The basic cleft of RPA70N is required for ATR-Chk1 activation. (A) Nuclear extracts from HEK293 cells expressing HA-ATRIP were incubated with GST, GST-RPA70N, or GST-RPA70N R41E R43E proteins bound to glutathione beads. Proteins bound to the beads were eluted, separated by SDS-PAGE, and immunoblotted with anti-HA antibody to detect ATRIP or stained with Coomassie blue (CB) to detect GST proteins. (B to D) HeLa cells were transfected with RPA70 siRNA or nontargeting (NT) siRNA as indicated. In addition, cells were cotransfected with expression vectors encoding siRNA-resistant wild-type RPA70 or RPA70 R41E R43E or with an empty vector control (VEC). (B) Transfected cells were left untreated (−) or UV irradiated (50 J/m²). Two hours after irradiation, cell lysates were separated by SDS-PAGE and immunoblotted with RPA70, CHK1-P-S317, and total CHK1 antibodies. Quantitations of phospho-CHK1 and CHK1 were measured by an infrared imaging system (Odyssey), and the ratios of phospho-CHK1 to CHK1 were normalized to those for the sample of cells transfected with the empty vector and RPA70 siRNA after DNA damage. (C) The percentage of successfully transfected cells in the population with an activated DNA damage response (in the absence of an added genotoxic agent) was determined by staining with the phosphopeptide-specific antibodies to the ATM substrate KAP1. At least 300 cells were scored. Error bars indicate standard errors (n = 3). (D) Transfected cells were stained with propidium iodide and analyzed by flow cytometry.

RAD9 and ATRIP compete for the same binding surface on RPA70N. (A) Nuclear extracts from HEK293 cells expressing HA-RAD9 were incubated with GST, GST-RPA70N, or GST-RPA70N R41E R43E protein bound to glutathione beads. Proteins bound to the beads were eluted, separated by SDS-PAGE, and immunoblotted with anti-HA antibody to detect RAD9 or stained with Coomassie blue (CB) to detect GST proteins. (B) Bovine serum albumin (1.6 nmol), purified ATRIP1-217 (0.8 nmol [1×] or 1.6 nmol [2×]), or purified ATRIP-crd (D58K D59K)1-217 (1.6 nmol) was added as indicated to nuclear extracts from HEK293 cells expressing HA-RAD9. The nuclear extracts were incubated with 0.8 nmol of GST-RPA70N or GST proteins bound to glutathione beads. Proteins bound to the beads were eluted, separated by SDS-PAGE, and immunoblotted with anti-HA antibody to detect RAD9 or stained with Coomassie blue to detect GST proteins.

The RAD9-RPA interaction promotes RAD9 localization to sites of DNA damage. (A and B) U2OS cells stably expressing HA-RAD9 or HA-RAD9-crd were not treated (NT) or treated with HU (10 mM for 4 h), UV (50 J/m², followed by 1 h of recovery), or IR (10 Gy, followed by 6 h recovery). Fixed cells were stained with anti-HA antibody and fluorescein isothiocyanate-conjugated secondary antibody. (A) Representative images are shown. (B) Cells were scored for RAD9 localization to DNA damage foci. Three hundred cells were scored per experiment. Error bars indicate standard errors (n = 3). P values were calculated using an unpaired, two-tailed t test. wt, wild type. (C) HA-RAD9- or HA-RAD9-crd-expressing U2OS cells were treated with low-dose IR (1 Gy) or left untreated, followed by 6 h of incubation. The number of RAD9 foci in each cell was scored. (D) The expression level of HA-RAD9 and HA-RAD9-crd in U2OS cells was analyzed by immunoblotting with anti-HA or anti-GAPDH antibody.

Simplified model of the RPA protein and DNA interactions that promote ATR signaling. RPA is a heterotrimer of three subunits, RPA70, RPA32, and RPA14, that contain six OB-fold domains. Four of these OB-fold domains can bind ssDNA in a specific orientation such that the amino-terminal RPA70 OB-fold domain (70N) is positioned near the 5′ primer-template junction. The RPA70N OB-fold domain binds checkpoint proteins, including ATRIP and RAD9. These interactions help to concentrate ATR-ATRIP and 9-1-1-TopBP1 complexes to promote TopBP1's activation of ATR. Many additional protein-protein and protein-nucleic acid interactions also participate but are not shown in this simplified diagram.

ATRIP, MRE11, and RAD9 interact with the same binding surface on RPA70N. (A) Sequence alignment of acidic peptides in RAD9, MRE11, ATRIP, and p53. (B to D) NMR ¹⁵N-¹H HSQC spectra of ¹⁵N-labeled RPA70N obtained in the absence (black) and presence (red) of ATRIP (B), RAD9 (C), or MRE11 (D) peptides. (E to G) RPA70N residues perturbed upon the addition of ATRIP (E), RAD9 (F), and MRE11 peptide (G) were mapped (in red) onto the crystal structure of RPA70N (Protein Data Bank accession no. 2B3G).

The RAD9 CRD interacts with RPA70N. (A) Diagram of the RAD9-crd protein illustrating the positions of the engineered mutations and two of the phosphorylated residues. (B) HA-wild-type RAD9 (wt) or HA-mutant RAD9-crd (crd) was expressed in HEK293 cells or rabbit reticulocyte lysates. Total cell lysates (TCL) or the in vitro transcription/translation reaction mixtures (IVT) were resolved by SDS-PAGE and immunoblotted with HA antibodies. (C) HA-wild-type RAD9 or the RAD9-crd mutant was expressed in HEK293 cells. Cells were left untreated (−) or treated with HU (10 mM), UV (50 J/m²), or IR (20 Gy). One hour after treatment, cells were harvested, lysates were resolved by SDS-PAGE, and immunoblots were probed with antibodies to RAD9 P-S272, RAD9 P-S387, or HA. The control was the lysate from untransfected cells. (D) Nuclear extracts from HEK293 cells expressing HA-RAD9 or HA-RAD9-crd (crd) and untransfected (−) cells were incubated with anti-HA agarose beads. The immunoprecipitated (IP) proteins were separated by SDS-PAGE, followed by immunoblotting with antibodies to RAD1, RAD17, TopBP1, and HA. The asterisk marks the position of an immunoglobulin G heavy-chain background band. (E and F) Nuclear extracts from HEK293 cells expressing HA-RAD9 (wt) or HA-RAD9-crd were incubated with GST-RPA70N or GST. Proteins bound to the beads were eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-HA antibody or staining with Coomassie blue to detect GST-RPA70N and GST. (F) The extracts were treated with lambda phosphatase (PPase) in the absence or presence of sodium vanadate (VO₄), as indicated, prior to incubation with GST proteins. (G) HEK293 cells expressing HA-RAD9 (wt) or HA-RAD9-crd (crd) were treated with IR (20 Gy), as indicated, followed by 2 h of incubation. Chromatin lysates were prepared and incubated with anti-HA agarose beads. The immunoprecipitated proteins were separated by SDS-PAGE, followed by immunoblotting with antibodies to HA and RPA70.

The RAD9-RPA interaction regulates DNA damage and replication stress responses. (A) Protein expression levels in two independent clones of RAD9^−/− ES cells stably expressing HA-RAD9 or HA-RAD9-crd were analyzed by immunoblotting total cell lysates. (B and C) RAD9^+/+ and RAD9^−/− cells complemented with the vector (−/−), HA-RAD9 (wt1 and wt2), or HA-RAD9-crd (crd1 and crd2) were treated with UV (50 J/m²), HU (10 mM), or left untreated as indicated. Following incubation for 1 h, lysates were prepared, resolved by SDS-PAGE, and immunoblotted with antibodies to CHK1-P-S345 or total CHK1. The phospho-CHK1 (P-CHK1) and CHK1 levels were quantitated with NIH Image software. The numbers are the ratios of phospho-CHK1 to CHK1 normalized to the ratio for the sample from cells complemented with the vector (−/−) after damage. (D and E) ES cell clones were treated with HU (10 mM for 12 h) (D) or UV (10 J/m²) (E) and grown for 7 days prior to the staining and scoring of surviving colonies. Percent viability was calculated relative to the viability of untreated cell populations. Error bars indicate standard error (n = 3). P values were calculated using an unpaired, two-tailed t test. vec, vector.