Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins

Abstract

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.ATR (ATM and Rad3-related) kinase is a protein kinase that coordinates cellular responses to genotoxic stress. ATR activation occurs primarily in S phase due to replication stress induced by DNA-damaging agents or replication inhibitors. More specifically, ATR activation is stimulated when the replication machinery encounters a DNA lesion and becomes uncoupled (the helicase continues to unwind DNA while the polymerase becomes stalled at the site of DNA damage) (9).The critical factor that promotes ATR activation is believed to be the accumulation of RPA (replication protein A)-coated single-stranded DNA (ssDNA) (11,33,43). At least two separate checkpoint complexes accumulate in distinct foci that colocalize with RPA. Rad17, a PCNA-like clamp loader protein, is recruited to RPA-ssDNA and loads the Rad9-Rad1-Hus1 checkpoint clamp at the junction of double-stranded and single-stranded DNA (4,14,53). Independently, ATR is recruited by ATR-interacting protein (ATRIP), which binds the RPA-ssDNA that accumulates at DNA lesions (3,15,37,52).ATRIP is required for ATR function, and mutation of either ATR or ATRIP causes the same phenotypes (3, 12). The strict requirement for ATRIP is conserved in Schizosaccharomyces pombe (Rad3 and Rad26), Saccharomyces cerevisiae (Mec1 and Ddc2/Lcd1/Pie1), and Xenopus laevis (xATR and xATRIP) (13,38,41,51). An N-terminal domain of ATRIP binds RPAssDNA and is necessary for stable ATR-ATRIP localization to damage-induced nuclear foci (3, 25).The ATR signaling pathway is currently viewed as an important target for the development of cancer therapies (10,22,24,32,34). However, the mechanism by which ATR is activated remains unclear. Localization to sites of DNA damage or replication stress has been suggested to be essential and perhaps sufficient to promote ATR signaling. However, mutations in ATRIP that disrupt the stable RPA-A...

Reconstitution of a human ATR-mediated checkpoint response to damaged DNA

“…1C). We found that in reaction mixtures that were of low ionic strength, ATR phosphorylated Chk1 in a manner virtually dependent on TopBP1, which is in agreement with previous reports (14,20) (Fig. 2, lane 3).…”

Human DNA Helicase B (HDHB) Binds to Replication Protein A and Facilitates Cellular Recovery from Replication Stress

“…4 and 5) are consistent with a role for RPA70N-HDHB interaction in recruiting HDHB to chromatin. Although technical difficulties have so far confounded our efforts to directly demonstrate the importance of these motifs in recruiting HDHB to chromatin, the interaction closely resembles that of RPA70N with ATRIP, Rad9, and Mre11 (28,33,50). Qualitatively, acidic peptides from each of these proteins have been shown to bind specifically to the same surface of RPA70N that binds the acidic peptide from HDHB (33).…”

“…Plasmids used for bacterial expression of His-tagged RPA truncation mutants were: pET15b-RPA70 (aa 1-120) for RPA70N (28) from Dr. C. Arrowsmith, pET15b-RPA32 (aa 172-270) for RPA32C (29), pET11d-RPA70 (aa 1-168) for RPA70NϩL (30) from Dr. M. Wold, pET15b-RPA70 (aa 181-422) for RPA70AB (31), and pET15b-RPA70 (aa 436 -616/32 (aa 43-171)/14) for RPA70C/ 32D/14 (32), both from Dr. A. Bochkarev.…”

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.…”