Flap endonuclease 1 (FEN1)-dependent long-patch repair has been considered a minor sub-pathway of DNA single-strand break repair (SSBR), activated only when short-patch repair is not feasible. However, the significance of long-patch repair in living cells remains unclear. Here, we employed human RPE-1 cells with FEN1 deletion to compare the requirements for short- and long-patch pathways for the rapid repair of various types of DNA single-strand breaks (SSBs). We found that SSBs arising from abortive topoisomerase 1 activity are repaired efficiently without FEN1. In contrast, the rapid repair of SSBs arising during base excision repair following treatment with methyl methanesulphonate (MMS) or following treatment with hydrogen peroxide (H2O2) exhibits an unexpectedly high dependence on FEN1. Indeed, in G1 phase, FEN1 deletion slows the rate of SSBR to a similar or even greater extent than deletion of the short-patch repair proteins XRCC1 or POLβ. As expected, the combined deletion of FEN1 with XRCC1 or POLβ has an additive or synergistic effect, severely attenuating SSBR rates after MMS or H2O2 exposure. These data highlight an unanticipated requirement for FEN1 in the rapid repair of SSBs in human cells, challenging the prevailing view that long-patch repair is a minor sub-pathway of SSBR.

Genome Damage and Stability Centre University of Sussex Falmer Brighton BN1 9RQ United Kingdom

Institute of Animal Pathology Vetsuisse Faculty University of Bern 3012 Bern Switzerland

Laboratory of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences 142 20 Prague 4 Czech Republic

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Caldecott  KW  Causes and consequences of DNA single-strand breaks. Trends Biochem Sci. 2024; 49:68–78. 10.1016/j.tibs.2023.11.001. PubMed DOI

Milano  L, Gautam  A, Caldecott  KW  DNA damage and transcription stress. Mol Cell. 2024; 84:711–3. 10.1016/j.molcel.2023.11.014. PubMed DOI

Caldecott  KW, Ward  ME, Nussenzweig  A  The threat of programmed DNA damage to neuronal genome integrity and plasticity. Nat Genet. 2022; 54:115–20. 10.1038/s41588-021-01001-y. PubMed DOI

Caldecott  KW  DNA single-strand break repair and human genetic disease. Trends Cell Biol. 2022; 32:733–45. 10.1016/j.tcb.2022.04.010. PubMed DOI

Gohil  D, Sarker  AH, Roy  R  Base excision repair: mechanisms and impact in biology, disease, and medicine. Int J Mol Sci. 2023; 24:14186. 10.3390/ijms241814186. PubMed DOI PMC

Abbotts  R, Wilson  DM  Coordination of DNA single strand break repair. Free Radic Biol Med. 2017; 107:228–44. 10.1016/j.freeradbiomed.2016.11.039. PubMed DOI PMC

Kubota  Y, Nash  RA, Klungland  A  et al.  Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 1996; 15:6662–70. 10.1002/j.1460-2075.1996.tb01056.x. PubMed DOI PMC

Singhal  RK, Prasad  R, Wilson  SH  DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem. 1995; 270:949–57. 10.1074/jbc.270.2.949. PubMed DOI

Sobol  RW, Horton  JK, Kühn  R  et al.  Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature. 1996; 379:183–6. 10.1038/379183a0. PubMed DOI

Nicholl  ID, Nealon  K, Kenny  MK  Reconstitution of human base excision repair with purified proteins. Biochemistry. 1997; 36:7557–66. 10.1021/bi962950w. PubMed DOI

Fortini  P, Pascucci  B, Parlanti  E  et al.  Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells. Biochemistry. 1998; 37:3575–80. 10.1021/bi972999h. PubMed DOI

Frosina  G, Fortini  P, Rossi  O  et al.  Two pathways for base excision repair in mammalian cells. J Biol Chem. 1996; 271:9573–8. 10.1074/jbc.271.16.9573. PubMed DOI

Klungland  A, Lindahl  T  Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 1997; 16:3341–8. 10.1093/emboj/16.11.3341. PubMed DOI PMC

Winters  TA, Russell  PS, Kohli  M  et al.  Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate. Nucleic Acids Res. 1999; 27:2423–33. 10.1093/nar/27.11.2423. PubMed DOI PMC

Caldecott  K, McKeown  C, Tucker  J  et al.  An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol. 1994; 14:68–76. PubMed PMC

Caldecott  KW, Aoufouchi  S, Johnson  P  et al.  XRCC1 polypeptide interacts with DNA polymerase β and possibly poly(ADP-ribose) polymerase, and DNA ligase III is a novel molecular “nick-sensor” PubMed DOI PMC

Cappelli  E, Taylor  R, Cevasco  M  et al.  Involvement of XRCC1 and DNA ligase III gene products in DNA base excision repair. J Biol Chem. 1997; 272:23970–5. 10.1074/jbc.272.38.23970. PubMed DOI

Whitehouse  CJ, Taylor  RM, Thistlethwaite  A  et al.  XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. Cell. 2001; 104:107–17. 10.1016/S0092-8674(01)00195-7. PubMed DOI

Ahel  I, Rass  U, El-Khamisy  SF  et al.  The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates. Nature. 2006; 443:713–6. 10.1038/nature05164. PubMed DOI

Kim  K, Biade  S, Matsumoto  Y  Involvement of flap endonuclease 1 in base excision DNA repair. J Biol Chem. 1998; 273:8842–8. 10.1074/jbc.273.15.8842. PubMed DOI

Levin  DS, McKenna  AE, Motycka  TA  et al.  Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair. Curr Biol. 2000; 10:919–S2. 10.1016/S0960-9822(00)00619-9. PubMed DOI

Matsumoto  Y, Kim  K, Bogenhagen  DF  Proliferating cell nuclear antigen-dependent abasic site repair in PubMed PMC

Pascucci  B, Stucki  M, Jónsson  ZO  et al.  Long patch base excision repair with purified human proteins: DNA ligase I as patch size mediator for DNA polymerases δ and ϵ. J Biol Chem. 1999; 274:33696–702. 10.1074/jbc.274.47.33696. PubMed DOI

Dianov  G, Price  A, Lindahl  T  Generation of single-nucleotide repair patches following excision of uracil residues from DNA. Mol Cell Biol. 1992; 12:1605–12. PubMed PMC

Dianov  G, Bischoff  C, Piotrowski  J  et al.  Repair pathways for processing of 8-oxoguanine in DNA by mammalian cell extracts. J Biol Chem. 1998; 273:33811–6. 10.1074/jbc.273.50.33811. PubMed DOI

Nealon  K, Nicholl  ID, Kenny  MK  Characterization of the DNA polymerase requirement of human base excision repair. Nucleic Acids Res. 1996; 24:3763–70. 10.1093/nar/24.19.3763. PubMed DOI PMC

Bennett  SE, Sung  J-S, Mosbaugh  DW  Fidelity of uracil-initiated base excision DNA repair in DNA polymerase β-proficient and -deficient mouse embryonic fibroblast cell extracts. J Biol Chem. 2001; 276:42588–600. 10.1074/jbc.M106212200. PubMed DOI

Sattler  U, Frit  P, Salles  B  et al.  Long-patch DNA repair synthesis during base excision repair in mammalian cells. EMBO Rep. 2003; 4:363–7. 10.1038/sj.embor.embor796. PubMed DOI PMC

Wang  D, Wu  W, Callen  E  et al.  Active DNA demethylation promotes cell fate specification and the DNA damage response. Science. 2022; 378:983–9. 10.1126/science.add9838. PubMed DOI PMC

Wu  W, Hill  SE, Nathan  WJ  et al.  Neuronal enhancers are hotspots for DNA single-strand break repair. Nature. 2021; 593:440–4. 10.1038/s41586-021-03468-5. PubMed DOI PMC

Fortini  P, Parlanti  E, Sidorkina  OM  et al.  The type of DNA glycosylase determines the base excision repair pathway in mammalian cells. J Biol Chem. 1999; 274:15230–6. 10.1074/jbc.274.21.15230. PubMed DOI

Matsumoto  Y, Kim  K  Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science. 1995; 269:699–702. 10.1126/science.7624801. PubMed DOI

Sung  J-S, DeMott  MS, Demple  B  Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA–protein cross-links with DNA polymerase β. J Biol Chem. 2005; 280:39095–103. 10.1074/jbc.M506480200. PubMed DOI

Vaitsiankova  A, Burdova  K, Sobol  M  et al.  PARP inhibition impedes the maturation of nascent DNA strands during DNA replication. Nat Struct Mol Biol. 2022; 29:329–38. 10.1038/s41594-022-00747-1. PubMed DOI PMC

Hrychova  K, Burdova  K, Polackova  Z  et al.  Dispensability of HPF1 for cellular removal of DNA single-strand breaks. Nucleic Acids Res. 2024; 52:10986–98. 10.1093/nar/gkae708. PubMed DOI PMC

Adamowicz  M, Hailstone  R, Demin  AA  et al.  XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair. Nat Cell Biol. 2021; 23:1287–98. 10.1038/s41556-021-00792-w. PubMed DOI PMC

Exell  JC, Thompson  MJ, Finger  LD  et al.  Cellularly active PubMed DOI PMC

Tsutakawa  SE, Classen  S, Chapados  BR  et al.  Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Cell. 2011; 145:198–211. 10.1016/j.cell.2011.03.004. PubMed DOI PMC

Kordon  MM, Zarębski  M, Solarczyk  K  et al.  STRIDE—a fluorescence method for direct, specific PubMed DOI PMC

Demin  AA, Hirota  K, Tsuda  M  et al.  XRCC1 prevents toxic PARP1 trapping during DNA base excision repair. Mol Cell. 2021; 81:3018–30. 10.1016/j.molcel.2021.05.009. PubMed DOI PMC

Hanzlikova  H, Kalasova  I, Demin  AA  et al.  The importance of poly(ADP-ribose) polymerase as a sensor of unligated Okazaki fragments during DNA replication. Mol Cell. 2018; 71:319–31. 10.1016/j.molcel.2018.06.004. PubMed DOI PMC

Pogozelski  WK, Tullius  TD  Oxidative strand scission of nucleic acids: routes initiated by hydrogen abstraction from the sugar moiety. Chem Rev. 1998; 98:1089–108. 10.1021/cr960437i. PubMed DOI

Henner  WD, Grunberg  SM, Haseltine  WA  Sites and structure of gamma radiation-induced DNA strand breaks. J Biol Chem. 1982; 257:11750–4. 10.1016/S0021-9258(18)33827-4. PubMed DOI

Henner  WD, Grunberg  SM, Haseltine  WA  Enzyme action at 3′ termini of ionizing radiation-induced DNA strand breaks. J Biological Chem. 1983; 258:15198–205. 10.1016/S0021-9258(17)43793-8. PubMed DOI

Henner  WD, Rodriguez  LO, Hecht  SM  et al.  γ Ray induced deoxyribonucleic acid strand breaks. 3′ Glycolate termini. J Biol Chem. 1983; 258:711–3. 10.1016/S0021-9258(18)33104-1. PubMed DOI