RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

Department of Cell Biology and Epigenetics Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 612 65 Brno Czech Republic

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Hirota K, Ooka M, Shimizu N, et al. XRCC1 counteracts poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib, and a clinical alkylating agent, temozolomide, by promoting the removal of trapped PARP1 from broken DNA. Genes Cells. 2022;27:331–344. PubMed PMC

Essers J, Theil AF, Baldeyron C, et al. Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol. 2005;25:9350–9359. PubMed 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;272:23970–23975. PubMed

Pascucci B, Stucki M, Jonsson ZO, et al. Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon. J Biol Chem. 1999;274:33696–33702. PubMed

Shuck SC, Short EA, Turchi JJ.. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res. 2008;18:64–72. PubMed PMC

G-M L. Mechanisms and functions of DNA mismatch repair. Cell Res. 2008;18(1):85–98. PubMed

Mao Z, Bozzella M, Seluanov A, et al. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst). 2008;7:1765–1771. PubMed PMC

Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand break repair pathway choice. Cell Res. 2008;18(1):134–147. PubMed

Isono M, Niimi A, Oike T, et al. BRCA1 directs the repair pathway to homologous recombination by promoting 53BP1 dephosphorylation. Cell Rep. 2017;18:520–532. PubMed

Li S, Chen P-L, Subramanian T, et al. Binding of ctIP to the BRCT repeats of BRCA1 involved in the transcription regulation of p21 is disrupted upon DNA Damage. J Biol Chem. 1999;274(16):11334–11338. PubMed

Shinohara M, Bishop DK, Shinohara A. Distinct functions in regulation of meiotic crossovers for DNA damage response clamp loader rad24(Rad17) and Mec1(ATR) kinase. Genetics. 2019;213(4):1255–1269. PubMed PMC

Hartlerode AJ, Guan Y, Rajendran A, et al. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks. PLoS One. 2012;7(11):e49211. PubMed PMC

Hsiao KY, Mizzen CA. Histone H4 deacetylation facilitates 53BP1 DNA damage signaling and double-strand break repair. J Mol Cell Biol. 2013;5:157–165. PubMed

Ataian Y, Krebs JE. Five repair pathways in one context: chromatin modification during DNA repair. Biochem Cell Biol. 2006;84:490–504. PubMed

Bao Y. Chromatin response to DNA double-strand break damage. Epigenomics. 2011;3:307–321. PubMed

Downs JA, Allard S, Jobin-Robitaille O, et al. Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol Cell. 2004;16:979–990. PubMed

Rogakou EP, Pilch DR, Orr AH, et al. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–5868. PubMed

Svobodova Kovarikova A, Legartova S, Krejci J, et al. H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions. Aging (Albany NY). 2018;10:2585–2605. PubMed PMC

Game JC, Chernikova SB. The role of RAD6 in recombinational repair, checkpoints and meiosis via histone modification. DNA Repair (Amst). 2009;8:470–482. PubMed

Game JC, Williamson MS, Baccari C. X-ray survival characteristics and genetic analysis for nine Saccharomyces deletion mutants that show altered radiation sensitivity. Genetics. 2005;169:51–63. PubMed PMC

Game JC, Williamson MS, Spicakova T, et al. The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18. Genetics. 2006;173:1951–1968. PubMed PMC

Grenon M, Costelloe T, Jimeno S, et al. Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain. Yeast. 2007;24:105–119. PubMed

Ikura T, Ogryzko VV, Grigoriev M, et al. Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell. 2000;102:463–473. PubMed

Murr R, Loizou JI, Yang YG, et al. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat Cell Biol. 2006;8:91–99. PubMed

Miller KM, Tjeertes JV, Coates J, et al. Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat Struct Mol Biol. 2010;17:1144–1151. PubMed PMC

Bartova E, Sustackova G, Stixova L, et al. Recruitment of Oct4 protein to UV-damaged chromatin in embryonic stem cells. PLoS One. 2011;6:e27281. PubMed PMC

Svobodova Kovarikova A, Stixova L, Kovarik A, et al. N(6)-adenosine methylation in RNA and a reduced m3G/TMG level in non-coding RNAs appear at microirradiation-induced DNA lesions. Cells. 2020;9:360. PubMed PMC

Xiang Y, Laurent B, Hsu CH, et al. RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response. Nature. 2017;543:573–576. PubMed PMC

Jimeno S, Balestra FR, Huertas P. The emerging role of RNA modifications in DNA double-strand break repair. Front Mol Biosci. 2021;8:664872. PubMed PMC

Zhang M, Wang L, Zhong D. Photolyase: dynamics and mechanisms of repair of sun-induced DNA damage. Photochem Photobiol. 2017;93:78–92. PubMed PMC

Zhang C, Chen L, Peng D, et al. METTL3 and N6-methyladenosine promote homologous recombination-mediated repair of DSBs by modulating DNA-RNA hybrid accumulation. Mol Cell. 2020;79:425–42 e7. PubMed

Costantino L, Koshland D. Genome-wide map of r-loop-induced damage reveals how a subset of r-loops contributes to genomic instability. Mol Cell. 2018;71:487–97 e3. PubMed PMC

Garcia-Muse T, Aguilera A. R loops: from physiological to pathological roles. Cell. 2019;179:604–618. PubMed

Ohle C, Tesorero R, Schermann G, et al. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell. 2016;167:1001–13 e7. PubMed

Paull TT. RNA-DNA hybrids and the convergence with DNA repair. Crit Rev Biochem Mol Biol. 2019;54:371–384. PubMed

Puget N, Miller KM, Legube G. Non-canonical DNA/RNA structures during transcription-coupled double-strand break repair: roadblocks or Bona fide repair intermediates? DNA Repair (Amst). 2019;81:102661. PubMed PMC

Zhang A, Peng B, Huang P, et al. The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response. J Biol Chem. 2017;292:6461–6467. PubMed PMC

Qu F, Tsegay PS, Liu Y. N(6)-methyladenosine, DNA repair, and genome stability. Front Mol Biosci. 2021;8:645823. PubMed PMC

Giessing AM, Jensen SS, Rasmussen A, et al. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria. RNA. 2009;15:327–336. PubMed 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 e5. PubMed PMC

Fisher AE, Hochegger H, Takeda S, et al. 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

Kutuzov MM, Belousova EA, Kurgina TA, et al. The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context. Sci Rep. 2021;11:4849. PubMed PMC

Ström CE, Johansson F, Uhlén M, et al. Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate. Nucleic Acids Res. 2010;39:3166–3175. PubMed PMC

Murai J, Huang SY, Das BB, et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 2012;72:5588–5599. PubMed PMC

Zhang J. Brothers in arms: emerging roles of RNA epigenetics in DNA damage repair. Cell Biosci. 2017;7:24. PubMed PMC

Bromberg KD, Mitchell TR, Upadhyay AK, et al. The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol. 2017;13:317–324. PubMed

Paquin KL, Howlett NG. Understanding the histone DNA repair code: H4K20me2 makes its mark. Mol Cancer Res. 2018;16:1335–1345. PubMed PMC

Suchankova J, Kozubek S, Legartova S, et al. Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of gammaH2AX- and NBS1-positive repair foci. Biol Cell. 2015;107:440–454. PubMed

Budhavarapu VN, Chavez M, Tyler JK. How is epigenetic information maintained through DNA replication? Epigenetics Chromatin. 2013;6:32. PubMed PMC

Bartova E, Legartova S, Dundr M, et al. A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair. Aging (Albany NY). 2019;11:2488–2511. PubMed PMC

Yankova E, Blackaby W, Albertella M, et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593:597–601. PubMed PMC

Poczta A, Rogalska A, Marczak A. Treatment of multiple myeloma and the role of melphalan in the era of modern therapies-current research and clinical approaches. J Clin Med. 2021;10:1841. PubMed PMC

Sehnalova P, Legartova S, Cmarko D, et al. Recruitment of HP1beta to UVA-induced DNA lesions is independent of radiation-induced changes in A-type lamins. Biol Cell. 2014;106:151–165. PubMed

San Martin Alonso M, Noordermeer SM. Untangling the crosstalk between BRCA1 and R-loops during DNA repair. Nucleic Acids Res. 2021;49:4848–4863. PubMed PMC

Suchankova J, Legartova S, Ruckova E, et al. Mutations in the TP53 gene affected recruitment of 53BP1 protein to DNA lesions, but level of 53BP1 was stable after gamma-irradiation that depleted MDC1 protein in specific TP53 mutants. Histochem Cell Biol. 2017;148:239–255. PubMed

Yu D, Horton JR, Yang J, et al. Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions. Nucleic Acids Res. 2021;49:11629–11642. PubMed PMC

Caron MC, Sharma AK, O’Sullivan J, et al. Poly(ADP-ribose) polymerase-1 antagonizes DNA resection at double-strand breaks. Nat Commun. 2019;10:2954. PubMed PMC

Trott DA, Porter AC. Hypothesis: transcript-templated repair of DNA double-strand breaks. Bioessays. 2006;28:78–83. PubMed

Vågbø CB, Slupphaug G. RNA in DNA repair. DNA Repair (Amst). 2020;95:102927. PubMed

Domingo-Prim J, Bonath F, Visa N. RNA at DNA double-strand breaks: the challenge of dealing with DNA:RNA hybrids. Bioessays. 2020;42:e1900225. PubMed

Lu S, Dong W, Zhao P, et al. lncRNA FAM83H-AS1 is associated with the prognosis of colorectal carcinoma and promotes cell proliferation by targeting the Notch signaling pathway. Oncol Lett. 2018;15:1861–1868. PubMed PMC

Noordermeer SM, Adam S, Setiaputra D, et al. The shieldin complex mediates 53BP1-dependent DNA repair. Nature. 2018;560:117–121. PubMed PMC

Abakir A, Giles TC, Cristini A, et al. N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat Genet. 2020;52:48–55. PubMed 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;23:1287–1298. PubMed PMC

Stixova L, Sehnalova P, Legartova S, et al. HP1beta-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs. Epigenetics Chromatin. 2014;7:39. PubMed PMC

Hegazy YA, Fernando CM, Tran EJ. The balancing act of R-loop biology: the good, the bad, and the ugly. J Biol Chem. 2020;295:905–913. PubMed PMC

Marnef A, Legube G. m(6)A RNA modification as a new player in R-loop regulation. Nat Genet. 2020;52:27–28. PubMed

Bartova E, Legartova S, Krejci J, et al. Depletion of A-type lamins and Lap2alpha reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2alpha protein is responsible for compactness of irradiated chromatin. J Cell Biochem. 2018;119:8146–8162. PubMed

Michelini F, Pitchiaya S, Vitelli V, et al. Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks. Nat Cell Biol. 2017;19:1400–1411. PubMed PMC

Zhu JK. Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet. 2009;43:143–166. PubMed PMC

Ito S, Shen L, Dai Q, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333:1300–1303. PubMed PMC

Zhao H, Zhu M, Limbo O, et al. RNase H eliminates R-loops that disrupt DNA replication but is nonessential for efficient DSB repair. EMBO Rep. 2018;19. DOI:10.15252/embr.201745335 PubMed DOI PMC

Horakova AH, Bartova E, Galiova G, et al. SUV39h-independent association of HP1 beta with fibrillarin-positive nucleolar regions. Chromosoma. 2010;119:227–241. PubMed

Sakaue-Sawano A, Ohtawa K, Hama H, et al. Tracing the silhouette of individual cells in S/G2/M phases with fluorescence. Chem Biol. 2008;15:1243–1248. PubMed

Komurkova D, Svobodova Kovarikova A, Bartova E. G-Quadruplex structures colocalize with transcription factories and nuclear speckles surrounded by acetylated and dimethylated histones H3. Int J Mol Sci. 2021;23:22. PubMed PMC

Hickson I, Zhao Y, Richardson CJ, et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004;64:9152–9159. PubMed

Yao TT, Mo SM, Liu LY, et al. 5-Aza-2’-deoxycytidine may influence the proliferation and apoptosis of cervical cancer cells via demethylation in a dose- and time-dependent manner. Genet Mol Res. 2013;12:312–318. PubMed

Stixova L, Bartova E, Matula P, et al. Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin. Epigenetics Chromatin. 2011;4:5. PubMed PMC

Prasad CB, Prasad SB, Yadav SS, et al. Olaparib modulates DNA repair efficiency, sensitizes cervical cancer cells to cisplatin and exhibits anti-metastatic property. Sci Rep. 2017;7:12876. PubMed PMC

Spies L, Koekemoer TC, Sowemimo AA, et al. Caspase-dependent apoptosis is induced by Artemisia afra Jacq. ex Willd in a mitochondria-dependent manner after G2/M arrest. S Afr J Bot. 2013;84:104–109.

Dimitrova N, Chen YC, Spector DL, et al. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature. 2008;456:524–528. PubMed PMC

Luijsterburg MS, Dinant C, Lans H, et al. Heterochromatin protein 1 is recruited to various types of DNA damage. J Cell Biol. 2009;185:577–586. PubMed PMC

Sustackova G, Kozubek S, Stixova L, et al. Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin. J Cell Physiol. 2012;227:1838–1850. PubMed

Dinant C, de Jager M, Essers J, et al. Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. J Cell Sci. 2007;120:2731–2740. PubMed

Jawad MM, Qader STA, Zaidan AA, et al. An overview of laser principle, laser-tissue interaction mechanisms and laser safety precautions for medical laser users. Int J Pharmacol. 2011;7:149–160.

Kotsantis P, Silva LM, Irmscher S, et al. Increased global transcription activity as a mechanism of replication stress in cancer. Nat Commun. 2016;7:13087. PubMed PMC

Legartova S, Lochmanova G, Zdrahal Z, et al. DNA damage changes distribution pattern and levels of HP1 protein isoforms in the nucleolus and increases phosphorylation of HP1beta-Ser88. Cells. 2019;1097:8. PubMed PMC

Kumar S, Alibhai D, Margineanu A, et al. FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ. Chemphyschem. 2011;12:609–626. PubMed PMC

Lakowicz JR, Gryczynski Z. High throughput screening with multiphoton excitation. J Biomol Screen. 1999;4:355–362. PubMed PMC

Sillen A, Engelborghs Y. The correct use of “average” fluorescence parameters. Photochem Photobiol. 1998;67:475–486.

Legartova S, Jugova A, Stixova L, et al. Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin. Biochimie. 2013;95:167–179. PubMed

Sedlackova H, Rask MB, Gupta R, et al. Equilibrium between nascent and parental MCM proteins protects replicating genomes. Nature. 2020;587:297–302. PubMed

RNA-related DNA damage and repair: The role of N7-methylguanosine in the cell nucleus exposed to UV light

PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin