In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.

Faculty of Science Charles University Prague Prague 2128 43 Czech Republic

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

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

Laboratory of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences Prague 4142 20 Czech Republic

Zobrazit více v PubMed

Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993; 362:709–715. PubMed

Caldecott K.W. DNA single-strand break repair and human genetic disease. Trends Cell Biol. 2022; 32:733–745. PubMed

Pommier Y., Sun Y., Huang S.N., Nitiss J.L.. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat. Rev. Mol. Cell Biol. 2016; 17:703–721. PubMed PMC

Demple B., Harrison L.. Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem. 1994; 63:915–948. PubMed

Caldecott K.W. Mammalian DNA base excision repair: dancing in the moonlight. DNA Repair (Amst.). 2020; 93:102921. PubMed

Hanzlikova H., Kalasova I., Demin A.A., Pennicott L.E., Cihlarova Z., Caldecott K.W.. The importance of poly(ADP-ribose) polymerase as a sensor of unligated Okazaki fragments during DNA replication. Mol. Cell. 2018; 71:319–331. PubMed PMC

Hanzlikova H., Caldecott K.W.. Perspectives on PARPs in S Phase. Trends Genet. 2019; 35:412–422. PubMed

Vaitsiankova A., Burdova K., Sobol M., Gautam A., Benada O., Hanzlikova H., Caldecott K.W.. PARP inhibition impedes the maturation of nascent DNA strands during DNA replication. Nat. Struct. Mol. Biol. 2022; 29:329–338. PubMed PMC

Langelier M.F., Eisemann T., Riccio A.A., Pascal J.M.. PARP family enzymes: regulation and catalysis of the poly(ADP-ribose) posttranslational modification. Curr. Opin. Struct. Biol. 2018; 53:187–198. PubMed PMC

Azarm K., Smith S.. Nuclear PARPs and genome integrity. Genes Dev. 2020; 34:285–301. PubMed PMC

Ame J.C., Rolli V., Schreiber V., Niedergang C., Apiou F., Decker P., Muller S., Hoger T., Menissier-de Murcia J., de Murcia G.. PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J. Biol. Chem. 1999; 274:17860–17868. PubMed

Johansson M. A human poly(ADP-ribose) polymerase gene family (ADPRTL): cDNA cloning of two novel poly(ADP-ribose) polymerase homologues. Genomics. 1999; 57:442–445. PubMed

Hanzlikova H., Gittens W., Krejcikova K., Zeng Z., Caldecott K.W.. Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin. Nucleic Acids Res. 2017; 45:2546–2557. PubMed PMC

Breslin C., Hornyak P., Ridley A., Rulten S.L., Hanzlikova H., Oliver A.W., Caldecott K.W.. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function. Nucleic Acids Res. 2015; 43:6934–6944. PubMed PMC

Langelier M.F., Planck J.L., Roy S., Pascal J.M.. Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1. Science. 2012; 336:728–732. PubMed PMC

Rack J.G.M., Palazzo L., Ahel I.. ADP-ribosyl)hydrolases: structure, function, and biology. Genes Dev. 2020; 34:263–284. PubMed PMC

Slade D., Dunstan M.S., Barkauskaite E., Weston R., Lafite P., Dixon N., Ahel M., Leys D., Ahel I.. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature. 2011; 477:616–620. PubMed PMC

Hanzlikova H., Prokhorova E., Krejcikova K., Cihlarova Z., Kalasova I., Kubovciak J., Sachova J., Hailstone R., Brazina J., Ghosh S.et al. .. Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair. Nat. Commun. 2020; 11:3391. PubMed PMC

Fontana P., Bonfiglio J.J., Palazzo L., Bartlett E., Matic I., Ahel I.. Serine ADP-ribosylation reversal by the hydrolase ARH3. eLife. 2017; 6:e28533. PubMed PMC

Sharifi R., Morra R., Appel C.D., Tallis M., Chioza B., Jankevicius G., Simpson M.A., Matic I., Ozkan E., Golia B.et al. .. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. EMBO J. 2013; 32:1225–1237. PubMed PMC

Gibbs-Seymour I., Fontana P., Rack J.G.M., Ahel I.. HPF1/C4orf27 is a PARP-1-interacting protein that regulates PARP-1 ADP-ribosylation activity. Mol. Cell. 2016; 62:432–442. PubMed PMC

Bonfiglio J.J., Fontana P., Zhang Q., Colby T., Gibbs-Seymour I., Atanassov I., Bartlett E., Zaja R., Ahel I., Matic I.. Serine ADP-ribosylation depends on HPF1. Mol. Cell. 2017; 65:932–940. PubMed PMC

Hendriks I.A., Buch-Larsen S.C., Prokhorova E., Elsborg J.D., Rebak A., Zhu K., Ahel D., Lukas C., Ahel I., Nielsen M.L.. The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome. Nat. Commun. 2021; 12:5893. PubMed PMC

Suskiewicz M.J., Zobel F., Ogden T.E.H., Fontana P., Ariza A., Yang J.C., Zhu K., Bracken L., Hawthorne W.J., Ahel D.et al. .. HPF1 completes the PARP active site for DNA damage-induced ADP-ribosylation. Nature. 2020; 579:598–602. PubMed PMC

Palazzo L., Leidecker O., Prokhorova E., Dauben H., Matic I., Ahel I.. Serine is the major residue for ADP-ribosylation upon DNA damage. eLife. 2018; 7:e34334. PubMed PMC

Buch-Larsen S.C., Rebak A., Hendriks I.A., Nielsen M.L.. Temporal and site-specific ADP-ribosylation dynamics upon different genotoxic stresses. Cells. 2021; 10:2927. PubMed PMC

Bartlett E., Bonfiglio J.J., Prokhorova E., Colby T., Zobel F., Ahel I., Matic I.. Interplay of histone marks with serine ADP-ribosylation. Cell Rep. 2018; 24:3488–3502. PubMed PMC

Prokhorova E., Zobel F., Smith R., Zentout S., Gibbs-Seymour I., Schutzenhofer K., Peters A., Groslambert J., Zorzini V., Agnew T.et al. .. Serine-linked PARP1 auto-modification controls PARP inhibitor response. Nat. Commun. 2021; 12:4055. PubMed PMC

Hoch N.C., Hanzlikova H., Rulten S.L., Tetreault M., Komulainen E., Ju L., Hornyak P., Zeng Z., Gittens W., Rey S.A.et al. .. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2017; 541:87–91. PubMed PMC

Polo L.M., Xu Y., Hornyak P., Garces F., Zeng Z., Hailstone R., Matthews S.J., Caldecott K.W., Oliver A.W., Pearl L.H.. Efficient single-strand break repair requires binding to both poly(ADP-ribose) and DNA by the central BRCT domain of XRCC1. Cell Rep. 2019; 26:573–581. PubMed PMC

Mistrik M., Vesela E., Furst T., Hanzlikova H., Frydrych I., Gursky J., Majera D., Bartek J.. Cells and stripes: a novel quantitative photo-manipulation technique. Sci. Rep. 2016; 6:19567. PubMed PMC

Nowak K., Rosenthal F., Karlberg T., Butepage M., Thorsell A.G., Dreier B., Grossmann J., Sobek J., Imhof R., Luscher B.et al. .. Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins. Nat. Commun. 2020; 11:5199. PubMed PMC

Serrano-Benitez A., Wells S.E., Drummond-Clarke L., Russo L.C., Thomas J.C., Leal G.A., Farrow M., Edgerton J.M., Balasubramanian S., Yang M.et al. .. Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity. EMBO J. 2023; 42:e113190. PubMed PMC

Leidecker O., Bonfiglio J.J., Colby T., Zhang Q., Atanassov I., Zaja R., Palazzo L., Stockum A., Ahel I., Matic I.. Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat. Chem. Biol. 2016; 12:998–1000. PubMed PMC

Larsen S.C., Hendriks I.A., Lyon D., Jensen L.J., Nielsen M.L.. Systems-wide analysis of serine ADP-ribosylation reveals widespread occurrence and site-specific overlap with phosphorylation. Cell Rep. 2018; 24:2493–2505. PubMed

Smith R., Zentout S., Rother M., Bigot N., Chapuis C., Mihut A., Zobel F.F., Ahel I., van Attikum H., Timinszky G.et al. .. HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage. Nat. Struct. Mol. Biol. 2023; 30:678–691. PubMed

Fisher A.E., Hochegger H., Takeda S., Caldecott K.W.. Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase. Mol. Cell. Biol. 2007; 27:5597–5605. PubMed PMC

El-Khamisy S.F., Masutani M., Suzuki H., Caldecott K.W.. A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage. Nucleic Acids Res. 2003; 31:5526–5533. PubMed PMC

Rudolph J., Roberts G., Muthurajan U.M., Luger K.. HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase. eLife. 2021; 10:e65773. PubMed PMC

Bacic L., Gaullier G., Sabantsev A., Lehmann L.C., Brackmann K., Dimakou D., Halic M., Hewitt G., Boulton S.J., Deindl S.. Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome. eLife. 2021; 10:e65773. PubMed PMC

Hewitt G., Borel V., Segura-Bayona S., Takaki T., Ruis P., Bellelli R., Lehmann L.C., Sommerova L., Vancevska A., Tomas-Loba A.et al. .. Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD. Mol. Cell. 2021; 81:767–783. PubMed PMC

Mohapatra J., Tashiro K., Beckner R.L., Sierra J., Kilgore J.A., Williams N.S., Liszczak G.. Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling. eLife. 2021; 10:e71502. PubMed PMC

Abplanalp J., Leutert M., Frugier E., Nowak K., Feurer R., Kato J., Kistemaker H.V.A., Filippov D.V., Moss J., Caflisch A.et al. .. Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase. Nat. Commun. 2017; 8:2055. PubMed PMC

Prokhorova E., Agnew T., Wondisford A.R., Tellier M., Kaminski N., Beijer D., Holder J., Groslambert J., Suskiewicz M.J., Zhu K.et al. .. Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease. Mol. Cell. 2021; 81:2640–2655. PubMed PMC