Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

Institute of Molecular Cancer Research University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland

Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Zobrazit více v PubMed

Zeman M.K., Cimprich K.A.. Causes and consequences of replication stress. Nat. Cell Biol. 2013; 16:2–9. PubMed PMC

Berti M., Cortez D., Lopes M.. The plasticity of DNA replication forks in response to clinically relevant genotoxic stress. Nat. Rev. Mol. Cell Biol. 2020; 21:633–651. PubMed

Brickner J.R., Garzon J.L., Cimprich K.A.. Walking a tightrope: the complex balancing act of R-loops in genome stability. Mol. Cell. 2022; 82:2267–2297. PubMed PMC

Hamperl S., Cimprich K.A.. The contribution of co-transcriptional RNA: DNA hybrid structures to DNA damage and genome instability. DNA Repair (Amst.). 2014; 19:84–94. PubMed PMC

Castillo-Guzman D., Chédin F.. Defining R-loop classes and their contributions to genome instability. DNA Repair (Amst.). 2021; 106:103182. PubMed PMC

De Magis A., Manzo S.G., Russo M., Marinello J., Morigi R., Sordet O., Capranico G.. DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2019; 116:816–825. PubMed PMC

Helmrich A., Ballarino M., Nudler E., Tora L.. Transcription-replication encounters, consequences and genomic instability. Nat. Struct. Mol. Biol. 2013; 20:412–418. PubMed

Hamperl S., Bocek M.J., Saldivar J.C., Swigut T., Cimprich K.A.. Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses. Cell. 2017; 170:774–786. PubMed PMC

Andrs M., Stoy H., Boleslavska B., Chappidi N., Kanagaraj R., Nascakova Z., Menon S., Rao S., Oravetzova A., Dobrovolna J.et al. .. Excessive reactive oxygen species induce transcription-dependent replication stress. Nat. Commun. 2023; 14:1791. PubMed PMC

Chappidi N., Nascakova Z., Boleslavska B., Zellweger R., Isik E., Andrs M., Menon S., Dobrovolna J., Balbo Pogliano C., Matos J.et al. .. Fork cleavage-religation cycle and active transcription mediate replication restart after Fork stalling at Co-transcriptional R-loops. Mol. Cell. 2020; 77:528–541. PubMed

Berti M., Chaudhuri A.R., Thangavel S., Gomathinayagam S., Kenig S., Vujanovic M., Odreman F., Glatter T., Graziano S., Mendoza-Maldonado R.et al. .. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 2013; 20:347–354. PubMed PMC

Di Marco S., Hasanova Z., Kanagaraj R., Chappidi N., Altmannova V., Menon S., Sedlackova H., Langhoff J., Surendranath K., Hühn D.et al. .. RECQ5 Helicase cooperates with MUS81 endonuclease in processing stalled replication forks at common fragile sites during mitosis. Mol. Cell. 2017; 66:658–671. PubMed

Isik E., Shukla K., Pospisilova M., König C., Andrs M., Rao S., Rosano V., Dobrovolna J., Krejci L., Janscak P.. MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops. Sci. Adv. 2024; 10:eadk2685. PubMed PMC

Boleslavska B., Oravetzova A., Shukla K., Nascakova Z., Ibini O.N., Hasanova Z., Andrs M., Kanagaraj R., Dobrovolna J., Janscak P.. DDX17 helicase promotes resolution of R-loop-mediated transcription – replication conflicts in human cells. Nucleic Acids Res. 2022; 50:12274–12290. PubMed PMC

Linder P., Jankowsky E.. From unwinding to clamping - the DEAD box RNA helicase family. Nat. Rev. Mol. Cell Biol. 2011; 12:505–516. PubMed

Stoy H., Zwicky K., Kuster D., Lang K.S., Krietsch J., Crossley M.P., Schmid J.A., Cimprich K.A., Merrikh H., Lopes M.. Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids. Nat. Struct. Mol. Biol. 2023; 30:348–359. PubMed PMC

Hasanova Z., Klapstova V., Porrua O., Stefl R., Sebesta M.. Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor. Nucleic Acids Res. 2023; 51:2818–2837. PubMed PMC

Groh M., Albulescu L.O., Cristini A., Gromak N.. Senataxin: genome guardian at the interface of transcription and Neurodegeneration. J. Mol. Biol. 2017; 429:3181–3195. PubMed

Gatti V., De Domenico S., Melino G., Peschiaroli A.. Senataxin and R-loops homeostasis: multifaced implications in carcinogenesis. Cell Death Discov. 2023; 9:145. PubMed PMC

Chen Y.Z., Bennett C.L., Huynh H.M., Blair I.P., Puls I., Irobi J., Dierick I., Abel A., Kennerson M.L., Rabin B.A.et al. .. DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am. J. Hum. Genet. 2004; 74:1128–1135. PubMed PMC

Moreira M.C., Klur S., Watanabe M., Németh A.H., Le Ber I., Moniz J.C., Tranchant C., Aubourg P., Tazir M., Schöls L.et al. .. Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2. Nat. Genet. 2004; 36:225–227. PubMed

Skourti-Stathaki K., Proudfoot N.J., Gromak N.. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol. Cell. 2011; 42:794–805. PubMed PMC

Kanagaraj R., Mitter R., Kantidakis T., Edwards M.M., Benitez A., Chakravarty P., Fu B., Becherel O., Yang F., Lavin M.F.et al. .. Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2. Proc. Natl. Acad. Sci. U.S.A. 2022; 119:2114314119. PubMed PMC

Hatchi E., Skourti-Stathaki K., Ventz S., Pinello L., Yen A., Kamieniarz-Gdula K., Dimitrov S., Pathania S., McKinney K.M., Eaton M.L.et al. .. BRCA1 Recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair. Mol. Cell. 2015; 57:636–647. PubMed PMC

Alzu A., Bermejo R., Begnis M., Lucca C., Piccini D., Carotenuto W., Saponaro M., Brambati A., Cocito A., Foiani M.et al. .. Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes. Cell. 2012; 151:835–846. PubMed PMC

Appanah R., Lones E.C., Aiello U., Libri D., De Piccoli G.. Sen1 Is recruited to replication forks via Ctf4 and Mrc1 and promotes genome stability. Cell Rep. 2020; 30:2094–2105. PubMed PMC

Yüce Ö., West S.C.. Senataxin, defective in the neurodegenerative disorder ataxia with oculomotor apraxia 2, lies at the interface of transcription and the DNA damage response. Mol. Cell. Biol. 2013; 33:406–417. PubMed PMC

Said M., Barra V., Balzano E., Talhaoui I., Pelliccia F., Giunta S., Naim V.. FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells. Commun. Biol. 2022; 5:1395. PubMed PMC

Richard P., Feng S., Manley J.L.. A SUMO-dependent interaction between Senataxin and the exosome, disrupted in the neurodegenerative disease AOA2, targets the exosome to sites of transcription-induced DNA damage. Genes Dev. 2013; 27:2227–2232. PubMed PMC

Ghodgaonkar M.M., Kehl P., Ventura I., Hu L., Bignami M., Jiricny J.. Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system. Nat. Commun. 2014; 5:4990. PubMed

Cottarel J., Frit P., Bombarde O., Salles B., Negrel A., Bernard S., Jeggo P.A., Lieber M.R., Modesti M., Calsou P.. A noncatalytic function of the ligation complex during nonhomologous end joining. J. Cell Biol. 2013; 200:173–186. PubMed PMC

Mischo H.E., Gómez-González B., Grzechnik P., Rondón A.G., Wei W., Steinmetz L., Aguilera A., Proudfoot N.J.. Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol. Cell. 2011; 41:21–32. PubMed PMC

Matsuoka S., Ballif B.A., Smogorzewska A., McDonald E.R., Hurov K.E., Luo J., Bakalarski C.E., Zhao Z., Solimini N., Lerenthal Y.et al. .. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science. 2007; 316:1160–1166. PubMed

Jackson D.A., Pombo A.. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in Human cells. J. Cell Biol. 1998; 140:1285–1295. PubMed PMC

Teloni F., Michelena J., Lezaja A., Kilic S., Ambrosi C., Menon S., Dobrovolna J., Imhof R., Janscak P., Baubec T.et al. .. Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability. Mol. Cell. 2019; 73:670–683. PubMed PMC

Minocherhomji S., Ying S., Bjerregaard V.A., Bursomanno S., Aleliunaite A., Wu W., Mankouri H.W., Shen H., Liu Y., Hickson I.D.. Replication stress activates DNA repair synthesis in mitosis. Nature. 2015; 528:286–290. PubMed

Wu W., Bhowmick R., Vogel I., Özer Ö., Ghisays F., Thakur R.S., de Leon E.S., Richter P.H., Ren L., Petrini J.H.et al. .. RTEL1 suppresses G-quadruplex-associated R-loops at difficult-to-replicate loci in the human genome. Nat. Struct. Mol. Biol. 2020; 27:424–437. PubMed

Krupina K., Goginashvili A., Cleveland D.W.. Causes and consequences of micronuclei. Curr. Opin. Cell Biol. 2021; 70:91–99. PubMed PMC

Lukas C., Savic V., Bekker-Jensen S., Doil C., Neumann B., Pedersen R.S., Grøhfte M., Chan K.L., Hickson I.D., Bartek J.et al. .. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 2011; 13:243–253. PubMed

Ray Chaudhuri A., Hashimoto Y., Herrador R., Neelsen K.J., Fachinetti D., Bermejo R., Cocito A., Costanzo V., Lopes M., Chaudhuri A.R.et al. .. Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat. Struct. Mol. Biol. 2012; 19:417–423. PubMed

Schlacher K., Christ N., Siaud N., Egashira A., Wu H., Jasin M.. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell. 2011; 145:529–542. PubMed PMC

Mijic S., Zellweger R., Chappidi N., Berti M., Jacobs K., Mutreja K., Ursich S., Ray Chaudhuri A., Nussenzweig A., Janscak P.et al. .. Replication fork reversal triggers fork degradation in BRCA2-defective cells. Nat. Commun. 2017; 8:859. PubMed PMC

Przetocka S., Porro A., Bolck H.A., Walker C., Lezaja A., Trenner A., von Aesch C., Himmels S.F., D’Andrea A.D., Ceccaldi R.et al. .. CtIP-mediated fork protection synergizes with BRCA1 to suppress genomic instability upon DNA replication stress. Mol. Cell. 2018; 72:568–582. PubMed

Liu W., Krishnamoorthy A., Zhao R., Cortez D.. Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors. Sci. Adv. 2020; 6:eabc3598. PubMed PMC

Miller M.S., Rialdi A., Ho J.S.Y., Tilove M., Martinez-Gil L., Moshkina N.P., Peralta Z., Noel J., Melegari C., Maestre A.M.et al. .. Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis. Nat. Immunol. 2015; 16:485–494. PubMed PMC

García-Rubio M.L., Pérez-Calero C., Barroso S.I., Tumini E., Herrera-Moyano E., Rosado I.V., Aguilera A.. The Fanconi anemia pathway protects genome integrity from R-loops. PLoS Genet. 2015; 11:1005674. PubMed PMC

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

San Martin-Alonso M., Soler-Oliva M.E., García-Rubio M., García-Muse T., Aguilera A.. Harmful R-loops are prevented via different cell cycle-specific mechanisms. Nat. Commun. 2021; 12:4451. PubMed PMC

Lemaçon D., Jackson J., Quinet A., Brickner J.R., Li S., Yazinski S., You Z., Ira G., Zou L., Mosammaparast N.et al. .. MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat. Commun. 2017; 8:860. PubMed PMC