Skip to main content
SpringerLink
Log in

Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s−1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Noer SL, Preus S, Gudnason D, Aznauryan M, Mergny JL, Birkedal V. Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy. Nucleic Acids Res. 2016;44(1):464–71. https://doi.org/10.1093/nar/gkv1320.

    Article  PubMed  CAS  Google Scholar 

  2. Li YY, Dubins DN, Le D, Leung K, Macgregor RB Jr. The role of loops and cation on the volume of unfolding of G-quadruplexes related to HTel. Biophys Chem. 2017;231:55–63. https://doi.org/10.1016/j.bpc.2016.12.003.

    Article  PubMed  CAS  Google Scholar 

  3. Lipps HJ, Rhodes D. G-quadruplex structures: in vivo evidence and function. Trends Cell Biol. 2009;19(8):414–22. https://doi.org/10.1016/j.tcb.2009.05.002.

    Article  PubMed  CAS  Google Scholar 

  4. De Cian A, Cristofari G, Reichenbach P, De Lemos E, Monchaud D, Teulade-Fichou MP, et al. Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action. P Natl Acad Sci USA. 2007;104(44):17347–52. https://doi.org/10.1073/pnas.0707365104.

    Article  Google Scholar 

  5. Maizels N. G4-associated human diseases. EMBO Rep. 2015;16(8):910–22. doi:https://doi.org/10.15252/embr.201540607.

  6. Wu YL, Shin-ya K, Brosh RM. FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol Cell Biol. 2008;28(12):4116–28.

    Article  CAS  Google Scholar 

  7. Tuesuwan B, Kern JT, Thomas PW, Rodriguez M, Li J, David WM, et al. Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents. Biochemistry-Us. 2008;47(7):1896–909. https://doi.org/10.1021/bi701747d.

    Article  CAS  Google Scholar 

  8. Plyler J, Jasheway K, Tuesuwan B, Karr J, Brennan JS, Kerwin SM, et al. Real-time investigation of SV40 large T-antigen helicase activity using surface plasmon resonance. Cell Biochem Biophys. 2009;53(1):43–52. https://doi.org/10.1007/s12013-008-9038-z.

    Article  PubMed  CAS  Google Scholar 

  9. Huber MD, Lee DC, Maizels N. G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition. Nucleic Acids Res. 2002;30(18):3954–61.

    Article  CAS  Google Scholar 

  10. Cheok CF, Wu L, Garcia PL, Janscak P, Hickson ID. The Bloom’s syndrome helicase promotes the annealing of complementary single-stranded DNA. Nucleic Acids Res. 2005;33(12):3932–41.

    Article  CAS  Google Scholar 

  11. Keller H, Kiosze K, Sachsenweger J, Haumann S, Ohlenschlager O, Nuutinen T, et al. The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding. Nucleic Acids Res. 2014;42(20):12614–27.

    Article  CAS  Google Scholar 

  12. Li JL, Harrison RJ, Reszka AP, Brosh RM Jr, Bohr VA, Neidle S, et al. Inhibition of the Bloom’s and Werner’s syndrome helicases by G-quadruplex interacting ligands. Biochemistry-Us. 2001;40(50):15194–202.

    Article  CAS  Google Scholar 

  13. Yangyuoru PM, Bradburn DA, Liu Z, Xiao TS, Russell R. The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism. J Biol Chem. 2018;293(6):1924–32. https://doi.org/10.1074/jbc.M117.815076.

    Article  PubMed  CAS  Google Scholar 

  14. Huber MD, Duquette ML, Shiels JC, Maizels N. A conserved G4 DNA binding domain in RecQ family helicases. J Mol Biol. 2006;358(4):1071–80.

    Article  CAS  Google Scholar 

  15. Byrd AK, Raney KD. A parallel quadruplex DNA is bound tightly but unfolded slowly by Pif1 helicase. J Biol Chem. 2015;290(10):6482–94. doi:DOI https://doi.org/10.1074/jbc.M114.630749.

  16. Gao J, Byrd AK, Zybailov BL, Marecki JC, Guderyon MJ, Edwards AD, et al. DEAD-box RNA helicases Dbp2, Ded1 and Mss116 bind to G-quadruplex nucleic acids and destabilize G-quadruplex RNA. Chem Commun (Camb). 2019;55(31):4467–70. https://doi.org/10.1039/c8cc10091h.

    Article  CAS  Google Scholar 

  17. Liu JQ, Chen CY, Xue Y, Hao YH, Tan Z. G-quadruplex hinders translocation of BLM helicase on DNA: a real-time fluorescence spectroscopic unwinding study and comparison with duplex substrates. J Am Chem Soc. 2010;132(30):10521–7. https://doi.org/10.1021/ja1038165.

    Article  PubMed  CAS  Google Scholar 

  18. Mendoza O, Gueddouda NM, Boule JB, Bourdoncle A, Mergny JL. A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands. Nucleic Acids Res. 2015;43(11):E71–U21.

    Article  CAS  Google Scholar 

  19. Duan XL, Liu NN, Yang YT, Li HH, Li M, Dou SX et al. G-quadruplexes significantly stimulate Pif1 helicase-catalyzed duplex DNA unwinding. J Biol Chem. 2015;290(12):7722–35. doi:DOI https://doi.org/10.1074/jbc.M114.628008.

  20. Wu G, Xing Z, Tran EJ, Yang D. DDX5 helicase resolves G-quadruplex and is involved in MYC gene transcriptional activation. Proc Natl Acad Sci U S A. 2019;116(41):20453–61. https://doi.org/10.1073/pnas.1909047116.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Karow JK, Chakraverty RK, Hickson ID. The Bloom’s syndrome gene product is a 3′-5′ DNA helicase. J Biol Chem. 1997;272(49):30611–4. https://doi.org/10.1074/jbc.272.49.30611.

    Article  PubMed  CAS  Google Scholar 

  22. Chen WF, Rety S, Guo HL, Dai YX, Wu WQ, Liu NN et al. Molecular mechanistic insights into Drosophila DHX36-mediated G-quadruplex unfolding: a structure-based model. Structure. 2018;26(3):403-+. doi:https://doi.org/10.1016/j.str.2018.01.008.

  23. Boule JB, Zakian VA. The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates. Nucleic Acids Res. 2007;35(17):5809–18. https://doi.org/10.1093/nar/gkm613.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Liu NN, Duan XL, Ai X, Yang YT, Li M, Dou SX, et al. The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase. Nucleic Acids Res. 2015;43(18):8942–54. https://doi.org/10.1093/nar/gkv916.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Sen D, Gilbert W. A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature. 1990;344(6265):410–4. https://doi.org/10.1038/344410a0.

    Article  PubMed  CAS  Google Scholar 

  26. Williamson JR, Raghuraman MK, Cech TR. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell. 1989;59(5):871–80.

    Article  CAS  Google Scholar 

  27. Yang WJ, Ruan YJ, Wu WH, Chen PP, Xu LJ, Fu FF. A “turn-on” and label-free fluorescent assay for the rapid detection of exonuclease III activity based on Tb3+-induced G-quadruplex conjugates. Anal Bioanal Chem. 2014;406(18):4535–40. https://doi.org/10.1007/s00216-014-7830-8.

    Article  PubMed  CAS  Google Scholar 

  28. Zhang AY, Balasubramanian S. The kinetics and folding pathways of intramolecular G-quadruplex nucleic acids. J Am Chem Soc. 2012;134(46):19297–308. https://doi.org/10.1021/ja309851t.

    Article  PubMed  CAS  Google Scholar 

  29. Takahashi S, Yamamoto J, Kitamura A, Kinjo M, Sugimoto N. Characterization of intracellular crowding environments with topology-based DNA quadruplex sensors. Anal Chem. 2019;91(4):2586–90. https://doi.org/10.1021/acs.analchem.8b04177.

    Article  PubMed  CAS  Google Scholar 

  30. Wang L, Wang QM, Wang YR, Xi XG, Hou XM. DNA-unwinding activity of Saccharomyces cerevisiae Pif1 is modulated by thermal stability, folding conformation, and loop lengths of G-quadruplex DNA. J Biol Chem. 2018;293(48):18504–13. https://doi.org/10.1074/jbc.RA118.005071.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Bugaut A, Balasubramanian S. A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes. Biochemistry-Us. 2008;47(2):689–97. https://doi.org/10.1021/bi701873c.

    Article  CAS  Google Scholar 

  32. Tippana R, Hwang H, Opresko PL, Bohr VA, Myong S. Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases. Proc Natl Acad Sci U S A. 2016;113(30):8448–53. https://doi.org/10.1073/pnas.1603724113.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Brown JA, Duym WW, Fowler JD, Suo Z. Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2′-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta. J Mol Biol. 2007;367(5):1258–69. https://doi.org/10.1016/j.jmb.2007.01.069.

    Article  PubMed  CAS  Google Scholar 

  34. Lucius AL, Wong CJ, Lohman TM. Fluorescence stopped-flow studies of single turnover kinetics of E.coli RecBCD helicase-catalyzed DNA unwinding. J Mol Biol. 2004;339(4):731–50. https://doi.org/10.1016/j.jmb.2004.04.009.

    Article  PubMed  CAS  Google Scholar 

  35. You HJ, Lattmann S, Rhodes D, Yan J. RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis. Nucleic Acids Res. 2017;45(1):206–14. https://doi.org/10.1093/nar/gkw881.

    Article  PubMed  CAS  Google Scholar 

  36. Giri B, Smaldino PJ, Thys RG, Creacy SD, Routh ED, Hantgan RR, et al. G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic Acids Res. 2011;39(16):7161–78. https://doi.org/10.1093/nar/gkr234.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Hou XM, Wu WQ, Duan XL, Liu NN, Li HH, Fu J, et al. Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase. Biochem J. 2015;466:189–99. https://doi.org/10.1042/bj20140997.

    Article  PubMed  CAS  Google Scholar 

  38. Zhou RB, Zhang JC, Bochman ML, Zakian VA, Ha T. Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA. Elife. 2014;3. doi:ARTN e02190 https://doi.org/10.7554/eLife.02190.

  39. Lu KY, Chen WF, Rety S, Liu NN, Wu WQ, Dai YX, et al. Insights into the structural and mechanistic basis of multifunctional S-cerevisiae Pif1p helicase. Nucleic Acids Res. 2018;46(3):1486–500. https://doi.org/10.1093/nar/gkx1217.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully thank Wei-Fei Chen at Northwest A&F University and Fang-Yuan Teng at the Affiliated Hospital of Southwest Medical University for critically reading the manuscript. We also thank X-G Xi’s laboratory members for insightful discussions.

Funding

This work was supported by the National Natural Science Foundation of China (no. 31870788).

Author information

Author notes

  1. Na-Nv Liu and Lei Ji contributed equally to this work as first authors.

Authors and Affiliations

  1. College of Life Sciences, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Na-Nv Liu, Lei Ji, Qian Guo, Yang-Xue Dai, Wen-Qiang Wu, Hai-Lei Guo, Ke-Yu Lu, Xiao-Mei Li & Xu-Guang Xi

  2. Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, Université Paris-Saclay, 61 Avenue du Président Wilson, 94235, Cachan, France

    Xu-Guang Xi

Authors
  1. Na-Nv Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lei Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qian Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yang-Xue Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wen-Qiang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hai-Lei Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ke-Yu Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiao-Mei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xu-Guang Xi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xu-Guang Xi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 205 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, NN., Ji, L., Guo, Q. et al. Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays. Anal Bioanal Chem 412, 7395–7404 (2020). https://doi.org/10.1007/s00216-020-02875-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02875-3

Keywords

Search

Navigation