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• W2029934390 abstract "Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways. Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways. Replication protein A (RPA) 1The abbreviations used are: RPA, replication protein A; ssDNA, single-stranded DNA; DBD, DNA-binding domain; DNA-PK, DNA-dependent protein kinase; ATM, ataxia-telangiectasia-mutated; ATR, ATM/Rad3-related; ATRIP, ATR-interacting protein; FACS, fluorescence-activated cell sorter; DSB, double-strand break; CREB, cyclic AMP-response element-binding protein; SCR, scrambled; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine; PI, propidium iodide; siRNA, small-interfering RNA. 1The abbreviations used are: RPA, replication protein A; ssDNA, single-stranded DNA; DBD, DNA-binding domain; DNA-PK, DNA-dependent protein kinase; ATM, ataxia-telangiectasia-mutated; ATR, ATM/Rad3-related; ATRIP, ATR-interacting protein; FACS, fluorescence-activated cell sorter; DSB, double-strand break; CREB, cyclic AMP-response element-binding protein; SCR, scrambled; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine; PI, propidium iodide; siRNA, small-interfering RNA. is a trimeric complex composed of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication in all organisms (1Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (391) Google Scholar). RPA represents the major cellular single-stranded DNA (ssDNA) binding activity in eukaryotic cells and coats ssDNA filaments stoichiometrically in vitro (1Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (391) Google Scholar). Through its binding and stabilization of ssDNA, RPA facilitates the unwinding and destabilization of double-stranded DNA, which represents a critical step during DNA replication, recombination, and repair. The major DNA binding activity of RPA resides within the 70-kDa RPA1 subunit, which contains a centrally positioned, high affinity, bipartite DNA-binding domain (DBD) and a low affinity carboxyl-terminal DBD (1Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (391) Google Scholar, 2Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1161) Google Scholar). The RPA2 protein also contains a DBD as well as a phosphorylation site-rich amino terminus that may regulate RPA activity in response to cell cycle phase transitions and DNA damage. Kinases implicated in the phosphorylation of RPA2 include cyclin-dependent kinases, and members of the PI 3-kinase-related kinase superfamily, including DNA-dependent protein kinase (DNA-PK), ataxia-telangiectasia-mutated (ATM), and ATM/Rad3-related (ATR) (3Liu V.F. Weaver D.T. Mol. Cell. Biol. 1993; 13: 7222-7231Crossref PubMed Scopus (186) Google Scholar, 4Barr S.M. Leung C.G. Chang E.E. Cimprich K.A. Curr. Biol. 2003; 13: 1047-1051Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Shao R.G. Cao C.X. Zhang H. Kohn K.W. Wold M.S. Pommier Y. EMBO J. 1999; 18: 1397-1406Crossref PubMed Scopus (302) Google Scholar, 6Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (220) Google Scholar, 7Niu H. Erdjument-Bromage H. Pan Z.Q. Lee S.H. Tempst P. Hurwitz J. J. Biol. Chem. 1997; 272: 12634-12641Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). DNA-PK, ATM, and ATR are serine/threonine-glutamine (Ser/Thr-Gln)-directed kinases with overlapping substrate specificities that regulate DNA repair, apoptosis, and cell cycle checkpoint responses to genotoxic stimuli (8Smith G.C.M. Jackson S.P. Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (764) Google Scholar, 9Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1648) Google Scholar). At least two Ser/Thr-Gln residues of RPA2 (Thr-21 and Ser-33) are phosphorylated by DNA-PK, and most likely ATM and ATR, in vitro and within intact cells in response to DNA damage (4Barr S.M. Leung C.G. Chang E.E. Cimprich K.A. Curr. Biol. 2003; 13: 1047-1051Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Niu H. Erdjument-Bromage H. Pan Z.Q. Lee S.H. Tempst P. Hurwitz J. J. Biol. Chem. 1997; 272: 12634-12641Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Gately D.P. Hittle J.C. Chan G.K. Yen T.J. Mol. Biol. Cell. 1998; 9: 2361-2374Crossref PubMed Scopus (163) Google Scholar, 11Zernik-Kobak M. Vasunia K. Connelly M. Anderson C.W. Dixon K. J. Biol. Chem. 1997; 272: 23896-23904Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The biochemical functions of individual RPA2 Ser/Thr-Gln sites are not well understood. However, DNA damage-induced phosphorylation of RPA2 correlates with decreased binding of RPA to p53 (12Abramova N.A. Russell J. Botchan M. Li R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7186-7191Crossref PubMed Scopus (94) Google Scholar, 13Liu J.S. Kuo S.R. McHugh M.M. Beerman T.A. Melendy T. J. Biol. Chem. 2000; 275: 1391-1397Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), suggesting that modulation of protein-protein interactions represents one functional end point. In addition to its role in the maintenance and processing of ssDNA, RPA has been implicated as a regulator of DNA damage-induced cell cycle checkpoints. Hypomorphic mutations in RPA1 are associated with hypersensitivity to DNA-damaging agents and cell cycle checkpoint defects in budding and fission yeasts (14Parker A.E. Clyne R.K. Carr A.M. Kelly T.J. Mol. Cell. Biol. 1997; 17: 2381-2390Crossref PubMed Scopus (41) Google Scholar, 15Longhese M.P. Neecke H. Paciotti V. Lucchini G. Plevani P. Nucleic Acids Res. 1996; 24: 3533-3537Crossref PubMed Scopus (80) Google Scholar). Immunodepletion of RPA from Xenopus oocytes abrogates an aphidicolin-induced DNA replication checkpoint, which functions to suppress mitosis in the presence of incompletely replicated DNA (16You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Using the Xenopus system it was also shown that RPA is required for the suppression of DNA synthesis in response to DNA strand breaks (17Costanzo V. Shechter D. Lupardus P.J. Cimprich K.A. Gottesman M. Gautier J. Mol. Cell. 2003; 11: 203-213Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The requirement of RPA for checkpoint activation in Xenopus extracts mirrors that for ATR (17Costanzo V. Shechter D. Lupardus P.J. Cimprich K.A. Gottesman M. Gautier J. Mol. Cell. 2003; 11: 203-213Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 18Hekmat-Nejad M. You Z.S. Yee M.C. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 19Guo Z.J. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (360) Google Scholar), implying a functional interaction between these two proteins during the initiation of checkpoint signals. Consistent with this notion, RPA is required for the association of ATR with chromatin in the Xenopus system, suggesting that RPA recruits ATR, either directly or indirectly, to sites of genetic damage (16You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Mammalian RPA was found to promote the chromatin association of ATR in vitro via the ATR-interacting protein, ATRIP (21Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2000) Google Scholar). Furthermore, in that study it was concluded that RPA is required for the ATR-mediated phosphorylation of its effector kinase, CHK1, after exposure to hydroxyurea or UV light. However, ATR also contains an intrinsic, RPA-independent, DNA binding activity (21Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2000) Google Scholar, 22Unsal-Kacmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (80) Google Scholar, 23Bomgarden R.D. Yean D. Yee M.C. Cimprich K.A. J. Biol. Chem. 2004; 279: 13346-13353Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and the quantitative requirement of RPA for ATR activation in vivo has not been established. In addition, although RPA has been extensively studied in vitro, the cellular consequences of RPA functional deficiency have yet to be explored in mammalian cells. In this report we have explored the genetic requirement of RPA for DNA replication, survival, and cell cycle checkpoint function in human cells. We demonstrate that RPA deficiency causes spontaneous DNA damage, apoptosis, and the induction of an ATM-dependent G2/M checkpoint. Surprisingly, we found that neither the RPA1 nor RPA2 subunits were absolutely required for ATR-dependent substrate phosphorylation in DNA-damaged HEK 293T cells. Our findings demonstrate a requirement for RPA in the maintenance of genomic integrity and suggest that ATR can be activated independently of RPA in response to genotoxic stress. Cell Culture and Antisera—HEK 293T and HeLa cells were maintained in Eagle's minimum essential medium containing 10% FCS. Antibody suppliers included: GeneTex (α-ATM), Oncogene Research (α-RPA1, α-RPA2, and α-BrdUrd), Santa Cruz Biotechnology (α-CHK1 (G-4)), Upstate Biotechnology (α-tubulin and α-pH2AX-139), Cell Signaling (α-CREB), Affinity Bioreagents (α-ATR), and R&D Systems (α-pATM-1981 and α-pCHK1–317). The α-pCREB-121 antibody has been published previously (24Shi Y. Venkataraman S.L. Dodson G.E. Mabb A.M. LeBlanc S.L. Tibbetts R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5898-5903Crossref PubMed Scopus (83) Google Scholar). Transfections and Protein Analysis—Transfection-ready small-interfering RNA (siRNA) duplexes were purchased from Dharmacon Research. siRNAs used in this study included: RPA1 (5′-AACUGGUUGACGAAAGUGGUG-3′), ATR (5′-AACCCGCGUUGGCGUGGUUGA-3′), RPA2, and ATM. The RPA2 and ATM siRNAs represented mixtures of four distinct RNA duplexes (SmartPool, Dharmacon). Three micrograms of siRNA was used for each transfection using the calcium phosphate DNA precipitation procedure. Cells were harvested 48–72 h later and extracts prepared as described (24Shi Y. Venkataraman S.L. Dodson G.E. Mabb A.M. LeBlanc S.L. Tibbetts R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5898-5903Crossref PubMed Scopus (83) Google Scholar). Seventy-five μg of total protein was separated on 10% SDS-PAGE gels and transferred to Immobilon PVDF membranes (Millipore). Membranes were blocked in Tris-buffered saline containing 0.2% Tween 20 (TBS-T) and 5% dried milk and incubated overnight at 4 °C with the indicated primary antibodies diluted in blocking solution. After washing, the blots were incubated with HRP-conjugated sheep anti-mouse or goat anti-rabbit secondary antibodies (Jackson ImmunoResearch) and developed using SuperSignal chemilumiscent substrate (Pierce). Immunofluorescence Microscopy—For the phospho-H2AX analysis, HeLa cells were transfected with scrambled control (SCR) or RPA1 siRNAs and fixed 72 h later in 4% paraformaldehyde at room temperature. The cells were then permeabilized in PBS, 0.2% Triton X-100 (PBS-T) for 10 min, washed once with PBS, and blocked for 30 min in PBS containing 3% BSA and 2% goat serum. The cells were then incubated overnight at 4 °C with 2 μg/ml α-phospho-H2AX antibody diluted in blocking solution. The cells were washed three times in PBS-T and incubated for 1 h at room temperature with 0.4 μg/ml FITC-conjugated goat anti-mouse IgG (Caltag). The cells were washed twice with PBS-T, once in PBS, and mounted using Vectashield containing DAPI (Vector Laboratories). A Carl Zeiss Axiovert 200 fluorescence microscrope was used to visualize all samples. Cell Cycle Analysis and Viability Assays—DNA synthesis was measured using a bromodeoxyuridine (BrdUrd) incorporation assay. HeLa cells were transfected with SCR or RPA1 siRNAs and then pulse-labeled 48 h later with 10 μm BrdUrd for 30 min. The cells were then cultured in BrdUrd-free medium and harvested at 4-h intervals over a 24-h period. Following fixation with ice-cold 70% ethanol, the cells were processed for immunostaining with α-BrdUrd as described (25Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar). The cells were subsequently stained with propidium iodide (PI) and analyzed by two-parameter flow cytometry. Profiles of the BrdUrd- and PI-stained populations were plotted using the WinMDI shareware package (Stanford University). Cell viability was directly measured via trypan blue uptake. Following trypsinization, cells were diluted 1:1 in trypan blue solution (Sigma) and examined under a microscope using a hemacytometer. For each treatment, 200 total cells were scored for trypan blue uptake, and the percent viability was calculated by dividing the number of trypan blue-positive cells by the total cell number. Phenotypic Characterization of RPA1-deficient Cells—We used an RPA1-specific siRNA to suppress RPA1 expression in HEK 293T or HeLa cells. Immunoblotting confirmed that the expression of RPA1 was reduced by more than 90% in both cell lines within 48 h of transfection, whereas the expression of RPA2 was unaffected (Fig. 1A). Transfection of a scrambled (SCR) siRNA had no effect on RPA1 expression. To examine the impact of RPA1 dosage suppression on DNA synthesis and cell cycle progression, we transfected HeLa cells with SCR or RPA1 siRNAs and then pulse-labeled the cells 48 h later with BrdUrd for 30 min. The cells were then incubated in BrdUrd-free medium and harvested at 4-h intervals for staining with α-BrdUrd and PI. FACS analysis of the stained cell populations revealed that RPA1-deficient cells incorporated BrdUrd at a reduced level in comparison with the control cells (Fig. 1B, zero time point). In addition to an acute DNA synthesis defect, RPA1-deficient cells also displayed abnormal S phase progression. By 8 h after BrdUrd pulse labeling, virtually all of the control cells accumulated 4 n DNA content, indicating the completion of S phase. In contrast, 60% of RPA1-deficient cells displayed a DNA content intermediate between 2 and 4 n at this time point. By 16 h post-labeling, the difference between the control and RPA1-deficient cell populations was even more pronounced. At this time point, 79% of the BrdUrd-positive control cells, but only 7% of RPA1-deficient cells, had completed mitosis and re-entered G1 phase. The majority of the RPA1-deficient cells retained a 4 n DNA content 16 h after BrdUrd pulse labeling, suggesting that the cells had arrested in G2/M phase. The G2/M arrest profile of RPA1-deficient HeLa cells was further examined over a 96-h time frame. HeLa cells were transfected with SCR or RPA1 siRNAs and then stained with PI either 48 or 96 h later to measure DNA content by flow cytometry. The cell cycle profiles of HeLa cells transfected with SCR or RPA1 siRNAs were comparable at 48 h post-transfection. However, by 96 h post-transfection, the RPA1-deficient cells displayed a robust G2/M arrest phenotype (Fig. 1C). In addition, a substantial fraction (60–70%) of the cells detached from the tissue culture dish and underwent apoptosis, as assessed by trypan blue staining and morphologic analysis (Fig. 1D). The remaining attached cells exhibited an abnormal, elongated morphology, possibly indicative of terminal G2/M arrest (see supplemental Fig. 1). In sum, we find that RPA1 functional deficiency causes a slowing of S phase, terminal G2/M arrest, and apoptosis in HeLa cells. RPA1 Deficiency Causes Spontaneous DNA Damage and Activates ATM—Based on the above findings, we hypothesized that insufficient levels of RPA1 resulted in the generation of unrepaired DNA damage during S phase, with subsequent activation of a G2/M cell cycle checkpoint. To test this hypothesis we first examined whether RPA1-deficient cells displayed increased levels of DNA damage. HeLa cells were transfected with SCR or RPA1 siRNAs and stained 72 h later with an antibody that recognizes phosphorylated histone γ-H2AX, a specific marker of DNA double-strand breaks (DSBs) (26Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (1931) Google Scholar). Control cells displayed only weak phospho-H2AX immunoreactivity, with less than 5% of cells displaying brightly stained phospho-H2AX nuclear foci that are characteristic of cells containing DSBs (Fig. 2A and data not shown). In contrast, 70% of RPA1 siRNA-transfected HeLa cells displayed intense phospho-H2AX staining (Fig. 2A). As noted above, the RPA1-deficient cells were also morphologically distinct from the controls, with most of the cells displaying an elongated nucleus. From these findings we conclude that RPA1 deficiency causes spontaneous DNA damage, which activates a terminal G2/M checkpoint in HeLa cells. It is well established that ATM is required for optimal G2/M checkpoint induction in response to IR and other agents that induce DSBs (27Beamish H. Williams R. Chen P. Lavin M.F. J. Biol. Chem. 1996; 271: 20486-20493Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 28Xu B. Kim S.T. Lim D.S. Kastan M.B. Mol. Cell. Biol. 2002; 22: 1049-1059Crossref PubMed Scopus (410) Google Scholar). To ascertain whether the G2/M arrest observed in RPA1-deficient HeLa cells was ATM-dependent, we transfected HeLa cells with RPA1 and ATM siRNAs and then examined the cell cycle profiles by PI staining 72 h later. Consistent with the earlier experiments, transfection of RPA1 siRNA alone caused a pronounced G2/M arrest, whereas transfection of an ATM siRNA did not substantially affect the HeLa cell cycle (Fig. 2B). Cells that were co-transfected with RPA1 and ATM siRNAs displayed an attenuated G2/M arrest phenotype relative to cells that were transfected with RPA1 siRNA alone (Fig. 2B), even though the RPA1 expression levels were comparably reduced in both cell populations (data not shown). This finding indicates that ATM contributes to the G2/M arrest observed in RPA1-deficient cells. However, the finding that the G2/M arrest was not completely abolished suggests that ATM-independent mechanisms may also contribute to checkpoint activation in response to RPA functional insufficiency. The above finding implied that ATM is activated as a consequence of RPA1 deficiency. To test this possibility, we measured the activation status of ATM in HeLa cells 48 or 72 h after transfection with SCR or RPA1 siRNAs. Activation of ATM was assessed using an antibody (α-pATM-1981) that recognizes an activation-specific autophosphorylation site at Ser-1981 (29Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2635) Google Scholar). This analysis revealed that ATM was autophosphorylated on Ser-1981 following transfection with RPA siRNA (Fig. 2C). Although autophosphorylation was detectable by 48 h, it was much more pronounced at 72 h post-transfection, which is consistent with the time course of DSB induction shown in Fig. 2A. The level of ATM autophosphorylation was about 3-fold less than that induced by 10 Gy of IR. From the combined findings, we conclude that prolonged RPA1 deficiency induces spontaneous DNA damage, activates ATM, and induces an ATM-dependent G2/M cell cycle checkpoint in HeLa cells. Effects of RPA siRNA on ATM and ATR Activation Following DNA Damage—We next sought to determine the relative requirement of RPA for the activation of ATM and ATR following DNA damage. The observation that ATM became autophosphorylated during prolonged RPA1 deficiency suggested that ATM activation does not require RPA. We formally tested this possibility and found that RPA1 siRNA had no effect on the autophosphorylation of ATM on Ser-1981 following exposure to 5 Gy of IR, validating that RPA is not required for the activation of ATM in response to this stimulus (Fig. 3). To determine the requirement of RPA for ATR activation, we examined the effects of siRNAs specific for RPA1 or RPA2 on the ATR-dependent phosphorylation of CHK1 on Ser-317 (30Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (191) Google Scholar, 31Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (845) Google Scholar). For comparative purposes, an ATR siRNA was employed as a side-by-side control. Interestingly, during the course of these experiments, we found that an RPA2 siRNA caused the coordinate down-regulation of RPA1, but not vice versa (Fig. 4A). The basis for this phenomenon is not known, but it was observed using several RPA2 siRNAs and may reflect a role for RPA2 as an RPA1 stability determinant. Immunoblot analysis using a CHK1 Ser-317 phospho-specific antibody revealed that neither RPA2 nor RPA1 siRNAs substantially inhibited the phosphorylation of CHK1 on Ser-317 following exposure of HEK 293T cells to hydroxyurea (3 mm), UV light (25 J/m2), or IR (10 Gy) (Fig. 4B). This lack of effect was probably not due to incomplete RPA1 or RPA2 suppression, as the levels of both proteins were drastically reduced 48 h after siRNA transfection (Fig. 4A). In the same experiment, an ATR siRNA suppressed CHK1 phosphorylation in response to all three stimuli (Fig. 4B). This experiment suggests that CHK1 phosphorylation on Ser-317 in response to hydroxyurea, UV light, and IR is ATR-dependent but RPA-independent. The requirement of RPA for the DNA damage-induced phosphorylation of other ATR substrates was also explored. The cyclic AMP-response element-binding protein (CREB) is a transcription factor that is phosphorylated by ATM in vivo on Ser-121 in response to IR (24Shi Y. Venkataraman S.L. Dodson G.E. Mabb A.M. LeBlanc S.L. Tibbetts R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5898-5903Crossref PubMed Scopus (83) Google Scholar). We have also shown that both ATM and ATR phosphorylate CREB on Ser-121 in vitro (Ref. 24Shi Y. Venkataraman S.L. Dodson G.E. Mabb A.M. LeBlanc S.L. Tibbetts R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5898-5903Crossref PubMed Scopus (83) Google Scholar and data not shown). In support of a role for ATR as a mediator of CREB phosphorylation in vivo, we found that an ATR siRNA abolished the hydroxyurea- and UV-induced phosphorylation of the Ser-121 residue (Fig. 4C). In contrast, transfection with RPA1 or RPA2 siRNAs had no effect on CREB phosphorylation in response to either stimulus. Thus, like CHK1, the UV- and hydroxyurea-induced phosphorylation of CREB is ATR-dependent but RPA-independent. Finally, in this experiment we also observed that the IR-induced phosphorylation of CREB was unaffected by an ATR siRNA, which contrasts with the CHK1 result (compare Fig. 4, B and C). However, this finding is consistent with our previous report demonstrating that IR-induced phosphorylation of CREB is ATM-dependent (24Shi Y. Venkataraman S.L. Dodson G.E. Mabb A.M. LeBlanc S.L. Tibbetts R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5898-5903Crossref PubMed Scopus (83) Google Scholar). In this study we have demonstrated that RPA is required for genomic integrity in mammalian cells; RPA insufficiency causes spontaneous DSBs, ATM-dependent G2/M arrest, and apoptotic cell death. The mechanism of DNA damage induction following RPA suppression is unknown but most likely involves the execution of an aberrant DNA replication cycle after RPA levels have declined below a critical threshold level. Consistent with this possibility, DSB induction and ATM activation were observed 72–96 h after RPA1 siRNA transfection, which is 24–48 h after RPA1 expression reached its minimum. It is possible that DSB induction reflects a structural role for RPA in protecting ssDNA filaments from spontaneous strand breakage. A non-exclusive possibility is that, in the prolonged absence of RPA, ssDNA present at replication forks is converted into DSBs through failed cycles of recombination. RPA functionally interacts with the enzymatic machinery of recombination, including RAD51, RAD52, and BLM, a helicase that contributes to the resolution of recombination intermediates present at stalled replication forks (32Brosh Jr., R.M. Li J.L. Kenny M.K. Karow J.K. Cooper M.P. Kureekattil R.P. Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 33Golub E.I. Gupta R.C. Haaf T. Wold M.S. Radding C.M. Nucleic Acids Res. 1998; 26: 5388-5393Crossref PubMed Scopus (158) Google Scholar, 34Hays S.L. Firmenich A.A. Massey P. Banerjee R. Berg P. Mol. Cell. Biol. 1998; 18: 4400-4406Crossref PubMed Scopus (121) Google Scholar, 35Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (862) Google Scholar). It is therefore conceivable that DSBs arise in RPA-deficient cells secondarily to gross DNA recombination abnormalities. In our hands an RPA1 siRNA did not substantially inhibit the IR-induced autophosphorylation of ATM nor did RPA1 or RPA2 siRNAs suppress ATR-dependent substrate phosphorylation in response to hydroxyurea or UV light. The RPA independence of ATR activation was somewhat surprising given several recent reports (16You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 21Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2000) Google Scholar, 36Wang Y. Qin J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15387-15392Crossref PubMed Scopus (148) Google Scholar) implicating RPA as an upstream regulator of the ATR pathway in Xenopus and mammalian cells. Nevertheless, we failed to observe inhibitory effects of RPA1 or RPA2 siRNAs on the hydroxyurea- or UV-induced phosphorylation of three ATR substrates: CHK1, CREB, and RAD17 (Fig. 4). 2G. E. Dodson, Y. Shi, and R. S. Tibbetts, unpublished results. In these same experiments, an ATR siRNA strongly inhibited substrate phosphorylation, even though the ATR siRNA was less efficacious than either the RPA1 or RPA2 siRNAs (Fig. 4). Because of inherent caveats associated with RNAi-based experiments, we cannot rule out the possibility that a low threshold level of RPA was sufficient to activate ATR in our experiments. It is also formally possible that ATM contributes to UV- and hydroxyurea-induced phosphorylation of CHK1 in the absence of RPA. However, the findings are most consistent with the conclusion that a reduction in RPA dosage does not cause a corresponding linear decrease in ATR activity, as assessed by phosphorylation of CHK1 on Ser-317 and CREB on Ser-121. Additional studies should clarify the role of RPA in the activation of ATR in response to genotoxic stress. We thank Dr. Kathryn Brumbaugh (R&D Systems) for providing the CHK1 phospho-Ser-317 antibody and Dr. Shigeki Miyamoto and Dr. David Wassarman for helpful comments regarding the manuscript. Download .pdf (.09 MB) Help with pdf files" @default.
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