Skip to main content
SpringerLink
Log in

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR

  • Communication
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific 13C/15N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.

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

Similar content being viewed by others

References

  • Alexeev DG, Lipanov AA, Skuratovskii I (1987a) Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove. Nature 325:821–823

    Article  ADS  Google Scholar 

  • Alexeev DG, Lipanov AA, Skuratovskii I (1987b) The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction. J Biomol Struct Dyn 4:989–1012

    Google Scholar 

  • Arnott S, Selsing E (1974) Structures for the polynucleotide complexes poly(dA) with poly (dT) and poly(dT) with poly(dA) with poly (dT). J Mol Biol 88:509–521

    Article  Google Scholar 

  • Bax A, Grishaev A (2005) Weak alignment NMR: a hawk-eyed view of biomolecular structure. Curr Opin Struct Biol 15:563–570

    Article  Google Scholar 

  • Beveridge DL, Dixit SB, Barreiro G, Thayer KM (2004) Molecular dynamics simulations of DNA curvature and flexibility: helix phasing and premelting. Biopolymers 73:380–403

    Article  Google Scholar 

  • Chan SS, Breslauer KJ, Hogan ME, Kessler DJ, Austin RH, Ojemann J, Passner JM, Wiles NC (1990) Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending. Biochemistry 29:6161–6171

    Google Scholar 

  • Chan SS, Breslauer KJ, Austin RH, Hogan ME (1993) Thermodynamics and premelting conformational changes of phased (dA)5 tracts. Biochemistry 32:11776–11784

    Article  Google Scholar 

  • Chen X, Mariappan SV, Kelley JJ 3rd, Bushweller JH, Bradbury EM, Gupta G (1998) A PCR-based method for uniform 13C/15N labeling of long DNA oligomers. FEBS Lett 436:372–376

    Article  Google Scholar 

  • Delagoutte E, von Hippel PH (2002) Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: structures and properties of isolated helicases. Q Rev Biophys 35:431–478

    Article  Google Scholar 

  • Duchardt E, Nilsson L, Schleucher J (2008) Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study. Nucleic Acids Res 36:4211–4219

    Article  Google Scholar 

  • Echodu D, Goobes G, Shajani Z, Pederson K, Meints G, Varani G, Drobny G (2008) Furanose dynamics in the HhaI methyltransferase target DNA studied by solution and solid-state NMR relaxation. J Phys Chem B 112:13934–13944

    Article  Google Scholar 

  • Fernandez AG, Anderson JN (2007) Nucleosome positioning determinants. J Mol Biol 371:649–668

    Article  Google Scholar 

  • Fujii S, Kono H, Takenaka S, Go N, Sarai A (2007) Sequence-dependent DNA deformability studied using molecular dynamics simulations. Nucleic Acids Res 35:6063–6074

    Article  Google Scholar 

  • Fuxreiter M, Luo N, Jedlovszky P, Simon I, Osman R (2002) Role of base flipping in specific recognition of damaged DNA by repair enzymes. J Mol Biol 323:823–834

    Article  Google Scholar 

  • Garcia HG, Grayson P, Han L, Inamdar M, Kondev J, Nelson PC, Phillips R, Widom J, Wiggins PA (2007) Biological consequences of tightly bent DNA: the other life of a macromolecular celebrity. Biopolymers 85:115–130

    Article  Google Scholar 

  • Getz MM, Andrews AJ, Fierke CA, Al-Hashimi HM (2007) Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation. RNA 13:251–266

    Article  Google Scholar 

  • Gimenes F, Takeda KI, Fiorini A, Gouveia FS, Fernandez MA (2008) Intrinsically bent DNA in replication origins and gene promoters. Genet Mol Res 7:549–558

    Article  Google Scholar 

  • Hagerman PJ (1985) Sequence dependence of the curvature of DNA: a test of the phasing hypothesis. Biochemistry 24:7033–7037

    Google Scholar 

  • Hansen AL, Al-Hashimi HM (2007) Dynamics of large elongated RNA by NMR carbon relaxation. J Am Chem Soc 129:16072–16082

    Article  Google Scholar 

  • Hansen AL, Nikolova EN, Casiano-Negroni A, Al-Hashimi HM (2009) Extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon R(1rho) NMR spectroscopy. J Am Chem Soc 131:3818–3819

    Article  Google Scholar 

  • Haran TE, Mohanty U (2009) The unique structure of A-tracts and intrinsic DNA bending. Q Rev Biophys 42:41–81

    Google Scholar 

  • Herrera JE, Chaires JB (1989) A premelting conformational transition in poly(dA)-poly(dT) coupled to daunomycin binding. Biochemistry 28:1993–2000

    Google Scholar 

  • Hud NV, Feigon J (2002) Characterization of divalent cation localization in the minor groove of the A(n)T(n) and T(n)A(n) DNA sequence elements by (1)H NMR spectroscopy and manganese(II). Biochemistry 41:9900–9910

    Google Scholar 

  • Isaacs RJ, Spielmann HP (2001) Relationship of DNA structure to internal dynamics: correlation of helical parameters from NOE-based NMR solution structures of d(GCGTACGC)(2) and d(CGCTAGCG)(2) with (13)C order parameters implies conformational coupling in dinucleotide units. J Mol Biol 307:525–540

    Article  Google Scholar 

  • Isaacs RJ, Spielmann HP (2004) A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility. DNA Repair (Amst) 3:455–464

    Google Scholar 

  • Jerkovic B, Bolton PH (2001) Magnesium increases the curvature of duplex DNA that contains dA tracts. Biochemistry 40:9406–9411

    Google Scholar 

  • Kaplan N, Moore IK, Fondufe-Mittendorf Y, Gossett AJ, Tillo D, Field Y, LeProust EM, Hughes TR, Lieb JD, Widom J et al (2009) The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458:362–366

    Article  ADS  Google Scholar 

  • Kojima C, Ono A, Kainosho M, James TL (1998) DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides. J Magn Reson 135:310–333

    Article  ADS  Google Scholar 

  • Koo HS, Wu HM, Crothers DM (1986) DNA bending at adenine · thymine tracts. Nature 320:501–506

    Article  ADS  Google Scholar 

  • Korzhnev DM, Orekhov VY, Kay LE (2005) Off-resonance R(1rho) NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain. J Am Chem Soc 127:713–721

    Article  Google Scholar 

  • Levene SD, Wu HM, Crothers DM (1986) Bending and flexibility of kinetoplast DNA. Biochemistry 25:3988–3995

    Google Scholar 

  • Lipari G, Szabo A (1981) Nuclear magnetic resonance relaxation in nucleic acid fragments: models for internal motion. Biochemistry 20:6250–6256

    Article  Google Scholar 

  • Louis JM, Martin RG, Clore GM, Gronenborn AM (1998) Preparation of uniformly isotope-labeled DNA oligonucleotides for NMR spectroscopy. J Biol Chem 273:2374–2378

    Article  Google Scholar 

  • Marini JC, Levene SD, Crothers DM, Englund PT (1982) Bent helical structure in kinetoplast DNA. Proc Natl Acad Sci USA 79:7664–7668

    Article  ADS  Google Scholar 

  • Marini JC, Effron PN, Goodman TC, Singleton CK, Wells RD, Wartell RM, Englund PT (1984) Physical characterization of a kinetoplast DNA fragment with unusual properties. J Biol Chem 259:8974–8979

    Google Scholar 

  • Masse JE, Bortmann P, Dieckmann T, Feigon J (1998) Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies. Nucleic Acids Res 26:2618–2624

    Article  Google Scholar 

  • Mittermaier A, Kay LE (2006) New tools provide new insights in NMR studies of protein dynamics. Science 312:224–228

    Article  ADS  Google Scholar 

  • Movileanu L, Benevides JM, Thomas GJ Jr (2002) Temperature dependence of the Raman spectrum of DNA. II. Raman signatures of premelting and melting transitions of poly(dA).poly(dT) and comparison with poly(dA-dT).poly(dA-dT). Biopolymers 63:181–194

    Article  Google Scholar 

  • Musselman C, Al-Hashimi HM, Andricioaei I (2007) iRED analysis of TAR RNA reveals motional coupling, long-range correlations, and a dynamical hinge. Biophys J 93:411–422

    Article  Google Scholar 

  • Palmer AG 3rd, Massi F (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev 106:1700–1719

    Article  Google Scholar 

  • Palmer AG 3rd, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238

    Article  Google Scholar 

  • Pelupessy P, Chiarparin E, Bodenhausen G (1999) Excitation of selected proton signals in NMR of isotopically labeled macromolecules. J Magn Reson 138:178–181

    Article  ADS  Google Scholar 

  • Prestegard JH, Bougault CM, Kishore AI (2004) Residual dipolar couplings in structure determination of biomolecules. Chem Rev 104:3519–3540

    Article  Google Scholar 

  • Schwieters CD, Clore GM (2007) A physical picture of atomic motions within the Dickerson DNA dodecamer in solution derived from joint ensemble refinement against NMR and large-angle X-ray scattering data. Biochemistry 46:1152–1166

    Article  Google Scholar 

  • Segal E, Widom J (2009) Poly(dA:dT) tracts: major determinants of nucleosome organization. Curr Opin Struct Biol 19:65–71

    Article  Google Scholar 

  • Segal E, Fondufe-Mittendorf Y, Chen L, Thastrom A, Field Y, Moore IK, Wang JP, Widom J (2006) A genomic code for nucleosome positioning. Nature 442:772–778

    Article  ADS  Google Scholar 

  • Shajani Z, Varani G (2005) 13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein. J Mol Biol 349:699–715

    Article  Google Scholar 

  • Shajani Z, Varani G (2007) NMR studies of dynamics in RNA and DNA by 13C relaxation. Biopolymers 86:348–359

    Article  Google Scholar 

  • Shajani Z, Varani G (2008) 13C relaxation studies of the DNA target sequence for hhai methyltransferase reveal unique motional properties. Biochemistry 47:7617–7625

    Article  Google Scholar 

  • Showalter SA, Baker NA, Tang C, Hall KB (2005) Iron responsive element RNA flexibility described by NMR and isotropic reorientational eigenmode dynamics. J Biomol NMR 32:179–193

    Article  Google Scholar 

  • Smith AL, Cekan P, Rangel DP, Sigurdsson ST, Mailer C, Robinson BH (2008) Theory for spin-lattice relaxation of spin probes on weakly deformable DNA. J Phys Chem B 112:9219–9236

    Article  Google Scholar 

  • Spielmann HP (1998) Dynamics in psoralen-damaged DNA by 1H-detected natural abundance 13C NMR spectroscopy. Biochemistry 37:5426–5438

    Article  Google Scholar 

  • Stellwagen NC, Magnusdottir S, Gelfi C, Righetti PG (2001) Preferential counterion binding to A-tract DNA oligomers. J Mol Biol 305:1025–1033

    Google Scholar 

  • Stivers JT (2008) Extrahelical damaged base recognition by DNA glycosylase enzymes. Chemistry 14:786–793

    Article  Google Scholar 

  • Sun X, Zhang Q, Al-Hashimi HM (2007) Resolving fast and slow motions in the internal loop containing stem-loop 1 of HIV-1 that are modulated by Mg2+ binding: role in the kissing-duplex structural transition. Nucleic Acids Res 35:1698–1713

    Article  Google Scholar 

  • Tchernaenko V, Radlinska M, Drabik C, Bujnicki J, Halvorson HR, Lutter LC (2003) Topological measurement of an A-tract bend angle: comparison of the bent and straightened states. J Mol Biol 326:737–749

    Article  Google Scholar 

  • Tchernaenko V, Halvorson HR, Lutter LC (2004) Topological measurement of an A-tract bend angle: effect of magnesium. J Mol Biol 341:55–63

    Article  Google Scholar 

  • Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH (1997) NMR evidence for slow collective motions in cyanometmyoglobin. Nat Struct Biol 4:292–297

    Article  Google Scholar 

  • Tolman JR, Al-Hashimi HM, Kay LE, Prestegard JH (2001) Structural and dynamic analysis of residual dipolar coupling data for proteins. J Am Chem Soc 123:1416–1424

    Article  Google Scholar 

  • Travers AA (2004) The structural basis of DNA flexibility. Philos Transact A Math Phys Eng Sci 362:1423–1438

    Article  MATH  MathSciNet  Google Scholar 

  • Ulanovsky L, Bodner M, Trifonov EN, Choder M (1986) Curved DNA: design, synthesis, and circularization. Proc Natl Acad Sci USA 83:862–866

    Article  ADS  Google Scholar 

  • Wu HM, Crothers DM (1984) The locus of sequence-directed and protein-induced DNA bending. Nature 308:509–513

    Article  ADS  Google Scholar 

  • Wu Z, Delaglio F, Tjandra N, Zhurkin VB, Bax A (2003) Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy. J Biomol NMR 26:297–315

    Article  Google Scholar 

  • Yan J, Bushweller JH (2001) An optimized PCR-based procedure for production of 13C/15N-labeled DNA. Biochem Biophys Res Commun 284:295–300

    Article  Google Scholar 

  • Ying J, Grishaev A, Bryce DL, Bax A (2006) Chemical shift tensors of protonated base carbons in helical RNA and DNA from NMR relaxation and liquid crystal measurements. J Am Chem Soc 128:11443–11454

    Article  Google Scholar 

  • Zhang Q, Al-Hashimi HM (2008) Extending the NMR spatial resolution limit for RNA by motional couplings. Nat Methods 5:243–245

    Article  Google Scholar 

  • Zhang Q, Throolin R, Pitt SW, Serganov A, Al-Hashimi HM (2003) Probing motions between equivalent RNA domains using magnetic field induced residual dipolar couplings: accounting for correlations between motions and alignment. J Am Chem Soc 125:10530–10531

    Article  Google Scholar 

  • Zhang Q, Sun X, Watt ED, Al-Hashimi HM (2006) Resolving the motional modes that code for RNA adaptation. Science 311:653–656

    Article  ADS  Google Scholar 

  • Zhang Q, Stelzer AC, Fisher CK, Al-Hashimi HM (2007) Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450:1263–1267

    Article  ADS  Google Scholar 

  • Zimmer DP, Crothers DM (1995) NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides. Proc Natl Acad Sci USA 92:3091–3095

    Article  ADS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Alexandar L. Hansen for stimulating discussions and for critically reading the manuscript and Dr. Alexander V. Kurochkin for maintenance of the NMR instruments. We gratefully acknowledge the Michigan Economic Development Cooperation and the Michigan Technology Tri-Corridor for support in the purchase of a 600 MHz spectrometer. This work was supported by a National Science Foundation CAREER award (MCB 0644278) received by H.M.A. E.N.N. was supported by a Rackham International Fellowship awarded by the University of Michigan.

Author information

Authors and Affiliations

  1. Department of Chemistry, Biophysics and Chemical Biology Program, University of Michigan, 930 North University Avenue, Ann Arbor, MI, 48109-1055, USA

    Evgenia N. Nikolova & Hashim M. Al-Hashimi

Authors
  1. Evgenia N. Nikolova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hashim M. Al-Hashimi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hashim M. Al-Hashimi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 2175 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nikolova, E.N., Al-Hashimi, H.M. Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR. J Biomol NMR 45, 9–16 (2009). https://doi.org/10.1007/s10858-009-9350-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-009-9350-y

Keywords

Search

Navigation