Recently published in vivo observations have highlighted the presence of cruciform structures within the genome, suggesting their potential significance in the rapid recognition of the target sequence for transcription factor binding. In this in vitro study, we investigate the organization and stability of the sense (coding) strand within the Serum Response Element of the c-Fos gene promoter (c-Fos SRE), specifically focusing on segments spanning 12 to 36 nucleotides, centered around the CArG-box. Through a thorough examination of UV absorption patterns with varying temperatures, we identified the emergence of a remarkably stable structure, which we conclusively characterized as a hairpin using complementary 1H NMR experiments. Our research decisively ruled out the formation of homoduplexes, as confirmed by supplementary fluorescence experiments. Utilizing molecular dynamics simulations with atomic distance constraints derived from NMR data, we explored the structural intricacies of the compact hairpin. Notably, the loop consisting of the six-membered A/T sequence demonstrated substantial stabilization through extensive stacking, non-canonical inter-base hydrogen bonding, and hydrophobic clustering of thymine methyl groups. These findings suggest the potential of the c-Fos SRE to adopt a cruciform structure (consisting of two opposing hairpins), potentially providing a topological recognition site for the SRF transcription factor under cellular conditions. Our results should inspire further biochemical and in vivo studies to explore the functional implications of these non-canonical DNA structures.

Department of Low Temperature Physics Faculty of Mathematics and Physics Charles University 5 Holešovičkách 2 180 00 Prague 8 Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 121 16 Prague 2 Czech Republic 420 95155 1471

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Gierer A. Model for DNA and Protein Interactions and the Function of the Operator. Nature. 1966;212:1480–1481. PubMed

Vologodskii A. V. Lukashin A. V. Anshelevich V. V. Frank-Kamenetskii M. D. Fluctuations in superhelical DNA. Nucleic Acids Res. 1979;6:967–982. PubMed PMC

Lilley D. M. The inverted repeat as a recognizable structural feature in supercoiled DNA molecules. Proc. Natl. Acad. Sci. U. S. A. 1980;77:6468–6472. PubMed PMC

Panayotatos N. Wells R. D. Cruciform structures in supercoiled DNA. Nature. 1981;289:466–470. PubMed

Brázda V. Laister R. C. Jagelská E. B. Arrowsmith C. Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol. Biol. 2011;12:33. PubMed PMC

Nag D. K. White M. A. Petes T. D. Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast. Nature. 1989;340:318–320. PubMed

Zhao J. Bacolla A. Wang G. Vasquez K. M. Non-B DNA structure-induced genetic instability and evolution. Cell. Mol. Life Sci. 2010;67:43–62. PubMed PMC

Amir-Aslani A. Mauffret O. Sourgen F. Neplaz S. Maroun R. G. Lescot E. Tevanian G. Fermandjian S. The Hairpin Structure of a Topoisomerase II Site DNA Strand Analyzed by Combined NMR and Energy Minimization Methods. J. Mol. Biol. 1996;263:776–788. PubMed

Belotserkovskii B. P. Mirkin S. M. Hanawalt P. C. DNA Sequences That Interfere with Transcription: Implications for Genome Function and Stability. Chem. Rev. 2013;113:8620–8637. PubMed

Egli M., in Nucleic Acids in Chemistry and Biology, ed. G. M. Blackburn, M. J. Gait, J. D. Loakes and D. M. Williams, Royal Society of Chemistry, Cambridge, UK, 3rd edn, 2006, pp. 13–75

Shlyakhtenko L. S. Hsieh P. Grigoriev M. Potaman V. N. Sinden R. R. Lyubchenko Y. L. A cruciform structural transition provides a molecular switch for chromosome structure and dynamics 1 1Edited by I. Tinoco. J. Mol. Biol. 2000;296:1169–1173. PubMed

Bikard D. Loot C. Baharoglu Z. Mazel D. Folded DNA in Action: Hairpin Formation and Biological Functions in Prokaryotes. Microbiol. Mol. Biol. Rev. 2010;74:570–588. PubMed PMC

Chasovskikh S. Dimtchev A. Smulson M. Dritschilo A. DNA transitions induced by binding of PARP-1 to cruciform structures in supercoiled plasmids. Cytometry, Part A. 2005;68A:21–27. PubMed

Mikheikin A. L. Lushnikov A. Y. Lyubchenko Y. L. Effect of DNA Supercoiling on the Geometry of Holliday Junctions. Biochemistry. 2006;45:12998–13006. PubMed PMC

Lushnikov A. Y. Potaman V. N. Lyubchenko Y. L. Site-specific labeling of supercoiled DNA. Nucleic Acids Res. 2006;34:e111. PubMed PMC

Mandal S. Selvam S. Cui Y. Hoque M. E. Mao H. Mechanical Cooperativity in DNA Cruciform Structures. ChemPhysChem. 2018;19:2627–2634. PubMed

Waga S. Mizuno S. Yoshida M. Chromosomal protein HMG1 removes the transcriptional block caused by the cruciform in supercoiled DNA. J. Biol. Chem. 1990;265:19424–19428. PubMed

Nag D. K. Petes T. D. Seven-base-pair inverted repeats in DNA form stable hairpins in vivo in Saccharomyces cerevisiae. Genetics. 1991;129:669–673. PubMed PMC

Coté A. G. Lewis S. M. Mus81-Dependent Double-Strand DNA Breaks at In Vivo-Generated Cruciform Structures in S. cerevisiae. Mol. Cell. 2008;31:800–812. PubMed

Inagaki H. Ohye T. Kogo H. Tsutsumi M. Kato T. Tong M. Emanuel B. S. Kurahashi H. Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations. Nat. Commun. 2013;4:1592. PubMed

Yamamoto Y. Miura O. Ohyama T. Cruciform Formable Sequences within Pou5f1 Enhancer Are Indispensable for Mouse ES Cell Integrity. Int. J. Mol. Sci. 2021;22:3399. PubMed PMC

Liu L. F. Wang J. C. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. U. S. A. 1987;84:7024–7027. PubMed PMC

Piechaczyk M. Blanchard J. M. c-fos proto-oncogene regulation and function. Crit. Rev. Oncol. Hematol. 1994;17:93–131. PubMed

Chiu R. Boyle W. J. Meek J. Smeal T. Hunter T. Karin M. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell. 1988;54:541–552. PubMed

Angel P. Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta, Rev. Cancer. 1991;1072:129–157. PubMed

Gilman M. Z. Wilson R. N. Weinberg R. A. Multiple protein-binding sites in the 5′-flanking region regulate c-fos expression. Mol. Cell. Biol. 1986;6:4305–4316. PubMed PMC

Prywes R. Roeder R. G. Inducible binding of a factor to the c-fos enhancer. Cell. 1986;47:777–784. PubMed

Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986;46:567–574. PubMed

Treisman R. The serum response element. Trends Biochem. Sci. 1992;17:423–426. PubMed

Treisman R. The SRE: a growth factor responsive transcriptional regulator. Semin. Cancer Biol. 1990;1:47–58. PubMed

Mericskay M. Parlakian A. Porteu A. Dandré F. Bonnet J. Paulin D. Li Z. An Overlapping CArG/Octamer Element Is Required for Regulation of desmin Gene Transcription in Arterial Smooth Muscle Cells. Dev. Biol. 2000;226:192–208. PubMed

Shore P. Sharrocks A. D. The MADS-box family of transcription factors. Eur. J. Biochem. 1995;229:1–13. PubMed

Profantová B. Coïc Y.-M. Y.-M. Profant V. Štěpánek J. Kopecký V. Turpin P.-Y. Alpert B. Zentz C. Organization of the MADS Box from Human SRF Revealed by Tyrosine Perturbation. J. Phys. Chem. B. 2015;119:1793–1801. PubMed

Huet A. Parlakian A. Arnaud M. Glandières J. Valat P. Fermandjian S. Paulin D. Alpert B. Zentz C. Mechanism of binding of serum response factor to serum response element. FEBS J. 2005;272:3105–3119. PubMed

Rivera V. M. Greenberg M. E. Growth factor-induced gene expression: the ups and downs of c-fos regulation. New Biol. 1990;2:751–758. PubMed

Treisman R. Ternary complex factors: growth factor regulated transcriptional activators. Curr. Opin. Genet. Dev. 1994;4:96–101. PubMed

Dalton S. Treisman R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell. 1992;68:597–612. PubMed

Pellegrini L. Tan S. Richmond T. J. Structure of serum response factor core bound to DNA. Nature. 1995;376:490–498. PubMed

Mo Y. Ho W. Johnston K. Marmorstein R. Crystal structure of a ternary SAP-1/SRF/c-fos SRE DNA complex. J. Mol. Biol. 2001;314:495–506. PubMed

West A. G. Shore P. Sharrocks A. D. DNA binding by MADS-box transcription factors: a molecular mechanism for differential DNA bending. Mol. Cell. Biol. 1997;17:2876–2887. PubMed PMC

West A. G. Sharrocks A. D. MADS-box transcription factors adopt alternative mechanisms for bending DNA. J. Mol. Biol. 1999;286:1311–1323. PubMed

Stepanek J. Vincent M. Turpin P.-Y. Paulin D. Fermandjian S. Alpert B. Zentz C. C→G base mutations in the CArG box of c-fos serum response element alter its bending flexibility. FEBS J. 2007;274:2333–2348. PubMed

Štěpánek J. Kopecký V. Mezzetti A. Turpin P.-Y. Paulin D. Alpert B. Zentz C. Structural and dynamic changes of the serum response element and the core domain of serum response factor induced by their association. Biochem. Biophys. Res. Commun. 2010;391:203–208. PubMed

Štěpánek J. Kopecký V. Turpin P.-Y. Li Z. Alpert B. Zentz C. DNA Electric Charge Oscillations Govern Protein–DNA Recognition. PLoS One. 2015;10:e0124444. PubMed PMC

Drewett V. DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences. Nucleic Acids Res. 2001;29:479–487. PubMed PMC

Kibbe W. A. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 2007;35:W43–W46. PubMed PMC

Hwang T. L. Shaka A. J. Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients. J. Magn. Reson. 1995;112:275–279.

Delaglio F. Grzesiek S. Vuister G. W. Zhu G. Pfeifer J. Bax A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 1995;6:277–293. PubMed

Goddard T. D. and Kneller D. G., Sparky 3, University of California, San Francisco, 2008

Lee W. Tonelli M. Markley J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics. 2015;31(8):1325–1327. PubMed PMC

Wemmer D. E., in NMR Spectroscopy and its Application to Biomedical Research, ed. S. K. Sarkar, Elsevier Science B.V., Amsterdam, 1996, pp. 281–312

Wijmenga S. S. van Buuren B. N. M. The use of NMR methods for conformational studies of nucleic acids. Prog. Nucl. Magn. Reson. Spectrosc. 1998;32:287–387.

Piotto M. Saudek V. Sklenář V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR. 1992;2:661–665. PubMed

van Dongen M. J. P. Wijmenga S. S. Eritja R. Azorín F. Hilbers C. W. Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy. J. Biomol. NMR. 1996;8:207–212. PubMed

Římal V. Štěpánková H. Štěpánek J. Analysis of NMR spectra in case of temperature-dependent chemical exchange between two unequally populated sites. Concepts Magn. Reson., Part A. 2011;38A:117–127.

Malinovski E. R., Factor Analysis in Chemistry, John Wiley & Sons, Inc., New York, 3rd edn, 2002

Jorgensen W. L. Chandrasekhar J. Madura J. D. Impey R. W. Klein M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935.

Humphrey W. Dalke A. Schulten K. VMD: Visual molecular dynamics. J. Mol. Graphics. 1996;14:33–38. PubMed

Vanommeslaeghe K. MacKerell A. D. CHARMM additive and polarizable force fields for biophysics and computer-aided drug design. Biochim. Biophys. Acta, Gen. Subj. 2015;1850:861–871. PubMed PMC

Phillips J. C. Braun R. Wang W. Gumbart J. Tajkhorshid E. Villa E. Chipot C. Skeel R. D. Kalé L. Schulten K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005;26:1781–1802. PubMed PMC

Ryckaert J.-P. Ciccotti G. Berendsen H. J. C. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977;23:327–341.

Roe D. R. Cheatham T. E. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013;9:3084–3095. PubMed

Pettersen E. F. Goddard T. D. Huang C. C. Couch G. S. Greenblatt D. M. Meng E. C. Ferrin T. E. UCSF Chimera – A visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. PubMed

Kwok C. K. Lam S. L. NMR proton chemical shift prediction of T·T mismatches in B-DNA duplexes. J. Magn. Reson. 2013;234:184–189. PubMed

Allawi H. T. SantaLucia J. Thermodynamics of internal C·T mismatches in DNA. Nucleic Acids Res. 1998;26:2694–2701. PubMed PMC

Allawi H. T. SantaLucia J. Thermodynamics and NMR of Internal G·T Mismatches in DNA. Biochemistry. 1997;36:10581–10594. PubMed

Lane A. Martin S. R. Ebel S. Brown T. Solution conformation of a deoxynucleotide containing tandem G.cntdot.A mismatched base pairs and 3’-overhanging ends in d(GTGAACTT)2. Biochemistry. 1992;31:12087–12095. PubMed

Zargarian L. Kanevsky I. Bazzi A. Boynard J. Chaminade F. Fossé P. Mauffret O. Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin. Nucleic Acids Res. 2009;37:4043–4054. PubMed PMC

Lam S. L. DSHIFT: a web server for predicting DNA chemical shifts. Nucleic Acids Res. 2007;35:W713–W717. PubMed PMC

Lam S. L. Lai K. F. Chi L. M. Proton chemical shift prediction of A·A mismatches in B-DNA duplexes. J. Magn. Reson. 2007;187:105–111. PubMed

Blake R. D. Delcourt S. G. Thermal stability of DNA. Nucleic Acids Res. 1998;26:3323–3332. PubMed PMC

Kaushik M. Kaushik S. Roy K. Singh A. Mahendru S. Kumar M. Chaudhary S. Ahmed S. Kukreti S. A bouquet of DNA structures: Emerging diversity. Biochem. Biophys. Rep. 2016;5:388–395. PubMed PMC

Lah J. Seručnik M. Vesnaver G. Influence of a Hairpin Loop on the Thermodynamic Stability of a DNA Oligomer. J. Nucleic Acids. 2011;2011:1–9. PubMed PMC

Portella G. Orozco M. Multiple Routes to Characterize the Folding of a Small DNA Hairpin. Angew. Chem. 2010;122:7839–7842. PubMed

Blose J. M. Lloyd K. P. Bevilacqua P. C. Portability of the GN(R)A Hairpin Loop Motif between RNA and DNA. Biochemistry. 2009;48:8787–8794. PubMed

Vologodskaia M. Y. Vologodskii A. V. Effect of Magnesium on Cruciform Extrusion in Supercoiled DNA. J. Mol. Biol. 1999;289:851–859. PubMed

Chou S.-H. Unusual DNA duplex and hairpin motifs. Nucleic Acids Res. 2003;31:2461–2474. PubMed PMC

Blommers M. J. J. Ven F. J. M. Marel G. A. Boom J. H. Hilbers C. W. The three-dimensional structure of a DNA hairpin in solution. Two-dimensional NMR studies and structural analysis of d(ATCCTATTTATAGGAT) Eur. J. Biochem. 1991;201:33–51. PubMed

Ying L. Wallace M. I. Klenerman D. Two-state model of conformational fluctuation in a DNA hairpin-loop. Chem. Phys. Lett. 2001;334:145–150.

Kuznetsov S. V. Ren C.-C. Woodson S. A. Ansari A. Loop dependence of the stability and dynamics of nucleic acid hairpins. Nucleic Acids Res. 2007;36:1098–1112. PubMed PMC

Lin M. M. Meinhold L. Shorokhov D. Zewail A. H. Unfolding and melting of DNA (RNA) hairpins: the concept of structure-specific 2D dynamic landscapes. Phys. Chem. Chem. Phys. 2008;10:4227. PubMed PMC

SantaLucia J. Hicks D. The Thermodynamics of DNA Structural Motifs. Annu. Rev. Biophys. Biomol. Struct. 2004;33:415–440. PubMed

SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. U. S. A. 1998;95:1460–1465. PubMed PMC

Markham N. R. Zuker M. DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res. 2005;33:W577–W581. PubMed PMC

Markham N. R. Zuker M. UNAFold: software for nucleic acid folding and hybridization. Methods Mol. Biol. 2008;453:3–31. PubMed

Wüthrich K., in NMR of Proteins and Nucleic Acids, John Wiley & Sons, Chichester, U.K., 1986, pp. 203–219

Feng X. Xie F.-Y. Ou X.-H. Ma J.-Y. Cruciform DNA in mouse growing oocytes: Its dynamics and its relationship with DNA transcription. PLoS One. 2020;15:e0240844. PubMed PMC

Matos-Rodrigues G. Hisey J. A. Nussenzweig A. Mirkin S. M. Detection of alternative DNA structures and its implications for human disease. Mol. Cell. 2023;83:3622–3641. PubMed

Wu T. Lyu R. You Q. He C. Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ. Nat. Methods. 2020;17:515–523. PubMed PMC

Matos-Rodrigues G. van Wietmarschen N. Wu W. Tripathi V. Koussa N. C. Pavani R. Nathan W. J. Callen E. Belinky F. Mohammed A. Napierala M. Usdin K. Ansari A. Z. Mirkin S. M. Nussenzweig A. S1-END-seq reveals DNA secondary structures in human cells. Mol. Cell. 2022;82:3538–3552. PubMed PMC

van Wietmarschen N. Sridharan S. Nathan W. J. Tubbs A. Chan E. M. Callen E. Wu W. Belinky F. Tripathi V. Wong N. Foster K. Noorbakhsh J. Garimella K. Cruz-Migoni A. Sommers J. A. Huang Y. Borah A. A. Smith J. T. Kalfon J. Kesten N. Fugger K. Walker R. L. Dolzhenko E. Eberle M. A. Hayward B. E. Usdin K. Freudenreich C. H. Brosh R. M. West S. C. McHugh P. J. Meltzer P. S. Bass A. J. Nussenzweig A. Repeat expansions confer WRN dependence in microsatellite-unstable cancers. Nature. 2020;586:292–298. PubMed PMC