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.

Figure 1.

Mapping the ATR–ATRIP interaction domains. (A) A library of ATR fragments fused to the Gal4 activation domain was transformed into yeast strain PJ694a containing full-length ATRIP fused to the Gal4 DNA binding domain. Cells were plated on media to select for positive-interacting fragments. Twenty plasmids containing ATR fragments that interacted with ATRIP were recovered and sequenced. A schematic diagram of where on the 2644-amino acid ATR protein these fragments are located is shown along with their starting and ending amino acid numbers. (B) A library of ATRIP fragments fused to the Gal4 activation domain was transformed into yeast strain PJ694a containing ATR amino acids 1–388 fused to the DNA binding domain of Gal4. Cells were plated on selective media, and 13 of the fragments that interacted with ATR1–388 were recovered and sequenced. In addition, two smaller fragments of ATRIP containing either amino acids 641–726 or 700–776 were tested directly for interaction with ATR1-388 in the PJ694a strain.

Figure 2.

An alternatively spliced ATRIP protein missing exon 11 is expressed in cells and cannot bind to ATR. (A) A schematic diagram of ATRIPΔ11 and wild-type ATRIP. (B) HA-ATRIP or HA-ATRIPΔ11 was stably expressed in HeLa cells. The total cell lysate (TCL) blots indicate equal expression levels. Immunoprecipitations (IP) from cell lysates were performed with either HA or ATR antibodies, separated by SDS-PAGE, and blotted with the indicated antibodies. ATRIP (long) is a longer exposure of the ATRIP blot to show the small amount of coimmunoprecipitated ATRIPΔ11 found in an ATR complex. (C) HCT116 or HeLa cells were treated with 50 J/m² of UV light and then incubated for the indicated number of hours. RNA was extracted and PCR was performed with primers specific to ATRIP or actin after reverse-transcription to generate cDNA. Cloning and sequencing confirmed the ATRIPΔ11 product.

Figure 3.

ATRIPΔ11 binds to RPA but does not localize to sites of DNA damage or replication stress efficiently. (A) Phoenix derivatives of human embryonic kidney 293 cells were transfected with retroviral vectors expressing HA-ATRIP or HA-ATRIPΔ11. Cells were lysed and incubated with beads bound to single-stranded DNA that had either been coated with recombinant RPA (+) or mock coated (-). After extensive washing, proteins bound to the beads were denatured and separated by SDS-PAGE followed by blotting with an HA antibody. Total cell lysate (TCL) is 5% of the lysate used in the pull down. (B) Indirect immunofluorescence was performed on U2OS cells stably expressing HA-ATRIP or HA-ATRIPΔ11 that had either been left untreated (none) or exposed to 8Gy of IR or 2 mM HU. Representative photographs of cells are shown. Bar, 10 μm. (C) Quantitation of HA–ATRIP or HA–ATRIPΔ11 foci formation. Cells containing five or more easily visualized foci were counted as positive. Cells were left untreated (-), exposed to HU for 5.5 h, or irradiated and then incubated for the indicated number of hours before staining. (D) Quantitation of HA–ATRIP foci formation in cells transfected with control or ATR-specific siRNA oligonucleotides. Cells were transfected with siRNA 3 d before treating with either 2 mM HU for 5.5 h or 8 Gy of IR 3 h before fixation. The inset is a Western blot using ATR antibodies to show the overall levels of ATR in the control and ATR siRNA-transfected cells. Error bars on all graphs indicate standard deviations where 100 cells were scored separately three times.

Figure 4.

ATRIPΔ11 cannot support ATR-dependent signaling. U2OS cells infected with retroviruses containing either no cDNA (vector), HA-ATRIP, or HA-ATRIPΔ11 were transfected with ATRIP siRNA (A) or nonspecific siRNA (C). The cDNAs were made immune to the ATRIP siRNA by mutations of wobble base pairs in the target sequence. Cells were either mock treated or treated with 30 J/m² of UV light and then incubated for 2 h. Cell lysates were separate by SDS-PAGE and blotted with antibodies to ATR, HA, ATRIP, Chk1, or phosphorylation-specific antibodies to Chk1 P-S345. The ATRIP blot is labeled to indicate the HA-ATRIP protein, endogenous ATRIP, and two bands that cross-react with the ATRIP antibody (^*). There is also a degradation product of the HA–ATRIP and HA–ATRIPΔ11 proteins in lanes 4–9.

Figure 5.

Interaction between ATRIP and RPA–ssDNA is required for accumulation of ATRIP–ATR into bright intranuclear foci. (A) HeLa cells were transfected with an HA–ATR expression vector and either nonspecific control or ATRIP siRNA. Indirect immunofluorescence was performed with antibodies to HA after exposing the cells to IR or HU. Cells were scored as having foci if there were five or more clearly defined foci per cell. (B) Phoenix derivatives of human embryonic kidney 293 cells were transfected with vectors expressing FLAG-ATRIP or FLAG-ATRIP missing either the first 107 amino acids (108–791) or the last 683 amino acids (1–107). These expression constructs also contained a heterologous nuclear localization signal to ensure that these proteins are localized to the nucleus. Cells were lysed and incubated with beads bound to single-stranded DNA that had either been coated with recombinant RPA (+) or mock coated (-). After extensive washing, proteins bound to the beads were denatured and separated by SDS-PAGE followed by blotting with the FLAG antibody. TCL is 5% of the lysate used in the pull down. (C) U20S cells stably expressing HA-ATRIP108-791 missing the first 107 amino acids were examined by indirect immunofluorescence by using antibodies to HA and counterstained with 4,6-diamidino-2-phenylindole to visualize the nucleus. (D) Cells expressing either NLS-HA-ATRIP or NLS-HA-ATRIP108-791, which contain a heterologous NLS, were left untreated, exposed to ionizing radiation, or treated with HU and then fixed and stained with HA antibodies followed by a FITC-conjugated secondary antibody. Representative cell images are shown.

Figure 6.

Binding to RPA–ssDNA is not essential for ATR–ATRIP-dependent Chk1 phosphorylation. U2OS cells expressing no cDNA (vector), NLS-HA-ATRIP (WT), or NLS-HA-ATRIP108-791 (108-791) were transfected with ATRIP siRNA to remove endogenous ATRIP. The wild-type cDNA was made immune to the ATRIP siRNA by mutations of wobble base pairs in the target sequence. The target sequence is completely deleted in the ATRIP108-791 mRNA. Cells were either mock treated (-) or treated with (A) 30 J/m² of UV light and then incubated for 2 h; (B) 0, 10, 30, or 60 J/m² of UV; (C) 8 Gy of IR; or (D) 1 mM HU for 0, 3, or 7 h. Cell lysates were separate by SDS-PAGE and blotted with antibodies to HA-ATRIP, Chk1, or phospho-S345-specific antibodies to Chk1. The bar graphs are quantitations of the Chk1 phosphorylation data in which the Chk1 P-S345 signal was divided by the total Chk1 signal. All values were compared with the value obtained in the damaged NLS–HA–ATRIP (WT)-expressing cells, which was set at 100%. Three independent sets of cell lines were created and analyzed in A to illustrate the variability in the assay.