The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
26 08 2024
26 08 2024
Historique:
received:
12
06
2024
accepted:
19
08
2024
medline:
27
8
2024
pubmed:
27
8
2024
entrez:
26
8
2024
Statut:
epublish
Résumé
The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein-protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.
Identifiants
pubmed: 39187637
doi: 10.1038/s41598-024-70589-y
pii: 10.1038/s41598-024-70589-y
doi:
Substances chimiques
Replication Protein A
0
Ataxia Telangiectasia Mutated Proteins
EC 2.7.11.1
ATR protein, human
EC 2.7.11.1
DNA, Single-Stranded
0
DNA-Binding Proteins
0
TOPBP1 protein, human
0
Carrier Proteins
0
Nuclear Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
19791Subventions
Organisme : National Science Foundation
ID : 1929346
Organisme : American Cancer Society
ID : RSG-21-028-01PMC
Organisme : NIH HHS
ID : CA257430
Pays : United States
Informations de copyright
© 2024. The Author(s).
Références
Kastan, M. B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).
pubmed: 15549093
doi: 10.1038/nature03097
Byun, T. S., Pacek, M., Yee, M., Walter, J. C. & Cimprich, K. A. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19, 1040–1052 (2005).
pubmed: 15833913
pmcid: 1091739
doi: 10.1101/gad.1301205
Bhat, K. P. & Cortez, D. RPA and RAD51: Fork reversal, fork protection, and genome stability. Nat. Struct. Mol. Biol. 25, 446–453 (2018).
pubmed: 29807999
pmcid: 6006513
doi: 10.1038/s41594-018-0075-z
Cybulla, E. & Vindigni, A. Leveraging the replication stress response to optimize cancer therapy. Nat. Rev. Cancer 23, 6–24 (2023).
pubmed: 36323800
doi: 10.1038/s41568-022-00518-6
Saldivar, J. C., Cortez, D. & Cimprich, K. A. The essential kinase ATR: Ensuring faithful duplication of a challenging genome. Nat. Rev. Mol. Cell Biol. 18, 622–636 (2017).
pubmed: 28811666
pmcid: 5796526
doi: 10.1038/nrm.2017.67
Reaper, P. M. et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat. Chem. Biol. 7, 428–430 (2011).
pubmed: 21490603
doi: 10.1038/nchembio.573
Charrier, J.-D. et al. Discovery of potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase as potential anticancer agents. J. Med. Chem. 54, 2320–2330 (2011).
pubmed: 21413798
doi: 10.1021/jm101488z
Yano, K. & Shiotani, B. Emerging strategies for cancer therapy by ATR inhibitors. Cancer Sci. 114, 2709–2721 (2023).
pubmed: 37189251
pmcid: 10323102
doi: 10.1111/cas.15845
Cong, K. & Cantor, S. B. Exploiting replication gaps for cancer therapy. Mol. Cell 82, 2363–2369 (2022).
pubmed: 35568026
pmcid: 9271608
doi: 10.1016/j.molcel.2022.04.023
Paes Dias, M. et al. Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps. Mol. Cell 81, 4692–4708 (2021).
pubmed: 34555355
doi: 10.1016/j.molcel.2021.09.005
Cong, K. et al. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Mol. Cell 81, 3128-3144.e7 (2021).
pubmed: 34216544
pmcid: 9089372
doi: 10.1016/j.molcel.2021.06.011
Panzarino, N. J. et al. Replication gaps underlie BRCA deficiency and therapy response. Cancer Res. 81, 1388–1397 (2021).
pubmed: 33184108
doi: 10.1158/0008-5472.CAN-20-1602
Maya-Mendoza, A. et al. High speed of fork progression induces DNA replication stress and genomic instability. Nature 559, 279–284 (2018).
pubmed: 29950726
doi: 10.1038/s41586-018-0261-5
Wong, R. P., García-Rodríguez, N., Zilio, N., Hanulová, M. & Ulrich, H. D. Processing of DNA polymerase-blocking lesions during genome replication is spatially and temporally segregated from replication forks. Mol. Cell 77, 3–16 (2020).
pubmed: 31607544
doi: 10.1016/j.molcel.2019.09.015
Zou, L. & Elledge, S. J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003).
pubmed: 12791985
doi: 10.1126/science.1083430
Ball, H. L. et al. Function of a conserved checkpoint recruitment domain in ATRIP proteins. Mol. Cell Biol. 27, 3367–3377 (2007).
pubmed: 17339343
pmcid: 1899971
doi: 10.1128/MCB.02238-06
Lee, J., Kumagai, A. & Dunphy, W. G. The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. J. Biol. Chem. 282, 28036–28044 (2007).
pubmed: 17636252
doi: 10.1074/jbc.M704635200
Delacroix, S., Wagner, J. M., Kobayashi, M., Yamamoto, K. & Karnitz, L. M. The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21, 1472–1477 (2007).
pubmed: 17575048
pmcid: 1891424
doi: 10.1101/gad.1547007
Kumagai, A., Lee, J., Yoo, H. Y. & Dunphy, W. G. TopBP1 activates the ATR-ATRIP complex. Cell 124, 943–955 (2006).
pubmed: 16530042
doi: 10.1016/j.cell.2005.12.041
Mordes, D. A., Glick, G. G., Zhao, R. & Cortez, D. TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. Genes Dev. 22, 1478–1489 (2008).
pubmed: 18519640
pmcid: 2418584
doi: 10.1101/gad.1666208
Bass, T. E. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nat. Cell Biol. 18, 1185–1195 (2016).
pubmed: 27723720
pmcid: 5245861
doi: 10.1038/ncb3415
Haahr, P. et al. Activation of the ATR kinase by the RPA-binding protein ETAA1. Nat. Cell Biol. 18, 1196–1207 (2016).
pubmed: 27723717
doi: 10.1038/ncb3422
Lee, Y.-C., Zhou, Q., Chen, J. & Yuan, J. RPA-binding protein ETAA1 is an ATR activator involved in DNA replication stress response. Curr. Biol. 26, 3257–3268 (2016).
pubmed: 27818175
pmcid: 5173396
doi: 10.1016/j.cub.2016.10.030
Bass, T. E. & Cortez, D. Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1. J. Cell Biol. 218, 1235–1249 (2019).
pubmed: 30755469
pmcid: 6446857
doi: 10.1083/jcb.201810058
Sørensen, C. S. et al. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 3, 247–258 (2003).
pubmed: 12676583
doi: 10.1016/S1535-6108(03)00048-5
Mailand, N. et al. Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J. 21, 5911–5920 (2002).
pubmed: 12411508
pmcid: 131064
doi: 10.1093/emboj/cdf567
Toledo, L. I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).
pubmed: 24267891
doi: 10.1016/j.cell.2013.10.043
Toledo, L., Neelsen, K. J. & Lukas, J. Replication catastrophe: When a checkpoint fails because of exhaustion. Mol. Cell 66, 735–749. https://doi.org/10.1016/j.molcel.2017.05.001 (2017).
doi: 10.1016/j.molcel.2017.05.001
pubmed: 28622519
Byrne, B. M. & Oakley, G. G. Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability. Semin. Cell Dev. Biol. 86, 112–120 (2019).
pubmed: 29665433
doi: 10.1016/j.semcdb.2018.04.005
Par, S. et al. OB-folds and genome maintenance: Targeting protein–DNA interactions for cancer therapy. Cancers 13, 3346 (2021).
pubmed: 34283091
pmcid: 8269290
doi: 10.3390/cancers13133346
Binz, S. K. & Wold, M. S. Regulatory functions of the N-terminal domain of the 70-kDa subunit of replication protein A (RPA). J. Biol. Chem. 283, 21559–21570 (2008).
pubmed: 18515800
pmcid: 2490791
doi: 10.1074/jbc.M802450200
Kim, C., Paulus, B. F. & Wold, M. S. Interactions of human replication protein A with oligonucleotides. Biochemistry 33, 14197–14206 (1994).
pubmed: 7947831
doi: 10.1021/bi00251a031
Takai, K. K., Kibe, T., Donigian, J. R., Frescas, D. & de Lange, T. Telomere protection by TPP1/POT1 requires tethering to TIN2. Mol. Cell 44, 647–659 (2011).
pubmed: 22099311
pmcid: 3222871
doi: 10.1016/j.molcel.2011.08.043
Patrick, S. M. & Turchi, J. J. Stopped-flow kinetic analysis of replication protein A-binding DNA. J. Biol. Chem. 276, 22630–22637 (2001).
pubmed: 11278662
doi: 10.1074/jbc.M010314200
Maréchal, A. & Zou, L. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response. Cell Res. 25, 9–23 (2015).
pubmed: 25403473
doi: 10.1038/cr.2014.147
Din, S., Brill, S. J., Fairman, M. P. & Stillman, B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 4, 968–977 (1990).
pubmed: 2200738
doi: 10.1101/gad.4.6.968
Zernik-Kobak, M., Vasunia, K., Connelly, M., Anderson, C. W. & Dixon, K. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells. J. Biol. Chem. 272, 23896–23904 (1997).
pubmed: 9295339
doi: 10.1074/jbc.272.38.23896
Block, W. D., Yu, Y. & Lees-Miller, S. P. Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21. Nucleic Acids Res. 32, 997–1005 (2004).
pubmed: 14872059
pmcid: 373400
doi: 10.1093/nar/gkh265
Cheng, X. et al. Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection. Proc. Natl. Acad. Sci. 115, 10028–10033 (2018).
pubmed: 30224481
pmcid: 6176631
doi: 10.1073/pnas.1806513115
Gan, X. et al. Proper RPA acetylation promotes accurate DNA replication and repair. Nucleic Acids Res. 51, 5565–5583 (2023).
pubmed: 37140030
pmcid: 10287905
doi: 10.1093/nar/gkad291
Zhao, M. et al. PCAF/GCN5-mediated acetylation of RPA1 promotes nucleotide excision repair. Cell Rep. 20, 1997–2009 (2017).
pubmed: 28854354
doi: 10.1016/j.celrep.2017.08.015
He, H., Wang, J. & Liu, T. UV-induced RPA1 acetylation promotes nucleotide excision repair. Cell Rep. 20, 2010–2025 (2017).
pubmed: 28854355
doi: 10.1016/j.celrep.2017.08.016
Ononye, O. E. et al. Biochemical impact of p300-mediated acetylation of replication protein A: Implications for DNA metabolic pathway choice. BioRxiv 15, 883 (2024).
Lopes, M., Foiani, M. & Sogo, J. M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27 (2006).
pubmed: 16387650
doi: 10.1016/j.molcel.2005.11.015
Ward, I. M., Minn, K. & Chen, J. UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress. J. Biol. Chem. 279, 9677–9680 (2004).
pubmed: 14742437
doi: 10.1074/jbc.C300554200
Shuck, S. C. & Turchi, J. J. Targeted inhibition of replication protein A reveals cytotoxic activity, synergy with chemotherapeutic DNA-damaging agents, and insight into cellular function. Cancer Res. 70, 3189–3198 (2010).
pubmed: 20395205
pmcid: 2882864
doi: 10.1158/0008-5472.CAN-09-3422
Mishra, A. K., Dormi, S. S., Turchi, A. M., Woods, D. S. & Turchi, J. J. Chemical inhibitor targeting the replication protein A–DNA interaction increases the efficacy of Pt-based chemotherapy in lung and ovarian cancer. Biochem. Pharmacol. 93, 25–33 (2015).
pubmed: 25449597
doi: 10.1016/j.bcp.2014.10.013
Gavande, N. S. et al. Structure-guided optimization of replication protein A (RPA)–DNA interaction inhibitors. ACS Med. Chem. Lett. 11, 1118–1124 (2020).
pubmed: 32550990
pmcid: 7294550
doi: 10.1021/acsmedchemlett.9b00440
VanderVere-Carozza, P. S. et al. In vivo targeting replication protein A for cancer therapy. Front. Oncol. 12, 6655 (2022).
doi: 10.3389/fonc.2022.826655
Glanzer, J. G., Liu, S. & Oakley, G. G. Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods. Bioorg. Med. Chem. 19, 2589–2595 (2011).
pubmed: 21459001
pmcid: 3399738
doi: 10.1016/j.bmc.2011.03.012
Glanzer, J. G. et al. A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A. Nucleic Acids Res. 41, 2047–2059 (2013).
pubmed: 23267009
doi: 10.1093/nar/gks1291
Glanzer, J. G. et al. RPA inhibition increases replication stress and suppresses tumor growth. Cancer Res. 74, 5165–5172 (2014).
pubmed: 25070753
pmcid: 4201622
doi: 10.1158/0008-5472.CAN-14-0306
Choi, J.-H., Lindsey-Boltz, L. A. & Sancar, A. Reconstitution of a human ATR-mediated checkpoint response to damaged DNA. Proc. Natl. Acad. Sci. 104, 13301–13306 (2007).
pubmed: 17686975
pmcid: 1941640
doi: 10.1073/pnas.0706013104
Choi, J.-H. et al. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling. Proc. Natl. Acad. Sci. 107, 13660–13665 (2010).
pubmed: 20616048
pmcid: 2922256
doi: 10.1073/pnas.1007856107
Lindsey-Boltz, L. A., Reardon, J. T., Wold, M. S. & Sancar, A. In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling. J. Biol. Chem. 287, 36123–36131 (2012).
pubmed: 22948311
pmcid: 3476280
doi: 10.1074/jbc.M112.407825
Choi, J.-H., Sancar, A. & Lindsey-Boltz, L. A. The human ATR-mediated DNA damage checkpoint in a reconstituted system. Methods 48, 3–7 (2009).
pubmed: 19245835
doi: 10.1016/j.ymeth.2009.02.006
Olson, E., Nievera, C. J., Klimovich, V., Fanning, E. & Wu, X. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint. J. Biol. Chem. 281, 39517–39533 (2006).
pubmed: 17035231
doi: 10.1074/jbc.M605121200
Vassin, V. M., Anantha, R. W., Sokolova, E., Kanner, S. & Borowiec, J. A. Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J. Cell Sci. 122, 4070–4080 (2009).
pubmed: 19843584
pmcid: 2776501
doi: 10.1242/jcs.053702
Greiner, J. V. & Glonek, T. Intracellular ATP concentration and implication for cellular evolution. Biology 10, 1166 (2021).
pubmed: 34827159
pmcid: 8615055
doi: 10.3390/biology10111166
Wu, Y., Fu, W., Zang, N. & Zhou, C. Structural characterization of human RPA70N association with DNA damage response proteins. Elife 12, 81639 (2023).
doi: 10.7554/eLife.81639
Oakley, G. G. et al. RPA phosphorylation in mitosis alters DNA binding and protein−protein interactions. Biochemistry 42, 3255–3264 (2003).
pubmed: 12641457
doi: 10.1021/bi026377u
Anantha, R. W., Sokolova, E. & Borowiec, J. A. RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage. Proc. Natl. Acad. Sci. 105, 12903–12908 (2008).
pubmed: 18723675
pmcid: 2529075
doi: 10.1073/pnas.0803001105
Vassin, V. M., Wold, M. S. & Borowiec, J. A. Replication protein A (RPA) phosphorylation prevents RPA association with replication centers. Mol. Cell Biol. 24, 1930–1943 (2004).
pubmed: 14966274
pmcid: 350552
doi: 10.1128/MCB.24.5.1930-1943.2004
Binz, S. K., Lao, Y., Lowry, D. F. & Wold, M. S. The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions: Evidence for an intersubunit interaction. J. Biol. Chem. 278, 35584–35591 (2003).
pubmed: 12819197
doi: 10.1074/jbc.M305388200
Hashimoto, Y., Tsujimura, T., Sugino, A. & Takisawa, H. The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1. Genes Cells 11, 993–1007 (2006).
pubmed: 16923121
doi: 10.1111/j.1365-2443.2006.00998.x
Yoo, H. Y., Kumagai, A., Shevchenko, A., Shevchenko, A. & Dunphy, W. G. Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM. J. Biol. Chem. 282, 17501–17506 (2007).
pubmed: 17446169
doi: 10.1074/jbc.M701770200
Klaeger, S. et al. The target landscape of clinical kinase drugs. Science 358, 1 (2017).
doi: 10.1126/science.aan4368
Davis, M. I. et al. Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1046–1051 (2011).
pubmed: 22037378
doi: 10.1038/nbt.1990
Ashley, A. K. et al. DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe. DNA Repair (Amst.) 21, 131–139 (2014).
pubmed: 24819595
doi: 10.1016/j.dnarep.2014.04.008
Anantha, R. W., Vassin, V. M. & Borowiec, J. A. Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair. J. Biol. Chem. 282, 35910–35923 (2007).
pubmed: 17928296
doi: 10.1074/jbc.M704645200
Liu, S. et al. Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress. Nucleic Acids Res. 40, 10780–10794 (2012).
pubmed: 22977173
pmcid: 3510507
doi: 10.1093/nar/gks849
Liu, S., Byrne, B. M., Byrne, T. N. & Oakley, G. G. Role of RPA phosphorylation in the ATR-dependent G2 cell cycle checkpoint. Genes 14, 2205 (2023).
pubmed: 38137027
pmcid: 10742774
doi: 10.3390/genes14122205
Lee, S., Heo, J. & Park, C.-J. Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide. J. Biol. Chem. 295, 18449–18458 (2020).
pubmed: 33127641
doi: 10.1074/jbc.RA120.016457
Soniat, M. M., Myler, L. R., Kuo, H.-C., Paull, T. T. & Finkelstein, I. J. RPA phosphorylation inhibits DNA resection. Mol. Cell 75, 145-153.e5 (2019).
pubmed: 31153714
pmcid: 6625828
doi: 10.1016/j.molcel.2019.05.005
Hasan, S. et al. Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol. Cell 7, 1221–1231 (2001).
pubmed: 11430825
doi: 10.1016/S1097-2765(01)00272-6
Balakrishnan, L., Stewart, J., Polaczek, P., Campbell, J. L. & Bambara, R. A. Acetylation of Dna2 endonuclease/helicase and flap endonuclease 1 by p300 promotes DNA stability by creating long flap intermediates. J. Biol. Chem. 285, 4398–4404 (2010).
pubmed: 20019387
doi: 10.1074/jbc.M109.086397
Balakrishnan, L. & Bambara, R. A. Okazaki fragment metabolism. Cold Spring Harb. Perspect. Biol. 5, a010173 (2013).
pubmed: 23378587
pmcid: 3552508
doi: 10.1101/cshperspect.a010173
Wanrooij, P. H. & Burgers, P. M. Yet another job for Dna2: Checkpoint activation. DNA Repair (Amst.) 32, 17–23 (2015).
pubmed: 25956863
doi: 10.1016/j.dnarep.2015.04.009
Sun, H. et al. Okazaki fragment maturation: DNA flap dynamics for cell proliferation and survival. Trends Cell Biol. 33, 221–234 (2023).
pubmed: 35879148
doi: 10.1016/j.tcb.2022.06.014
Choi, J.-H., Lindsey-Boltz, L. A. & Sancar, A. Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA. Nucleic Acids Res. 37, 1501–1509 (2009).
pubmed: 19139065
pmcid: 2655664
doi: 10.1093/nar/gkn1075