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• W3026131189 abstract "•HLTF mediates fork reversal in vivo, associated with DSB formation•HLTF prevents unrestrained replication driven by PRIMPOL or the TLS protein REV1•Unrestrained DNA synthesis promotes S phase progression under replication stress•HLTF loss increases cellular resistance to replication stress and ATR inhibition DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy. DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy. A variety of DNA-damaging agents, protein-DNA complexes, and DNA secondary structures can threaten genome stability by slowing replication fork progression, a condition defined as replication stress (Zeman and Cimprich, 2014Zeman M.K. Cimprich K.A. Causes and consequences of replication stress.Nat. Cell Biol. 2014; 16: 2-9Crossref PubMed Scopus (1162) Google Scholar). Nucleotide depletion induced by oncogene activation or hydroxyurea (HU) treatment also causes replication stress (Kotsantis et al., 2018Kotsantis P. Petermann E. Boulton S.J. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place.Cancer Discov. 2018; 8: 537-555Crossref PubMed Scopus (173) Google Scholar). Cells initiate a complex response to replication fork stalling that allows them to maintain fork stability and, ultimately, complete DNA replication (Cortez, 2019Cortez D. Replication-Coupled DNA Repair.Mol. Cell. 2019; 74: 866-876Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This response is tightly regulated and coordinated by the checkpoint kinase ATR, which is activated by single-stranded DNA (ssDNA)-containing DNA structures that form when replication forks stall (Saldivar et al., 2017Saldivar J.C. Cortez D. Cimprich K.A. The essential kinase ATR: ensuring faithful duplication of a challenging genome.Nat. Rev. Mol. Cell Biol. 2017; 18: 622-636Crossref PubMed Scopus (396) Google Scholar). Unresolved or persistent stalled forks are vulnerable structures susceptible to nucleolytic processing and double-strand break (DSB) formation and, ultimately, cause genome instability (Cortez, 2019Cortez D. Replication-Coupled DNA Repair.Mol. Cell. 2019; 74: 866-876Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, Pasero and Vindigni, 2017Pasero P. Vindigni A. Nucleases Acting at Stalled Forks: How to Reboot the Replication Program with a Few Shortcuts.Annu. Rev. Genet. 2017; 51: 477-499Crossref PubMed Scopus (67) Google Scholar). DNA-damage tolerance (DDT) pathways are another crucial response to replication stress (Branzei and Szakal, 2017Branzei D. Szakal B. Building up and breaking down: mechanisms controlling recombination during replication.Crit. Rev. Biochem. Mol. Biol. 2017; 52: 381-394Crossref PubMed Scopus (57) Google Scholar). Replication fork reversal is one form of DDT proposed to protect fork integrity during replication stress (Neelsen and Lopes, 2015Neelsen K.J. Lopes M. Replication fork reversal in eukaryotes: from dead end to dynamic response.Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220Crossref PubMed Scopus (285) Google Scholar). By reannealing the nascent DNA strands on each sister chromatid to form a fourth regressed arm, fork reversal actively converts the three-armed fork into a Holliday junction (HJ)-like structure. Different kinds of genotoxic stress can lead to helicase-polymerase uncoupling and ssDNA accumulation, but fork reversal restrains replication fork progression and is thought to prevent ssDNA accumulation at the fork (Neelsen and Lopes, 2015Neelsen K.J. Lopes M. Replication fork reversal in eukaryotes: from dead end to dynamic response.Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220Crossref PubMed Scopus (285) Google Scholar, Ray Chaudhuri et al., 2012Ray 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. 2012; 19: 417-423Crossref PubMed Scopus (327) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (400) Google Scholar). Fork reversal may also promote template switching and error-free lesion bypass (Cortez, 2019Cortez D. Replication-Coupled DNA Repair.Mol. Cell. 2019; 74: 866-876Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, Neelsen and Lopes, 2015Neelsen K.J. Lopes M. Replication fork reversal in eukaryotes: from dead end to dynamic response.Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220Crossref PubMed Scopus (285) Google Scholar, Saugar et al., 2014Saugar I. Ortiz-Bazán M.A. Tercero J.A. Tolerating DNA damage during eukaryotic chromosome replication.Exp. Cell Res. 2014; 329: 170-177Crossref PubMed Scopus (26) Google Scholar). Thus, it is proposed to protect and resolve stalled replication forks. Two other forms of DDT are also possible in mammalian cells. Specialized translesion synthesis (TLS) polymerases can directly bypass DNA lesions in order to resume DNA synthesis, preventing persistent replication fork stalling and, ultimately, DSB formation (Sale, 2013Sale J.E. Translesion DNA synthesis and mutagenesis in eukaryotes.Cold Spring Harb. Perspect. Biol. 2013; 5: a012708Crossref PubMed Scopus (189) Google Scholar, Saugar et al., 2014Saugar I. Ortiz-Bazán M.A. Tercero J.A. Tolerating DNA damage during eukaryotic chromosome replication.Exp. Cell Res. 2014; 329: 170-177Crossref PubMed Scopus (26) Google Scholar). Alternatively, repriming can restart DNA synthesis downstream of a stalled polymerase. In higher eukaryotes, a central effector of this process is the primase-polymerase PRIMPOL, which can utilize its DNA primase activity to reprime DNA synthesis downstream of the lesion, leaving a ssDNA gap behind the fork (Bianchi et al., 2013Bianchi J. Rudd S.G. Jozwiakowski S.K. Bailey L.J. Soura V. Taylor E. Stevanovic I. Green A.J. Stracker T.H. Lindsay H.D. et al.PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication.Mol. Cell. 2013; 52: 566-573Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, Garcia-Gómez et al., 2013Garcia-Gómez S. Reyes A. Martínez-Jiménez M.I. Chocrón E.S. Mourón S. Terrados G. Powell C. Salido E. Méndez J. Holt I.J. et al.PrimPol, an archaic primase/polymerase operating in human cells.Mol. Cell. 2013; 52: 541-553Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, Keen et al., 2014Keen B.A. Jozwiakowski S.K. Bailey L.J. Bianchi J. Doherty A.J. Molecular dissection of the domain architecture and catalytic activities of human PrimPol.Nucleic Acids Res. 2014; 42: 5830-5845Crossref PubMed Scopus (72) Google Scholar, Kobayashi et al., 2016Kobayashi K. Guilliam T.A. Tsuda M. Yamamoto J. Bailey L.J. Iwai S. Takeda S. Doherty A.J. Hirota K. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.Cell Cycle. 2016; 15: 1997-2008Crossref PubMed Scopus (63) Google Scholar, Mourón et al., 2013Mourón S. Rodriguez-Acebes S. Martínez-Jiménez M.I. Garcia-Gómez S. Chocrón S. Blanco L. Méndez J. Repriming of DNA synthesis at stalled replication forks by human PrimPol.Nat. Struct. Mol. Biol. 2013; 20: 1383-1389Crossref PubMed Scopus (190) Google Scholar, Pilzecker et al., 2016Pilzecker B. Buoninfante O.A. Pritchard C. Blomberg O.S. Huijbers I.J. van den Berk P.C. Jacobs H. PrimPol prevents APOBEC/AID family mediated DNA mutagenesis.Nucleic Acids Res. 2016; 44: 4734-4744Crossref PubMed Scopus (25) Google Scholar, Schiavone et al., 2016Schiavone D. Jozwiakowski S.K. Romanello M. Guilbaud G. Guilliam T.A. Bailey L.J. Sale J.E. Doherty A.J. PrimPol Is Required for Replicative Tolerance of G Quadruplexes in Vertebrate Cells.Mol. Cell. 2016; 61: 161-169Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Šviković et al., 2019Šviković S. Crisp A. Tan-Wong S.M. Guilliam T.A. Doherty A.J. Proudfoot N.J. Guilbaud G. Sale J.E. R-loop formation during S phase is restricted by PrimPol-mediated repriming.EMBO J. 2019; 38: e99793Crossref PubMed Scopus (56) Google Scholar, Wan et al., 2013Wan L. Lou J. Xia Y. Su B. Liu T. Cui J. Sun Y. Lou H. Huang J. hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.EMBO Rep. 2013; 14: 1104-1112Crossref PubMed Scopus (132) Google Scholar). After PRIMPOL extends the DNA primer by a few nucleotides using its polymerase activity, the replicative polymerase can continue nascent DNA synthesis. How mammalian cells choose between the alternative forms of DDT—fork reversal, TLS, and repriming—is not clear, although several proteins have been implicated in regulating these processes. Proliferating cell nuclear antigen (PCNA) is a central regulator of DDT. In yeast and higher eukaryotes, PCNA monoubiquitination promotes TLS polymerase recruitment and lesion bypass in a potentially error-prone manner (Hoege et al., 2002Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1736) Google Scholar, Sale, 2013Sale J.E. Translesion DNA synthesis and mutagenesis in eukaryotes.Cold Spring Harb. Perspect. Biol. 2013; 5: a012708Crossref PubMed Scopus (189) Google Scholar). PCNA polyubiquitination, mediated by the E3 ligase Rad5 in yeast, promotes template switching, which uses the sister chromatid as a template for error-free lesion bypass (Branzei and Szakal, 2017Branzei D. Szakal B. Building up and breaking down: mechanisms controlling recombination during replication.Crit. Rev. Biochem. Mol. Biol. 2017; 52: 381-394Crossref PubMed Scopus (57) Google Scholar, Hoege et al., 2002Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1736) Google Scholar). In mammalian cells, the E3 ubiquitin ligases HLTF and SHPRH contribute to PCNA polyubiquitination, although polyubiquitination is still observed upon the loss of both proteins (Saugar et al., 2014Saugar I. Ortiz-Bazán M.A. Tercero J.A. Tolerating DNA damage during eukaryotic chromosome replication.Exp. Cell Res. 2014; 329: 170-177Crossref PubMed Scopus (26) Google Scholar, Unk et al., 2010Unk I. Hajdu I. Blastyák A. Haracska L. Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance.DNA Repair (Amst.). 2010; 9: 257-267Crossref PubMed Scopus (133) Google Scholar). This implies that additional factors are likely involved and that DDT processes are more complex in mammalian cells. In higher eukaryotes, multiple proteins participate in fork remodeling via replication fork reversal, although the distinct contributions of each are not known (Neelsen and Lopes, 2015Neelsen K.J. Lopes M. Replication fork reversal in eukaryotes: from dead end to dynamic response.Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220Crossref PubMed Scopus (285) Google Scholar). Three regulators of the process—SMARCAL1, ZRANB3, and HLTF—are members of the SWI/SNF2 family. Each of these remodelers is capable of fork reversal in vitro (Achar et al., 2011Achar Y.J. Balogh D. Haracska L. Coordinated protein and DNA remodeling by human HLTF on stalled replication fork.Proc. Natl. Acad. Sci. USA. 2011; 108: 14073-14078Crossref PubMed Scopus (61) Google Scholar, Bansbach et al., 2009Bansbach C.E. Betous R. Lovejoy C.A. Glick G.G. Cortez D. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.Genes Dev. 2009; 23: 2405-2414Crossref PubMed Scopus (174) Google Scholar, Betous et al., 2012Betous R. Mason A.C. Rambo R.P. Bansbach C.E. Badu-Nkansah A. Sirbu B.M. Eichman B.F. Cortez D. SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.Genes Dev. 2012; 26: 151-162Crossref PubMed Scopus (196) Google Scholar, Blastyák et al., 2010Blastyák A. Hajdu I. Unk I. Haracska L. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.Mol. Cell. Biol. 2010; 30: 684-693Crossref PubMed Scopus (134) Google Scholar, Ciccia et al., 2009Ciccia A. Bredemeyer A.L. Sowa M.E. Terret M.E. Jallepalli P.V. Harper J.W. Elledge S.J. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.Genes Dev. 2009; 23: 2415-2425Crossref PubMed Scopus (151) Google Scholar, Ciccia et al., 2012Ciccia A. Nimonkar A.V. Hu Y. Hajdu I. Achar Y.J. Izhar L. Petit S.A. Adamson B. Yoon J.C. Kowalczykowski S.C. et al.Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress.Mol. Cell. 2012; 47: 396-409Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, Couch et al., 2013Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Betous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. et al.ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar, Yuan et al., 2012Yuan J. Ghosal G. Chen J. The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.Mol. Cell. 2012; 47: 410-421Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Yusufzai et al., 2009Yusufzai T. Kong X. Yokomori K. Kadonaga J.T. The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA.Genes Dev. 2009; 23: 2400-2404Crossref PubMed Scopus (105) Google Scholar), and each is recruited to the replication fork through distinct interactions (Poole and Cortez, 2017Poole L.A. Cortez D. Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability.Crit. Rev. Biochem. Mol. Biol. 2017; 52: 696-714Crossref PubMed Scopus (75) Google Scholar). Electron microscopy (EM) studies also indicate that SMARCAL1 and ZRANB3 are required for fork reversal in vivo (Kolinjivadi et al., 2017Kolinjivadi A.M. Sannino V. De Antoni A. Zadorozhny K. Kilkenny M. Techer H. Baldi G. Shen R. Ciccia A. Pellegrini L. et al.Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.Mol. Cell. 2017; 67: 867-881. e7Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, Vujanovic et al., 2017Vujanovic M. Krietsch J. Raso M.C. Terraneo N. Zellweger R. Schmid J.A. Taglialatela A. Huang J.W. Holland C.L. Zwicky K. et al.Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.Mol. Cell. 2017; 67: 882-890.e5Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (400) Google Scholar), but whether HLTF is needed in vivo has not been addressed. HLTF, like its yeast ortholog Rad5, contains an ATPase domain and an E3 ubiquitin ligase domain (Unk et al., 2010Unk I. Hajdu I. Blastyák A. Haracska L. Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance.DNA Repair (Amst.). 2010; 9: 257-267Crossref PubMed Scopus (133) Google Scholar). Both proteins also contain a HIRAN domain, which binds specifically to 3′-OH ssDNA ends. HLTF’s ATPase and 3′ ssDNA binding activities are needed for fork reversal in vitro (Achar et al., 2015Achar Y.J. Balogh D. Neculai D. Juhasz S. Morocz M. Gali H. Dhe-Paganon S. Venclovas Č. Haracska L. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.Nucleic Acids Res. 2015; 43: 10277-10291PubMed Google Scholar, Chavez et al., 2018Chavez D.A. Greer B.H. Eichman B.F. The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression.J. Biol. Chem. 2018; 293: 8484-8494Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, Hishiki et al., 2015Hishiki A. Hara K. Ikegaya Y. Yokoyama H. Shimizu T. Sato M. Hashimoto H. Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.J. Biol. Chem. 2015; 290: 13215-13223Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Kile et al., 2015Kile A.C. Chavez D.A. Bacal J. Eldirany S. Korzhnev D.M. Bezsonova I. Eichman B.F. Cimprich K.A. HLTF’s Ancient HIRAN Domain Binds 3′ DNA Ends to Drive Replication Fork Reversal.Mol. Cell. 2015; 58: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In vivo, HLTF slows replication fork progression upon nucleotide depletion, and in its absence, forks fail to slow and progress unrestrained. As the HIRAN domain is needed to restrain replication fork progression, fork reversal and fork slowing may be linked (Kile et al., 2015Kile A.C. Chavez D.A. Bacal J. Eldirany S. Korzhnev D.M. Bezsonova I. Eichman B.F. Cimprich K.A. HLTF’s Ancient HIRAN Domain Binds 3′ DNA Ends to Drive Replication Fork Reversal.Mol. Cell. 2015; 58: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Indeed, the loss of two other proteins involved in fork reversal, RAD51 and ZRANB3, also leads to unrestrained fork progression upon replication stress (Vujanovic et al., 2017Vujanovic M. Krietsch J. Raso M.C. Terraneo N. Zellweger R. Schmid J.A. Taglialatela A. Huang J.W. Holland C.L. Zwicky K. et al.Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.Mol. Cell. 2017; 67: 882-890.e5Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (400) Google Scholar). How unrestrained replication fork progression is sustained in the absence of HLTF is unknown. Increased endogenous replication stress is a hallmark of cancer cells and can be induced by nucleotide depletion or conditions that perturb DNA replication, including oncogene activation and deregulation of origin firing (Kotsantis et al., 2018Kotsantis P. Petermann E. Boulton S.J. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place.Cancer Discov. 2018; 8: 537-555Crossref PubMed Scopus (173) Google Scholar). Interestingly, HLTF is frequently silenced in colorectal cancer (Moinova et al., 2002Moinova H.R. Chen W.D. Shen L. Smiraglia D. Olechnowicz J. Ravi L. Kasturi L. Myeroff L. Plass C. Parsons R. et al.HLTF gene silencing in human colon cancer.Proc. Natl. Acad. Sci. USA. 2002; 99: 4562-4567Crossref PubMed Scopus (141) Google Scholar), and its deficiency accelerates tumorigenesis in a mouse model (Sandhu et al., 2012Sandhu S. Wu X. Nabi Z. Rastegar M. Kung S. Mai S. Ding H. Loss of HLTF function promotes intestinal carcinogenesis.Mol. Cancer. 2012; 11: 18Crossref PubMed Scopus (35) Google Scholar). This suggests that HLTF is a tumor suppressor (Dhont et al., 2016Dhont L. Mascaux C. Belayew A. The helicase-like transcription factor (HLTF) in cancer: loss of function or oncomorphic conversion of a tumor suppressor?.Cell. Mol. Life Sci. 2016; 73: 129-147Crossref PubMed Scopus (22) Google Scholar). Given HLTF’s ability to restrain DNA replication and its potential role in cancer, we sought to understand how HLTF affects the replication stress response and the role of HLTF-mediated fork remodeling in this process. Here, we report that HLTF loss limits DSB formation and promotes increased resistance to replication stress, allowing cells to continue DNA replication using PRIMPOL. Surprisingly, a specific defect in HLTF’s HIRAN domain also leads to unrestrained DNA replication and replication stress resistance, but, in this case, via REV1-mediated TLS. Our results suggest that HLTF’s activities are central to regulate replication fork reversal and to prevent alternative mechanisms of stress-resistant DNA replication that promote DNA synthesis, S phase progression, and cellular resistance to replication stress. They also demonstrate the remarkable plasticity of the replication fork in tolerating replication stress when fork reversal is disrupted. Therefore, we propose that HLTF loss may promote tumorigenesis by unleashing alternative, and potentially more mutagenic, modes of replication stress tolerance. HLTF promotes fork reversal in vitro on model replication fork structures (Achar et al., 2011Achar Y.J. Balogh D. Haracska L. Coordinated protein and DNA remodeling by human HLTF on stalled replication fork.Proc. Natl. Acad. Sci. USA. 2011; 108: 14073-14078Crossref PubMed Scopus (61) Google Scholar, Blastyák et al., 2010Blastyák A. Hajdu I. Unk I. Haracska L. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.Mol. Cell. Biol. 2010; 30: 684-693Crossref PubMed Scopus (134) Google Scholar). To test whether HTLF can also promote fork reversal in vivo, we used EM to monitor fork reversal in HLTF-KO (knockout) cell lines generated using CRISPR targeting (Figure S1A) (Kile et al., 2015Kile A.C. Chavez D.A. Bacal J. Eldirany S. Korzhnev D.M. Bezsonova I. Eichman B.F. Cimprich K.A. HLTF’s Ancient HIRAN Domain Binds 3′ DNA Ends to Drive Replication Fork Reversal.Mol. Cell. 2015; 58: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). After exposing control and HLTF-KO cells to a low dose of HU (50 μM), we isolated replication intermediates and analyzed their structure using in vivo psoralen crosslinking and EM. Reversed fork structures represented approximately 23% of the replication intermediates we observed in HU-treated wild-type (WT) cells (Figures 1A and 1B ), consistent with the number of reversed forks observed following other types of treatment (Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (400) Google Scholar). By contrast, both HLTF-KO cell lines exhibited a significant 2- to 3-fold reduction in reversed fork frequency. This finding demonstrates that HLTF is a bona fide fork-reversal protein in human cells. A lack of fork reversal in vivo is associated with unrestrained fork progression (Vujanovic et al., 2017Vujanovic M. Krietsch J. Raso M.C. Terraneo N. Zellweger R. Schmid J.A. Taglialatela A. Huang J.W. Holland C.L. Zwicky K. et al.Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.Mol. Cell. 2017; 67: 882-890.e5Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (400) Google Scholar), and our previous data suggest that HLTF loss also leads to this phenotype (Kile et al., 2015Kile A.C. Chavez D.A. Bacal J. Eldirany S. Korzhnev D.M. Bezsonova I. Eichman B.F. Cimprich K.A. HLTF’s Ancient HIRAN Domain Binds 3′ DNA Ends to Drive Replication Fork Reversal.Mol. Cell. 2015; 58: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To confirm and extend this finding, we monitored fork progression using the dose of HU used in the fork reversal assay and a dose of the DNA crosslinker, mitomycin C (MMC), which induces fork reversal in vivo (Vujanovic et al., 2017Vujanovic M. Krietsch J. Raso M.C. Terraneo N. Zellweger R. Schmid J.A. Taglialatela A. Huang J.W. Holland C.L. Zwicky K. et al.Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.Mol. Cell. 2017; 67: 882-890.e5Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Briefly, we pulse-labeled cells with the thymidine analogiododeoxyuridine (IdU), added the drug during a second chlorodeoxyuridine (CldU) pulse, and examined fork progression using DNA spreading (Figure 1C). In contrast to WT cells in which replication tracts were shortened by about 30% upon drug treatment, replication tracts in both HLTF-KO clones were unaffected and, thus, exhibited unrestrained fork progression (Figure 1D). We also observed this phenotype in chronic myelogenous leukemia K562 cells and non-cancerous retina pigmented epithelium RPE1 HLTF-KO cell lines (Figures S1A and S1B; Table S1). These findings suggest that HLTF’s ability to restrain fork progression is not cell type specific and occurs in response to multiple types of replication stress. Intrigued by the nature of the unrestrained fork progression, we next asked whether the replication observed in HLTF-KOs was continuous or whether forks might use another mode of DNA synthesis in these cells. In fact, recent studies suggest that the unrestrained replication observed in HLTF-deficient cells may be associated with discontinuous DNA replication (Peng et al., 2018Peng M. Cong K. Panzarino N.J. Nayak S. Calvo J. Deng B. Zhu L.J. Morocz M. Hegedus L. Haracska L. et al.Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress.Cell Rep. 2018; 24: 3251-3261Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To test whether replication is discontinuous in our HLTF-KO cells, we treated cells with 50 μM HU and then incubated permeabilized cells with and without S1 nuclease. This ssDNA-specific nuclease cleaves replication intermediates that contain ssDNA formed at gaps or DNA secondary structures (Quinet et al., 2016Quinet A. Martins D.J. Vessoni A.T. Biard D. Sarasin A. Stary A. Menck C.F. Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells.Nucleic Acids Res. 2016; 44: 5717-5731Crossref PubMed Scopus (38) Google Scholar, Quinet et al., 2017Quinet A. Carvajal-Maldonado D. Lemacon D. Vindigni A. DNA Fiber Analysis: Mind the Gap!.Methods Enzymol. 2017; 591: 55-82Crossref PubMed Scopus (82) Google Scholar). We found that S1 treatment specifically shortened replication tracts produced in HLTF-KO cells under HU-induced replication stress (Figure S1C). This finding strongly suggests that replication proceeds in a discontinuous way when HLTF is lost, with the production of ssDNA gaps. In higher eukaryotes, de novo priming mediated by PRIMPOL facilitates fork progression by allowing the replisome to skip over barriers, leaving a ssDNA gap behind the fork (Garcia-Gómez et al., 2013Garcia-Gómez S. Reyes A. Martínez-Jiménez M.I. Chocrón E.S. Mourón S. Terrados G. Powell C. Salido E. Méndez J. Holt I.J. et al.PrimPol, an archaic primase/polymerase operating in human cells.Mol. Cell. 2013; 52: 541-553Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, Wan et al., 2013Wan L. Lou J. Xia Y. Su B. Liu T. Cui J. Sun Y. Lou H. Huang J. hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.EMBO Rep. 2013; 14: 1104-1112Crossref PubMed Scopus (132) Google Scholar). To determine whether PRIMPOL mediates discontinuous replication in HLTF-KO cells under conditions of nucleotide d" @default.
• W3026131189 created "2020-05-29" @default.
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• W3026131189 date "2020-06-01" @default.
• W3026131189 modified "2023-10-17" @default.
• W3026131189 title "HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis" @default.
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• W3026131189 doi "https://doi.org/10.1016/j.molcel.2020.04.031" @default.

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