR-CHK1 checkpoint signalling pathway during replication stress (Zou and Elledge, 2003), it remains unclear that whether RPA is also involved in ATM-dependent checkpoint cascades. Here I showed that the ATM inhibitor is able to suppress RPA Ser4/8 phosphorylation directly under these conditions, suggesting that the ATM signalling pathway may participate in this phosphorylation although the deficiency of RPA Ser4/8 phosphorylation is most likely due to the failure such cells to enter S-phase.…”

“…CHK1 is largely activated in response to ssDNA formation due to occurrence of functional uncoupled DNA polymerase and MCM helicase following replication inhibition (Byun et al, 2005). This ssDNA is rapidly coated by RPA which then recruits ATR through the ATR interacting protein (ATRIP) (Zou and Elledge, 2003). The formation of this complex activates downstream CHK1 through phosphorylation at Ser345 and Ser317 (Green et al, 2000;Zhao and Piwnica-Worms, 2001).…”

“…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.…”

ERK activity facilitates activation of the S-phase DNA damage checkpoint by modulating ATR function

“…Disruption or attenuation of this response plays an essential role in promoting tumorigenesis (Lengauer et al, 1998;Hoeijmakers, 2001;Schar, 2001;Rouse and Jackson, 2002). Stalled replication forks are sensed by replication protein A (RPA)-mediated ATR (ATM-and Rad3-related kinase) recruitment to DNA damage-induced nuclear foci (Zou and Elledge, 2003). ATR and its related ATM kinases play central roles in the amplification of double-stranded break (DSB) signals, coordination of DNA damage repair and activation of cell cycle checkpoints (Zhou and Elledge, 2000;Shiloh, 2003).…”