Helicases in R-loop Formation and Resolution
2023; Elsevier BV; Volume: 299; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2023.105307
ISSN1083-351X
AutoresShizhuo Yang, Lacey Winstone, Sohaumn Mondal, Yuliang Wu,
Tópico(s)Chromosomal and Genetic Variations
ResumoWith the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA–DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA–DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA–DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases. With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA–DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA–DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA–DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases. R-loops are three-stranded nucleic acid structures consisting of an RNA–DNA hybrid and a displaced single-stranded (ss) DNA. They play critical roles in various biological processes, including transcriptional regulation and replication, genomic instability, class switch recombination in B cells, DNA damage and repair, and telomere maintenance (1Brickner 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: 2267-2297Google Scholar, 2Petermann E. Lan L. Zou L. Sources, resolution and physiological relevance of R-loops and RNA-DNA hybrids.Nat. Rev. Mol. Cell Biol. 2022; 23: 521-540Google Scholar, 3Hegazy Y.A. Fernando C.M. Tran E.J. The balancing act of R-loop biology: the good, the bad, and the ugly.J. Biol. Chem. 2020; 295: 905-913Google Scholar, 4Niehrs C. Luke B. Regulatory R-loops as facilitators of gene expression and genome stability.Nat. Rev. Mol. Cell Biol. 2020; 21: 167-178Google Scholar, 5Garcia-Muse T. Aguilera A. R loops: from physiological to pathological roles.Cell. 2019; 179: 604-618Google Scholar). Helicases are a group of molecular motors that utilize the energy from nucleoside triphosphate hydrolysis to unwind and remodel DNA and RNA molecules, or protein–nucleic acid interactions (6Brosh Jr., R.M. Bohr V.A. Human premature aging, DNA repair and RecQ helicases.Nucleic Acids Res. 2007; 35: 7527-7544Google Scholar, 7Dillingham M.S. Superfamily I helicases as modular components of DNA-processing machines.Biochem. Soc. Trans. 2011; 39: 413-423Google Scholar, 8Bernstein K.A. Gangloff S. Rothstein R. The RecQ DNA helicases in DNA repair.Annu. Rev. Genet. 2010; 44: 393-417Google Scholar, 9Jankowsky E. RNA helicases at work: binding and rearranging.Trends Biochem. Sci. 2011; 36: 19-29Google Scholar). Notably, some helicases also possess strand annealing activity (10Wu Y. Unwinding and rewinding: double faces of helicase?.J. Nucleic Acids. 2012; 2012140601Google Scholar). This unique property allows them to play crucial roles in R-loop biogenesis. They can utilize their unwinding activity to separate double-stranded (ds) DNA, facilitate RNA invasion, or use their strand annealing activity to promote RNA to hybridize to ssDNA, enabling R-loop formation. Conversely, helicases can also utilize their unwinding activity to separate RNA–DNA hybrids to resolve R-loops. Indeed, various helicases have been implicated in R-loop assembly and disassembly. For example, SETX (Senataxin), AQR (Aquarius), WRN (Werner syndrome), BLM (Bloom syndrome), RTEL1 (Regulator of telomere elongation helicase 1), PIF1 (Petite integration factor 1), FANCM (Fanconi anemia complementation group M), ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG (CRISPR-associated DinG protein), and several DEAD/H-box proteins are reported to resolve R-loops. In contrast, helicases such as Cas3 and UPF1 (Up-frameshift protein 1) are reported to stimulate R-loop formation. Moreover, helicases such as DDX1, DDX17, and DHX9 are involved in both R-loop formation and resolution. Several excellent reviews are available for R-loops and their biological functions (1Brickner 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: 2267-2297Google Scholar, 2Petermann E. Lan L. Zou L. Sources, resolution and physiological relevance of R-loops and RNA-DNA hybrids.Nat. Rev. Mol. Cell Biol. 2022; 23: 521-540Google Scholar, 3Hegazy Y.A. Fernando C.M. Tran E.J. The balancing act of R-loop biology: the good, the bad, and the ugly.J. Biol. Chem. 2020; 295: 905-913Google Scholar, 4Niehrs C. Luke B. Regulatory R-loops as facilitators of gene expression and genome stability.Nat. Rev. Mol. Cell Biol. 2020; 21: 167-178Google Scholar, 5Garcia-Muse T. Aguilera A. R loops: from physiological to pathological roles.Cell. 2019; 179: 604-618Google Scholar). Specific enzymes in R-loop metabolism, including nuclease RNase H (11Cerritelli S.M. Sakhuja K. Crouch R.J. RNase H1, the gold standard for R-loop detection.Methods Mol. Biol. 2022; 2528: 91-114Google Scholar, 12Silva S. Guillen-Mendoza C. Aguilera A. RNase H1 hybrid-binding domain-based tools for cellular biology studies of DNA-RNA hybrids in mammalian cells.Methods Mol. Biol. 2022; 2528: 115-125Google Scholar, 13Wulfridge P. Yan Q. Sarma K. Targeted nuclease approaches for mapping native R-loops.Methods Mol. Biol. 2022; 2528: 373-380Google Scholar) and topoisomerase (14Patel P.S. Krishnan R. Hakem R. Emerging roles of DNA topoisomerases in the regulation of R-loops.Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2022; 876-877503450Google Scholar, 15Saha S. Pommier Y. R-loops, type I topoisomerases and cancer.NAR Cancer. 2023; 5: zcad013Google Scholar), have been discussed extensively. However, a comprehensive understanding of helicases in R-loop biogenesis is missing. In this review, we will summarize the latest knowledge of the roles of helicases in R-loop metabolism. In addition, this review will address the challenges related to drug discovery efforts targeting helicases and R-loops. While R-loops were first observed in vitro in 1976 (16Thomas M. White R.L. Davis R.W. Hybridization of RNA to double-stranded DNA: formation of R-loops.Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2294-2298Google Scholar), the existence of an R-loop in vivo was not reported until 1995 in bacteria (17Drolet M. Phoenix P. Menzel R. Masse E. Liu L.F. Crouch R.J. Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Google Scholar). R-loops were initially considered a by-product of transcription where the nascent RNA transcribed by RNA polymerase remains base paired with its template DNA, leaving the non-template ssDNA. Now, it is known that R-loops occur genome-wide and are present in all organisms, from bacteria to humans (18Jiang Y. Huang F. Chen L. Gu J.H. Wu Y.W. Jia M.Y. et al.Genome-wide map of R-loops reveals its interplay with transcription and genome integrity during germ cell meiosis.J Adv Res. 2022; 51: 45-57Google Scholar, 19Jauregui-Lozano J. Cottingham K. Hall H. Tissue-specific, genome-wide mapping of R-loops in Drosophila using MapR.Bio Protoc. 2022; 12e4516Google Scholar, 20Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chedin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Google Scholar, 21Scheuren M. Mohner J. Zischler H. R-loop landscape in mature human sperm: regulatory and evolutionary implications.Front. Genet. 2023; 141069871Google Scholar). Indeed, the development and wide use of CRISPR techniques demonstrate that R-loop structures exist naturally in cells. In the context of the CRISPR system, an ssRNA (guide RNA) hybrids with a targeted ssDNA, displaces an ssDNA, and forms an R-loop structure; an endonuclease Cas protein then cleaves the targeted DNA (5Garcia-Muse T. Aguilera A. R loops: from physiological to pathological roles.Cell. 2019; 179: 604-618Google Scholar, 22Crossley M.P. Bocek M. Cimprich K.A. R-loops as cellular regulators and genomic threats.Mol. Cell. 2019; 73: 398-411Google Scholar). R-loops and RNA–DNA hybrids can also form during transcription, DNA replication, double-strand breaks (DSBs) repair, and at telomeres (Fig. 1). Generally, R-loops are categorized as either physiological or pathological (1Brickner 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: 2267-2297Google Scholar, 5Garcia-Muse T. Aguilera A. R loops: from physiological to pathological roles.Cell. 2019; 179: 604-618Google Scholar). The physiological R-loops are programmed, whereas the pathological R-loops are nonprogrammed. Pathological R-loops can threaten genomic stability in various ways, such as generating transcription-replication collisions, single-stranded DNA breaks (SSBs), and DSBs (23Li X. Manley J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.Cell. 2005; 122: 365-378Google Scholar, 24Paulsen R.D. Soni D.V. Wollman R. Hahn A.T. Yee M.C. Guan A. et al.A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.Mol. Cell. 2009; 35: 228-239Google Scholar). On the other hand, the physiological functions of R-loops comprise immunoglobulin class switching of B cells in vertebrates (25Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Google Scholar), gene editing using CRISPR-Cas9 (26Jiang F. Taylor D.W. Chen J.S. Kornfeld J.E. Zhou K. Thompson A.J. et al.Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.Science. 2016; 351: 867-871Google Scholar), mitochondrial DNA replication (27Holt I.J. R-loops and mitochondrial DNA metabolism.Methods Mol. Biol. 2022; 2528: 173-202Google Scholar), specific regulatory steps in transcription (28Huertas P. Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.Mol. Cell. 2003; 12: 711-721Google Scholar), DSB repair (29Ouyang J. Yadav T. Zhang J.M. Yang H. Rheinbay E. Guo H. et al.RNA transcripts stimulate homologous recombination by forming DR-loops.Nature. 2021; 594: 283-288Google Scholar), CGG repeat contraction (30Lee H.G. Imaichi S. Kraeutler E. Aguilar R. Lee Y.W. Sheridan S.D. et al.Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation.Cell. 2023; 186: 2593-2609.e18Google Scholar), and maintaining telomere homeostasis (31Ngo G.H.P. Grimstead J.W. Baird D.M. UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.Nat. Commun. 2021; 12: 3849Google Scholar). Furthermore, RNA polymerase III can catalyze transcription at DSBs, forming a transient RNA–DNA hybrid to protect the 3ʹ overhang from degradation before replication protein A (RPA) binding, demonstrating the RNA–DNA hybrid at DSB is an essential repair intermediate in the process of homologous recombination (HR)-mediated DSB repair (32Liu S. Hua Y. Wang J. Li L. Yuan J. Zhang B. et al.RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination.Cell. 2021; 184: 1314-1329.e1310Google Scholar). These findings indicate R-loops can both cause DSB and facilitate DSB repair, leading to negative or positive outcomes depending on the molecular environment of the R-loop. Thus, R-loop homeostasis must be tightly regulated to balance its physiological and pathological roles properly. Several neurodegenerative disorders and various cancers are associated with dysregulated R-loops. For example, more than half of patients with the neuroinflammatory Aicardi-Goutières syndrome (AGS) have biallelic mutations in RNase H2 (33Crow Y.J. Chase D.S. Lowenstein Schmidt J. Szynkiewicz M. Forte G.M. Gornall H.L. et al.Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.Am. J. Med. Genet. A. 2015; 167A: 296-312Google Scholar, 34Lim Y.W. Sanz L.A. Xu X. Hartono S.R. Chedin F. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutieres syndrome.Elife. 2015; 4e08007Google Scholar). R-loop is also implicated in ataxia with oculomotor apraxia type 2 (AOA2) (35Moreira M.C. Klur S. Watanabe M. Nemeth A.H. Le Ber I. Moniz J.C. et al.Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2.Nat. Genet. 2004; 36: 225-227Google Scholar) and juvenile amyotrophic lateral sclerosis (ALS4) (36Chen Y.Z. Bennett C.L. Huynh H.M. Blair I.P. Puls I. Irobi J. et al.DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4).Am. J. Hum. Genet. 2004; 74: 1128-1135Google Scholar). AOA2 is an autosomal recessive disease associated with SETX loss of function, while ALS4 is an autosomal dominant disease provoked by toxic gain-of-function mutations in SETX (37Mersaoui S.Y. Yu Z. Coulombe Y. Karam M. Busatto F.F. Masson J.Y. et al.Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids.EMBO J. 2019; 38e100986Google Scholar). In fragile X syndrome, the expansion of CGG trinucleotide repeats in the FMR1 gene leads to R–loop formation that contributes to DNA damage and chromosomal instability (38Chakraborty A. Jenjaroenpun P. McCulley A. Li J. Hilali S.E. Haarer B. et al.Fragile X mental retardation protein regulates R-loop formation and prevents global chromosome fragility.bioRxiv. 2019; ([preprint])https://doi.org/10.1101/601906Google Scholar). Moreover, mutations in the RNA binding protein TDP-43 can cause aberrant R-loop formation, damaging DNA and contributing to ALS (39Wood M. Quinet A. Lin Y.L. Davis A.A. Pasero P. Ayala Y.M. et al.TDP-43 dysfunction results in R-loop accumulation and DNA replication defects.J. Cell Sci. 2020; 133jcs244129Google Scholar, 40Giannini M. Bayona-Feliu A. Sproviero D. Barroso S.I. Cereda C. Aguilera A. TDP-43 mutations link amyotrophic lateral sclerosis with R-loop homeostasis and R loop-mediated DNA damage.PLoS Genet. 2020; 16e1009260Google Scholar). In the context of tumorigenesis, mutations in DDX41 are associated with myeloid neoplasms myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (41Truong P. Pimanda J.E. DDX41: the poster child for familial MDS/AML grows up.Blood. 2023; 141: 447-449Google Scholar), and excessive R-loops are found in DDX41 mutated cells (42Weinreb J.T. Ghazale N. Pradhan K. Gupta V. Potts K.S. Tricomi B. et al.Excessive R-loops trigger an inflammatory cascade leading to increased HSPC production.Dev. Cell. 2021; 56: 627-640.e625Google Scholar, 43Shinriki S. Hirayama M. Nagamachi A. Yokoyama A. Kawamura T. Kanai A. et al.DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.Leukemia. 2022; 36: 2605-2620Google Scholar). Similarly, EWS-FIL1 fusion in Ewing sarcoma also induces R-loop accumulation and perturbs HR repair, potentially mediating chemosensitivity (44Gorthi A. Romero J.C. Loranc E. Cao L. Lawrence L.A. Goodale E. et al.EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma.Nature. 2018; 555: 387-391Google Scholar). Several groups have employed affinity pull-down and BioID (proximity-dependent biotin identification) techniques to identify R-loop binding proteins (45Cristini A. Groh M. Kristiansen M.S. Gromak N. RNA/DNA hybrid interactome identifies DXH9 as a molecular player in transcriptional termination and R-loop-associated DNA damage.Cell Rep. 2018; 23: 1891-1905Google Scholar, 46Wu T. Nance J. Chu F. Fazzio T.G. Characterization of R-loop-interacting proteins in embryonic stem cells reveals roles in rRNA processing and gene expression.Mol. Cell. Proteomics. 2021; 20100142Google Scholar, 47Wang I.X. Grunseich C. Fox J. Burdick J. Zhu Z. Ravazian N. et al.Human proteins that interact with RNA/DNA hybrids.Genome Res. 2018; 28: 1405-1414Google Scholar, 48Mosler T. Conte F. Longo G.M.C. Mikicic I. Kreim N. Mockel M.M. et al.R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.Nat. Commun. 2021; 12: 7314Google Scholar, 49Yan Q. Wulfridge P. Doherty J. Fernandez-Luna J.L. Real P.J. Tang H.Y. et al.Proximity labeling identifies a repertoire of site-specific R-loop modulators.Nat. Commun. 2022; 13: 53Google Scholar, 50Shinriki S. Hirayama M. Nagamachi A. Yokoyama A. Kawamura T. Kanai A. et al.DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.Leukemia. 2022; 36: 2605Google Scholar) (Table S1). Kumar et al. (51Kumar A. Fournier L.A. Stirling P.C. Integrative analysis and prediction of human R-loop binding proteins.G3 (Bethesda). 2022; 12jkac142Google Scholar) analyzed the five datasets and found only 12 common R-loop binding proteins: DDX5, NAT10, NPM1, NOP2, DDX18, NOP58, ALYREF, U2AF1, ILF3, RBM14, PDCD11, and MYBBP1A. More recently, Marchena-Cruz et al. used siRNA screening coupled with AID-induced DSBs as a readout and identified 46 proteins that affect R-loop homeostasis (52Marchena-Cruz E. Camino L.P. Bhandari J. Silva S. Marqueta-Gracia J.J. Amdeen S.A. et al.DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops.Cell Rep. 2023; 42112148Google Scholar) (Table S1). These seven datasets revealed varying results, with no single protein present in all datasets. Using similar approaches, such as affinity pull-down with S9.6 antibody by Cristini et al. (45Cristini A. Groh M. Kristiansen M.S. Gromak N. RNA/DNA hybrid interactome identifies DXH9 as a molecular player in transcriptional termination and R-loop-associated DNA damage.Cell Rep. 2018; 23: 1891-1905Google Scholar) and Wu et al. (46Wu T. Nance J. Chu F. Fazzio T.G. Characterization of R-loop-interacting proteins in embryonic stem cells reveals roles in rRNA processing and gene expression.Mol. Cell. Proteomics. 2021; 20100142Google Scholar), affinity pull-down with Myc-tagged RNase H (50Shinriki S. Hirayama M. Nagamachi A. Yokoyama A. Kawamura T. Kanai A. et al.DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.Leukemia. 2022; 36: 2605Google Scholar), or BioID with RNase H as a bait by Mosler et al. (48Mosler T. Conte F. Longo G.M.C. Mikicic I. Kreim N. Mockel M.M. et al.R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.Nat. Commun. 2021; 12: 7314Google Scholar) and Yan et al. (49Yan Q. Wulfridge P. Doherty J. Fernandez-Luna J.L. Real P.J. Tang H.Y. et al.Proximity labeling identifies a repertoire of site-specific R-loop modulators.Nat. Commun. 2022; 13: 53Google Scholar), their datasets are largely different (Table S1). Collectively, these results suggest R-loop binding proteins are highly variable depending on study conditions and thus additional investigations are required to fully elucidate the identity of R-loop binding proteins. The diversity of identified R-loop binding proteins can be attributed to several factors. Firstly, different methods employed in these studies may lead to different R-loop binding proteins. For instance, affinity pull-down can capture stable protein complexes but not proteins with weak or transient interactions. While BioID can capture weak or transient binding partners, the increased sensitivity may also detect potential indirect interactions with R-loops. Secondly, flaws in different methods may yield false positive—non-specific binding proteins. Indeed, the S9.6 antibody has a non-specific binding with dsRNA (53Smolka J.A. Sanz L.A. Hartono S.R. Chedin F. Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.J. Cell Biol. 2021; 220e202004079Google Scholar, 54Crossley M.P. Brickner J.R. Song C. Zar S.M.T. Maw S.S. Chedin F. et al.Catalytically inactive, purified RNase H1: a specific and sensitive probe for RNA-DNA hybrid imaging.J. Cell Biol. 2021; 220e202101092Google Scholar), RNase H consists of two enzymes (H1 and H2) with specificities in the cell cycle and subcellular location (55Holmes J.B. Akman G. Wood S.R. Sakhuja K. Cerritelli S.M. Moss C. et al.Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 9334-9339Google Scholar, 56Lockhart A. Pires V.B. Bento F. Kellner V. Luke-Glaser S. Yakoub G. et al.RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids.Cell Rep. 2019; 29: 2890-2900.e2895Google Scholar), and AID-based screening may detect ssDNA other than R-loops (52Marchena-Cruz E. Camino L.P. Bhandari J. Silva S. Marqueta-Gracia J.J. Amdeen S.A. et al.DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops.Cell Rep. 2023; 42112148Google Scholar). Thirdly, the different cell types used in these studies such as HeLa, HEK293, U2OS, human B cells, and mouse embryonic stem cells may contribute to variable results. Finally, there may be a dynamic change of R-loops in cells and thus the associated R-loop binding proteins may also undergo dynamic changes, resulting in increased diversity amongst different datasets. Nevertheless, despite the different observations in R-loop binding proteins, helicases are consistently identified in all studies (Table S1, highlighted in yellow). RNase H1 and H2 are nucleases that specifically target and degrade the RNA molecule within RNA–DNA hybrid of R-loops (57Hyjek M. Figiel M. Nowotny M. RNases H: structure and mechanism.DNA Repair (Amst). 2019; 84102672Google Scholar). RNase H1 is primarily localized in the mitochondria (55Holmes J.B. Akman G. Wood S.R. Sakhuja K. Cerritelli S.M. Moss C. et al.Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 9334-9339Google Scholar), whereas RNase H2 displays G2/M-phase specific expression (56Lockhart A. Pires V.B. Bento F. Kellner V. Luke-Glaser S. Yakoub G. et al.RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids.Cell Rep. 2019; 29: 2890-2900.e2895Google Scholar). We believe a large number of nucleases that target and degrade ssDNA or RNA released from RNA–DNA hybrids by helicases would be more robust in eliminating R-loops. Consistent with the initial observation that R-loops are a threat to genome stability, many helicases have been reported to prevent R-loop accumulation, including SETX, AQR, WRN, BLM, FANCM, SMARCAL, PIF1, DDX5, ATRX, and CasDinG (Table 1).Table 1Helicases involved in R-loop metabolismHelicase familyNameRole in R-loopDisease or main phenotype/biological functionNoteReferencesForm R-loopResolve R-loopHumanYeastSF1SETXSen1√AOA2 and ALS4(58Mischo H.E. Gomez-Gonzalez B. Grzechnik P. Rondon A.G. Wei W. Steinmetz L. et al.Yeast Sen1 helicase protects the genome from transcription-associated instability.Mol. Cell. 2011; 41: 21-32Google Scholar, 149Alzu A. Bermejo R. Begnis M. Lucca C. Piccini D. Carotenuto W. et al.Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes.Cell. 2012; 151: 835-846Google Scholar)AQR√Splicing factorAka IBP160(150Sollier J. Stork C.T. Garcia-Rubio M.L. Paulsen R.D. Aguilera A. Cimprich K.A. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability.Mol. Cell. 2014; 56: 777-785Google Scholar)UPF1Upf1√Nonsense mRNA degradation surveillance(31Ngo G.H.P. Grimstead J.W. Baird D.M. UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.Nat. Commun. 2021; 12: 3849Google Scholar)PIF1Rrm3, ScPif1, SpPfh1√Genomic instability in the nucleus and mitochondriaS. cerevisiae has two members: Rrm3 and ScPif1; S. pombe has one: Pfh1(151Tran P.L.T. Pohl T.J. Chen C.F. Chan A. Pott S. Zakian V.A. PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes.Nat. Commun. 2017; 815025Google Scholar)SF2WRN√Werner syndrome(63Marabitti V. Lillo G. Malacaria E. Palermo V. Sanchez M. Pichierri P. et al.ATM pathway activation limits R-loop-associated genomic instability in Werner syndrome cells.Nucleic Acids Res. 2019; 47: 3485-3502Google Scholar)BLMSgs1√Bloom syndrome(65Chang E.Y. Novoa C.A. Aristizabal M.J. Coulombe Y. Segovia R. Chaturvedi R. et al.RECQ-like helicases Sgs1 and BLM regulate R-loop-associated genome instability.J. Cell Biol. 2017; 216: 3991-4005Google Scholar)RTEL1√Telomere shorteningInteracts with Poldip3(68Bjorkman A. Johansen S.L. Lin L. Schertzer M. Kanellis D.C. Katsori A.M. et al.Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.Genes Dev. 2020; 34: 1065-1074Google Scholar)FANCMMph1√Congenital abnormalities, pancytopenia, infertility, and cancer proneness(70Schwab R.A. Nieminuszczy J. Shah F. Langton J. Lopez Martinez D. Liang C.C. et al.The fanconi anemia pathway maintains genome stability by coordinating replication and transcription.Mol. Cell. 2015; 60: 351-361Google Scholar, 71Hodson C. van Twest S. Dylewska M. O'Rourke J.J. Tan W. Murphy V.J. et al.Branchpoint translocation by fork remodelers as a general mechanism of R-loop removal.Cell Rep. 2022; 41111749Google Scholar, 152Lafuente-Barquero J. 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