Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.

Genome Damage and Stability Centre School of Life Sciences University of Sussex Brighton UK

Laboratory of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences Prague 4 Czech Republic

Laboratory of Molecular Structure Characterization Institute of Microbiology of the Czech Academy of Sciences Prague 4 Czech Republic

Zobrazit více v PubMed

Hottiger MO, Hassa PO, Lüscher B, Schüler H, Koch-Nolte F. Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem. Sci. 2010;35:208–219. doi: 10.1016/j.tibs.2009.12.003. PubMed DOI

Amé J-C, Spenlehauer C, de Murcia G. The PARP superfamily. Bioessays. 2004;26:882–893. doi: 10.1002/bies.20085. PubMed DOI

Azarm K, Smith S. Nuclear PARPs and genome integrity. Gene Dev. 2020;34:285–301. doi: 10.1101/gad.334730.119. PubMed DOI PMC

Pandey N, Black BE. Rapid detection and signaling of DNA damage by PARP-1. Trends Biochem. Sci. 2021;46:744–757. doi: 10.1016/j.tibs.2021.01.014. PubMed DOI PMC

Chaudhuri AR, Nussenzweig A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat. Rev. Mol. Cell Bio. 2017;18:610–621. doi: 10.1038/nrm.2017.53. PubMed DOI PMC

Rack JGM, Palazzo L, Ahel I. (ADP-ribosyl)hydrolases: structure, function, and biology. Gene Dev. 2020;34:263–284. doi: 10.1101/gad.334631.119. PubMed DOI PMC

Lin W, Amé J-C, Aboul-Ela N, Jacobson EL, Jacobson MK. Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase. J. Biol. Chem. 1997;272:11895–11901. doi: 10.1074/jbc.272.18.11895. PubMed DOI

Davidovic L, Vodenicharov M, Afar EB, Poirier GG. Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism. Exp. Cell. Res. 2001;268:7–13. doi: 10.1006/excr.2001.5263. PubMed DOI

Caldecott KW. Protein ADP-ribosylation and the cellular response to DNA strand breaks. DNA Repair. 2014;19:108–113. doi: 10.1016/j.dnarep.2014.03.021. PubMed DOI

Hanzlikova H, Caldecott KW. Perspectives on PARPs in S phase. Trends Genet. 2019;35:412–422. doi: 10.1016/j.tig.2019.03.008. PubMed DOI

Hanzlikova H, et al. The importance of poly(ADP-ribose) polymerase as a sensor of unligated Okazaki fragments during DNA replication. Mol. Cell. 2018;71:319–331. doi: 10.1016/j.molcel.2018.06.004. PubMed DOI PMC

Bryant HE, et al. PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 2009;28:2601–2615. doi: 10.1038/emboj.2009.206. PubMed DOI PMC

Sugimura K, Takebayashi S-I, Taguchi H, Takeda S, Okumura K. PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA. J. Cell Biol. 2008;183:1203–1212. doi: 10.1083/jcb.200806068. PubMed DOI PMC

Hochegger H, et al. Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells. EMBO J. 2006;25:1305–1314. doi: 10.1038/sj.emboj.7601015. PubMed DOI PMC

Somyajit K, Mishra A, Jameei A, Nagaraju G. Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase. Carcinogenesis. 2015;36:13–24. doi: 10.1093/carcin/bgu211. PubMed DOI

Patel AG, Sarkaria JN, Kaufmann SH. Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proc. Natl Acad. Sci. USA. 2011;108:3406–3411. doi: 10.1073/pnas.1013715108. PubMed DOI PMC

Haince J-F, et al. PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites. J. Biol. Chem. 2008;283:1197–1208. doi: 10.1074/jbc.M706734200. PubMed DOI

Berti M, et al. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 2013;20:347–354. doi: 10.1038/nsmb.2501. PubMed DOI PMC

Chaudhuri, A. R. et al. Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat. Struct. Mol. Biol.19, 417–423 (2012). PubMed

Zheng L, Shen B. Okazaki fragment maturation: nucleases take centre stage. J. Mol. Cell. Biol. 2011;3:23–30. doi: 10.1093/jmcb/mjq048. PubMed DOI PMC

Kao H-I, Bambara RA. The protein components and mechanism of eukaryotic Okazaki fragment maturation. Crit. Rev. Biochem Mol. 2008;38:433–452. doi: 10.1080/10409230390259382. PubMed DOI

Stodola JL, Burgers PM. Mechanism of lagging-strand DNA replication in eukaryotes. Adv. Exp. Med. Biol. 2017;1042:117–133. doi: 10.1007/978-981-10-6955-0_6. PubMed DOI

Balakrishnan L, Bambara RA. Okazaki fragment metabolism. Csh Perspect. Biol. 2013;5:a010173–a010173. PubMed PMC

Burgers, P. M. J. & Kunkel, T. A. Eukaryotic DNA replication fork. Annu. Rev. Biochem.10.1146/annurev-biochem-061516-044709 (2017). PubMed PMC

Kahli, M., Osmundson, J. S., Yeung, R. & Smith, D. J. Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo. Nucleic Acids Res. 47, 1814–1822 (2018). PubMed PMC

Hedglin M, Pandey B, Benkovic SJ. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis. Proc. Natl Acad. Sci. USA. 2016;113:E1777–E1786. doi: 10.1073/pnas.1523653113. PubMed DOI PMC

Arakawa H, et al. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells. Nucleic Acids Res. 2012;40:2599–2610. doi: 10.1093/nar/gkr1024. PubMed DOI PMC

Kumamoto, S. et al. HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation. Nucleic Acids Res.10.1093/nar/gkab269 (2021). PubMed PMC

Matsuzaki Y, Adachi N, Koyama H. Vertebrate cells lacking FEN‐1 endonuclease are viable but hypersensitive to methylating agents and H2O2. Nucleic Acids Res. 2002;30:3273–3277. doi: 10.1093/nar/gkf440. PubMed DOI PMC

Bryant HE, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 2005;434:913–917. doi: 10.1038/nature03443. PubMed DOI

Farmer H, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005;434:917–921. doi: 10.1038/nature03445. PubMed DOI

Pommier Y, O’Connor MJ, de Bono J. Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action. Sci. Transl. Med. 2016;8:362ps17. doi: 10.1126/scitranslmed.aaf9246. PubMed DOI

Murai J, et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 2012;72:5588–5599. doi: 10.1158/0008-5472.CAN-12-2753. PubMed DOI PMC

Fugger K, et al. Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors. Science. 2021;372:156–165. doi: 10.1126/science.abb4542. PubMed DOI PMC

Zimmermann M, et al. CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions. Nature. 2018;559:285–289. doi: 10.1038/s41586-018-0291-z. PubMed DOI PMC

Hewitt G, et al. Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD. Mol. Cell. 2021;81:767–783. doi: 10.1016/j.molcel.2020.12.006. PubMed DOI PMC

Giovannini S, et al. ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition. Nucleic Acids Res. 2020;48:4928–4939. doi: 10.1093/nar/gkaa255. PubMed DOI PMC

Cong K, et al. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Mol. Cell. 2021;81:3128–3144.e7. doi: 10.1016/j.molcel.2021.06.011. PubMed DOI PMC

Bae S-H, Bae K-H, Kim J-A, Seo Y-S. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature. 2001;412:456–461. doi: 10.1038/35086609. PubMed DOI

Liu, B., Hu, J., Wang, J. & Kong, D. Direct visualization of RNA-DNA primer removal from Okazaki fragments provides support for flap cleavage and exonucleolytic pathways in eukaryotic cells. J. Biol. Chem.292, jbc.M116.758599 (2017). PubMed PMC

Arakawa H, Iliakis G. Alternative Okazaki fragment ligation pathway by DNA ligase III. Genes. 2015;6:385–398. doi: 10.3390/genes6020385. PubMed DOI PMC

Sriramachandran AM, et al. Genome-wide nucleotide-resolution mapping of DNA replication patterns, single-strand breaks, and lesions by GLOE-seq. Mol. Cell. 2020;78:975–985.e7. doi: 10.1016/j.molcel.2020.03.027. PubMed DOI PMC

Balakrishnan L, Bambara RA. Flap endonuclease 1. Annu. Rev. Biochem. 2013;82:119–138. doi: 10.1146/annurev-biochem-072511-122603. PubMed DOI PMC

Sparks JL, et al. RNase H2-initiated ribonucleotide excision repair. Mol. Cell. 2012;47:980–986. doi: 10.1016/j.molcel.2012.06.035. PubMed DOI PMC

Maya-Mendoza A, et al. High speed of fork progression induces DNA replication stress and genomic instability. Nature. 2018;18:3059–3284. PubMed

Wojtaszek JL, et al. A small molecule targeting mutagenic translesion synthesis improves chemotherapy. Cell. 2019;178:152–159.e11. doi: 10.1016/j.cell.2019.05.028. PubMed DOI PMC

Zellweger R, et al. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J. Cell Biol. 2015;208:563–579. doi: 10.1083/jcb.201406099. PubMed DOI PMC

Taglialatela A, et al. REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps. Mol. Cell. 2021;81:4008–4025.e7. doi: 10.1016/j.molcel.2021.08.016. PubMed DOI PMC

Tirman S, et al. Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells. Mol. Cell. 2021;81:4026–4040.e8. doi: 10.1016/j.molcel.2021.09.013. PubMed DOI PMC

Panzarino NJ, et al. Replication gaps underlie BRCA deficiency and therapy response. Cancer Res. 2021;81:1388–1397. doi: 10.1158/0008-5472.CAN-20-1602. PubMed DOI PMC

Simoneau A, Xiong R, Zou L. The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells. Gene Dev. 2021;35:1271–1289. doi: 10.1101/gad.348479.121. PubMed DOI PMC

Tumey LN, et al. The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors. Bioorg. Med. Chem. Lett. 2005;15:277–281. doi: 10.1016/j.bmcl.2004.10.086. PubMed DOI

Hoch NC, et al. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2017;541:87–91. doi: 10.1038/nature20790. PubMed DOI PMC

Reijns MAM, et al. Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell. 2012;149:1008–1022. doi: 10.1016/j.cell.2012.04.011. PubMed DOI PMC

Lingeman E, Jeans C, Corn JE. Production of purified CasRNPs for efficacious genome editing. Curr. Protoc. Mol. Biol. 2017;120:31.10.1–31.10.19. doi: 10.1002/cpmb.43. PubMed DOI

Breslin C, et al. Measurement of chromosomal DNA single-strand breaks and replication fork progression rates. Methods Enzymol. 2006;409:410–425. doi: 10.1016/S0076-6879(05)09024-5. PubMed DOI

Neelsen KJ, Chaudhuri AR, Follonier C, Herrador R, Lopes M. Functional analysis of DNA and chromatin. Methods Mol. Biol. 2013;1094:177–208. doi: 10.1007/978-1-62703-706-8_15. PubMed DOI

Zellweger R, Lopes M. Dynamic architecture of eukaryotic DNA replication forks in vivo, visualized by electron microscopy. Methods Mol. Biol. 2018;1672:261–294. doi: 10.1007/978-1-4939-7306-4_19. PubMed DOI

Dispensability of HPF1 for cellular removal of DNA single-strand breaks

DNA polymerase α-primase facilitates PARP inhibitor-induced fork acceleration and protects BRCA1-deficient cells against ssDNA gaps

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair