Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSβ, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSβ, MutLβ, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.

Department of Biology and National Centre for Biomolecular Research Masaryk University Kamenice 5 A7 Brno 62500 Czech Republic

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 143 00 Prague Czech Republic

International Clinical Research Center St Anne's University Hospital Pekarska 53 Brno 656 91 Czech Republic

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Zeman M. K., Cimprich K. A., Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014). PubMed PMC

Garcia-Muse T., Aguilera A., Transcription-replication conflicts: How they occur and how they are resolved. Nat. Rev. Mol. Cell Biol. 17, 553–563 (2016). PubMed

Macheret M., Halazonetis T. D., Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress. Nature 555, 112–116 (2018). PubMed PMC

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 170, 774–786.e19 (2017). PubMed PMC

Lang K. S., Hall A. N., Merrikh C. N., Ragheb M., Tabakh H., Pollock A. J., Woodward J. J., Dreifus J. E., Merrikh H., Replication-transcription conflicts generate R-Loops that orchestrate bacterial stress survival and pathogenesis. Cell 170, 787–799.e18 (2017). PubMed PMC

Chappidi N., Nascakova Z., Boleslavska B., Zellweger R., Isik E., Andrs M., Menon S., Dobrovolna J., Balbo Pogliano C., Matos J., Porro A., Lopes M., Janscak P., Fork cleavage-religation cycle and active transcription mediate replication restart after fork stalling at co-transcriptional R-loops. Mol. Cell 77, 528–541.e8 (2020). 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 82, 2267–2297 (2022). PubMed PMC

Hamperl S., Cimprich K. A., The contribution of co-transcriptional RNA: DNA hybrid structures to DNA damage and genome instability. DNA Repair 19, 84–94 (2014). 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. 21, 633–651 (2020). PubMed

Castillo-Guzman D., Chédin F., Defining R-loop classes and their contributions to genome instability. DNA Repair 106, 103182 (2021). 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. 116, 816–825 (2019). PubMed PMC

Duquette M. L., Handa P., Vincent J. A., Taylor A. F., Maizels N., Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev. 18, 1618–1629 (2004). PubMed PMC

Castillo Bosch P., Segura-Bayona S., Koole W., Heteren J. T., Dewar J. M., Tijsterman M., Knipscheer P., FANCJ promotes DNA synthesis through G-quadruplex structures. EMBO J. 33, 2521–2533 (2014). PubMed PMC

Mendoza O., Bourdoncle A., Boulé J. B., Brosh R. M. Jr., Mergny J. L., G-quadruplexes and helicases. Nucleic Acids Res. 44, 1989–2006 (2016). PubMed PMC

Sakellariou D., Bak S. T., Isik E., Barroso S. I., Porro A., Aguilera A., Bartek J., Janscak P., Peña-Diaz J., MutSβ regulates G4-associated telomeric R-loops to maintain telomere integrity in ALT cancer cells. Cell Rep. 39, 110602 (2022). PubMed

Jiricny J., The multifaceted mismatch-repair system. Nat. Rev. Mol. Cell Biol. 7, 335–346 (2006). PubMed

Habraken Y., Sung P., Prakash L., Prakash S., Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3. Curr. Biol. 6, 1185–1187 (1996). PubMed

Modrich P., Mechanisms in eukaryotic mismatch repair. J. Biol. Chem. 281, 30305–30309 (2006). PubMed PMC

Kadyrov F. A., Dzantiev L., Constantin N., Modrich P., Endonucleolytic function of mutlα in human mismatch repair. Cell 126, 297–308 (2006). PubMed

Räschle M., Marra G., Nyström-Lahti M., Schär P., Jiricny J., Identification of hMutLβ, a heterodimer of hMLH1 and hPMS1. J. Biol. Chem. 274, 32368–32375 (1999). PubMed

Cannavo E., Marra G., Sabates-Bellver J., Menigatti M., Lipkin S. M., Fischer F., Cejka P., Jiricny J., Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair. Cancer Res. 65, 10759–10766 (2005). PubMed

Lipkin S. M., Moens P. B., Wang V., Lenzi M., Shanmugarajah D., Gilgeous A., Thomas J., Cheng J., Touchman J. W., Green E. D., Schwartzberg P., Collins F. S., Cohen P. E., Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat. Genet. 31, 385–390 (2002). PubMed

Pinto R. M., Dragileva E., Kirby A., Lloret A., Lopez E., St. Claire J., Panigrahi G. B., Hou C., Holloway K., Gillis T., Guide J. R., Cohen P. E., Li G. M., Pearson C. E., Daly M. J., Wheeler V. C., Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in huntington’s disease mice: Genome-wide and candidate approaches. PLOS Genet. 9, e1003930 (2013). PubMed PMC

Zhao X., Zhang Y., Wilkins K., Edelmann W., Usdin K., MutLγ promotes repeat expansion in a Fragile X mouse model while EXO1 is protective. PLOS Genet. 14, e1007719 (2018). PubMed PMC

Teloni F., Michelena J., Lezaja A., Kilic S., Ambrosi C., Menon S., Dobrovolna J., Imhof R., Janscak P., Baubec T., Altmeyer M., Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability. Mol. Cell 73, 670–683.e12 (2019). PubMed PMC

Bauer M., Nascakova Z., Mihai A. I., Cheng P. F., Levesque M. P., Lampart S., Hurwitz R., Pfannkuch L., Dobrovolna J., Jacobs M., Bartfeld S., Dohlman A., Shen X., Gall A. A., Salama N. R., Töpfer A., Weber A., Meyer T. F., Janscak P., Müller A., The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress. Nat. Commun. 11, 5117 (2020). PubMed PMC

Lossaint G., Larroque M., Ribeyre C., Bec N., Larroque C., Decaillet C., Gari K., Constantinou A., FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling. Mol. Cell 51, 678–690 (2013). PubMed

Ying S., Minocherhomji S., Chan K. L., Palmai-Pallag T., Chu W. K., Wass T., Mankouri H. W., Liu Y., Hickson I. D., MUS81 promotes common fragile site expression. Nat. Cell Biol. 15, 1001–1007 (2013). PubMed

Naim V., Wilhelm T., Debatisse M., Rosselli F., ERCC1 and MUS81-EME1 promote sister chromatid separation by processing late replication intermediates at common fragile sites during mitosis. Nat. Cell Biol. 15, 1008–1015 (2013). PubMed

Berti M., Chaudhuri A. R., Thangavel S., Gomathinayagam S., Kenig S., Vujanovic M., Odreman F., Glatter T., Graziano S., Mendoza-Maldonado R., Marino F., Lucic B., Biasin V., Gstaiger M., Aebersold R., Sidorova J. M., Monnat R. J., Lopes M., Vindigni A., Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 20, 347–354 (2013). PubMed PMC

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

Kotsantis P., Segura-Bayona S., Margalef P., Marzec P., Ruis P., Hewitt G., Bellelli R., Patel H., Goldstone R., Poetsch A. R., Boulton S. J., RTEL1 regulates G4/R-loops to avert replication-transcription collisions. Cell Rep. 33, 108546 (2020). PubMed PMC

Gupta S., Gellert M., Yang W., Mechanism of mismatch recognition revealed by human MutSβ bound to unpaired DNA loops. Nat. Struct. Mol. Biol. 19, 72–78 (2012). PubMed PMC

Burdova K., Mihaljevic B., Sturzenegger A., Chappidi N., Janscak P., The mismatch-binding factor MutSβ can mediate ATR activation in response to DNA double-strand breaks. Mol. Cell 59, 603–614 (2015). PubMed

Johnson J., Okyere R., Joseph A., Musier-Forsyth K., Kankia B., Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs. Nucleic Acids Res. 41, 220–228 (2013). PubMed PMC

Barroso S., Herrera-Moyano E., Muñoz S., García-Rubio M., Gómez-González B., Aguilera A., The DNA damage response acts as a safeguard against harmful DNA–RNA hybrids of different origins. EMBO Rep. 20, 47250 (2019). PubMed PMC

Cannavo E., Gerrits B., Marra G., Schlapbach R., Jiricny J., Characterization of the interactome of the human MutL homologues MLH1, PMS1, and PMS2. J. Biol. Chem. 282, 2976–2986 (2007). PubMed

Peng M., Litman R., Xie J., Sharma S., Brosh R. M. Jr., Cantor S. B., The FANCJ/MutLα interaction is required for correction of the cross-link response in FA-J cells. EMBO J. 26, 3238–3249 (2007). PubMed PMC

Odermatt D. C., Lee W. T. C., Wild S., Jozwiakowski S. K., Rothenberg E., Gari K., Cancer-associated mutations in the ironsulfur domain of FANCJ affect G-quadruplex metabolism. PLOS Genet. 16, 1008740 (2020). PubMed PMC

Wu Y., Shin-ya K., Brosh R. M. Jr., FANCJ Helicase defective in fanconia anemia and Breast Cancer unwinds G-Quadruplex DNA to defend genomic stability. Mol. Cell. Biol. 28, 4116–4128 (2008). PubMed PMC

Suhasini A. N., Sommers J. A., Muniandy P. A., Coulombe Y., Cantor S. B., Masson J.-Y., Seidman M. M., Brosh R. M. Jr., Fanconi anemia group J helicase and MRE11 nuclease interact to facilitate the DNA damage response. Mol. Cell. Biol. 33, 2212–2227 (2013). PubMed PMC

Guillemette S., Branagan A., Peng M., Dhruva A., Scharer O. D., Cantor S. B., FANCJ localization by mismatch repair is vital to maintain genomic integrity after UV irradiation. Cancer Res. 74, 932–944 (2014). 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. 50, 12274–12290 (2022). PubMed PMC

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

Wu C. G., Spies M., G-quadruplex recognition and remodeling by the FANCJ helicase. Nucleic Acids Res. 44, 8742–8753 (2016). PubMed PMC

Peng M., Cong K., Panzarino N. J., Nayak S., Calvo J., Deng B., Zhu L. J., Morocz M., Hegedus L., Haracska L., Cantor S. B., Opposing roles of FANCJ and HLTF protect forks and restrain replication during stress. Cell Rep. 24, 3251–3261 (2018). PubMed PMC

Andrs M., Stoy H., Boleslavska B., Chappidi N., Kanagaraj R., Nascakova Z., Menon S., Rao S., Oravetzova A., Dobrovolna J., Surendranath K., Lopes M., Janscak P., Excessive reactive oxygen species induce transcription-dependent replication stress. Nat. Commun. 14, 1791 (2023). PubMed PMC

Lee W. T. C., Yin Y., Morten M. J., Tonzi P., Gwo P. P., Odermatt D. C., Modesti M., Cantor S. B., Gari K., Huang T. T., Rothenberg E., Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling. Nat. Commun. 12, 2525 (2021). 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. 11, e1005674 (2015). PubMed PMC

Vujanovic M., Krietsch J., Raso M. C., Terraneo N., Zellweger R., Schmid J. A., Taglialatela A., Huang J. W., Holland C. L., Zwicky K., Herrador R., Jacobs H., Cortez D., Ciccia A., Penengo L., Lopes M., Replication Fork slowing and reversal upon DNA damage require PCNA polyubiquitination and ZRANB3 DNA translocase activity. Mol. Cell 67, 882–890.e5 (2017). 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. 5, 4990 (2014). PubMed

Tighe A., Staples O., Taylor S., Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores. J. Cell Biol. 181, 893–901 (2008). PubMed PMC

Henricksen L. A., Umbricht C. B., Wold M. S., Recombinant replication protein A: Expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994). PubMed

Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation