Oxidative charge transfer To repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metallointercalators

Potent oxidants which intercalate in DNA serve as tools to probe DNA-mediated electron-transfer reactions. A photoexcited rhodium intercalator, Rh(phi)2DMB3+ (phi = 9,10-phenanthrenequinone diimine and DMB = 4,4'-dimethyl-2,2'-bipyridine), tethered to DNA, promotes both oxidative damage to 5'-GG-3' doublets in DNA and the repair of thymine dimers from a remote site on the DNA duplex. DNA-mediated repair of a thymine dimer lesion by charge transfer from the tethered rhodium intercalator is quantitative, albeit with low photoefficiency, occurs in an intraduplex reaction over long range (36 A), and requires that the intervening bases be paired. When both oxidative reactions, repair and oxidative damage, are monitored on the same duplex, competition is evident; the presence of both a 5'-GG-3' site and the thymine dimer diminished the dimer repair efficiency by 20-40% and decreased damage at the 5'-GG-3' sites 2-fold compared to similar sequences lacking either the guanine doublet or thymine dimer, respectively. In addition to damage at the 5'-G of 5'-GG-3' sites, we also observe oxidation at the 3'-G of the 5'-GT<>TG-3' tetrad only in the presence of thymine dimer. Overall, the yield of repaired thymine strand was at least 10 times higher than the yield of oxidized guanine in the same sequences. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Dipyridophenanzine (dppz) complexes of ruthenium(III), less potent oxidants which intercalate in DNA, oxidize 5'-GG-3' doublets efficiently but cannot trigger the repair of the thymine dimer lesion. Oxidative damage to DNA from a distance, mediated by the DNA base pair stack, can, however, be utilized to probe the disruption in the base stack generated by the thymine dimer. The presence of the dimer does not diminish oxidation by a Ru(III) intercalator at a distal guanine doublet, suggesting that the disruption caused by the dimer does not block charge transfer through the DNA duplex. DNA-mediated electron-transfer reactions of metallointercalators therefore serve to illustrate important aspects of radical migration and its consequence with respect to reactions at a distance through the DNA base pair stack.     

Structural characterization of the 1:1 adduct formed between the antitumor antibiotic hedamycin and the oligonucleotide duplex d(CACGTG)2 by 2D NMR spectroscopy

2D NMR spectroscopic methods have been used to determine the structure of the adduct formed between the antitumor antibiotic hedamycin and the oligodeoxyribonucleotide duplex d(CACGTG)2. Evidence for both intercalation and alkylation in the adduct was observed, and a model for the binding interaction was constructed based on intermolecular NOEs and distance-restrained molecular dynamics. In our computationally refined model, the anthrapyrantrione chromophore of hedamycin is intercalated between the 5'-CG-3' bases with the two aminosugar groups placed in the minor groove and the six carbon bisepoxide side chain located in the major groove. The anglosamine sugar attached at C8 is oriented in the 3' direction relative to the intercalation site, while the N,N-dimethylvancosamine attached at C10 is oriented to the 5' side, with each aminosugar wedged between a guanine exocyclic amino group and one of the groove walls. The terminal epoxide carbon C18 is covalently bound to the N7 atom of the central guanine, as evidenced by lability of the C8 hydrogen of this purine upon reaction with hedamycin. Our binding model places the C10-attached N,N-dimethylvancosamine of hedamycin in van der Waals contact with the alkylated strand. A strong NOE contact verifies the close proximity of the terminal methyl group (C19) of the bisepoxide side chain to the methyl group of the thymine on the 3' side of the alkylated guanine. This, in conjunction with other data, suggests hydrophobic interactions between the bisepoxide chain and the floor of the major groove may contribute to sequence recognition. Furthermore, it is proposed that the 5'-CGT sequence selectivity of hedamycin arises, in part, from complementarity in shape between the chromophore substituents and the major and minor groove at the binding site.     

Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics

An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.     

The high resolution crystal structure of the DNA decamer d(AGGCATGCCT)

The crystal structure of the DNA decamer d(AGGCATGCCT) has been determined to a resolution of 1.3 A and R factor of 13.9%. The structure has a unique conformation with each of the decamer single strands forming base-pairing interactions with two symmetry-related strands. The central eight bases of the decamer form an A-DNA octamer duplex with one symmetry-related strand whilst the terminal 5'-A and T-3' bases are flipped out and away from the octamer helix axis to form base-pairing interactions with a second symmetry-related strand. These A.T base-pairs lie perpendicular to the crystallographic c axis and pack within the unit cell in conjunction with a symmetry-related A.T base-pair displaced by 3.4 A degrees along the c axis. A novel base triplet interaction of the type A*(G.C) is present in the structure with interaction from the major groove side of the terminal 5'-A base to the minor groove of the central A-DNA octamer. This structure reports the first example of cobalt hexammine binding to a right-handed DNA duplex. The crystallographic asymmetric unit contains two cobalt hexammine ligands with one site in the major groove coordinating via hydrogen bonds to the 5'-AGG bases, and the second site located between DNA molecules and interacting with the oxygen atoms of phosphate groups.     

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.     

Site-specific endonucleases RspLKI and RspLKII from Rhodococcus species LK2 are isoschizomers of SphI and BamHI

Two site-specific endonucleases, RspLKI and RspLKII, have been isolated and purified to functional homogeneity from the soil bacterium Rhodococcus species LK2. RspLKI recognizes the 5'-GCATG decreases C-3' DNA sequence and RspLKII recognizes the 5'-G decreases GATCC-3' sequence (arrows indicate DNA cleavage sites). The isolated enzymes are class II site specific endonucleases and are isoschizomers of endonucleases SphI and BamHI, respectively.     

Dynamics of a bis-intercalator DNA complex by 1H-detected natural abundance 13C NMR spectroscopy

The dynamics of the DNA oligomer d(CGCTAGCG)2 (CTSYM) and its complex with the dye 1,1-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1, 3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO) (CTSYMTOTO) bis-intercalated at the 5'-CT-3' sequence steps have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data were acquired and have been analyzed in the context of the Lipari and Szabo model-free formalism. The overall rotational correlation time for the CTSYM is 3.44 ns and the CTSYMTOTO is 3.48 ns. The generalized order parameters (S2) for methine carbons in the CTSYM and CTSYMTOTO are relatively high but nonuniform for the molecules and show sequence context and conformation-dependent variations. Average values of S2 = 0.79 +/- 0.02 for the CTSYM, S2 = 0.80 +/- 0.04 for the CTSYMTOTO aromatic spins, S2 = 0.76 +/- 0.02 for the CTSYM, and S2 = 0.83 +/- 0.05 for the CTSYMTOTO deoxyribose spins were found. The S2 values for the 5'-terminal deoxyribose are lower than for the other residues. The DNA backbone in CTSYMTOTO is distorted and elongated at the site of intercalation, and the C3' atom of the C3 deoxyribose residue has a very low S2 = 0.57 +/- 0.06. The low order for this spin is interpreted in terms of exchange between the C2'-endo and O1'-endo conformations of the C3 deoxyribose. Significant chemical exchange processes were found for most of the aromatic spins in CTSYM that are interpreted in terms of microsecond to millisecond time scale dynamics. The microsecond to millisecond dynamics of the bases in CTSYM are quenched upon TOTO complex formation due to unwinding of the helix and an increase in the surface area of the bases in mutual contact and the large surface area in contact with the intercalated dye. The derived order parameters combined with the solution structure provide motional models for conformational changes induced in the backbone in response to the ligand binding.     

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.     

Neomycin, spermine and hexaamminecobalt (III) share common structural motifs in converting B- to A-DNA

The (dG)n.(dC)n-containing 34mer DNA duplex [d(A2G15C15T2)]2 can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH3)6(3+). Conversion is demonstrated by a characteristic red shift in the circular dichroism spectra and dramatic NMR spectral changes in chemical shifts. Additional support comes from the substantially stronger CH6/GH8-H3'NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are consistent with a deoxyribose pucker transition from the predominate C2'-endo (S-type) to the C3'-endo (N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH3)6(3+)/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent positively charged amine functions of these ligands. In addition, ligands like spermine or Co(NH3)6(3+) can adhere to guanine bases in the deep major groove of the double helix, as is evident from the significant direct NOE cross-peaks from the protons of Co(NH3)6(3+) to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future directions in preparing more potent derivatives of Co(NH3)6(3+) for RNA binding or the induction of A-DNA.     

Stochastic model for a wavelike exothermal reaction in condensed heterogeneous systems

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.     

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.     

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.     

Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid motif "Dx2SLYP" is critical for synthetic activities

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif "Dx2SLYP" are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3' to 5' exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.     

Solution-state structure of a DNA dodecamer duplex containing a Cis-syn thymine cyclobutane dimer, the major UV photoproduct of DNA

The solution structures of a duplex DNA dodecamer containing a cis-syn cyclobutane thymine dimer d(GCACGAAT[cs]TAAG).d(CTTAATTCG TGC) and its native parent sequence were determined using NMR data collected at 750 MHz. The dodecamer sequence corresponds to the section of a site-specific cis-syn dimer containing 49-mer that was found to be the binding site for the dimer-specific T4 denV endonuclease V repair enzyme by chemical and enzymatic footprinting experiments. Structures of both sequences were derived from NOE restrained molecular dynamics/simulated annealing calculations using a fixed outer layer of water and an inner dynamic layer of water with sodium counterions. The resulting structures reveal a subtle distortion to the phosphodiester backbone in the dimer-containing sequence which includes a BII phosphate at the T9pA10 junction immediately 3' to the dimer. The BII phosphate, established experimentally by analysis of the 31P chemical shifts and interpretation of the 3JP-H3' values using an optimized Karplus relationship, enables the DNA helix to accommodate the dimer by destacking the base 3' to the dimer. Furthermore, the structures provide explanations for the unusually shifted T8-N3H imino, A16-H2 and T8-Me proton resonances and T9pA10 (31)P NMR resonance and are consistent with bending, unwinding, and thermodynamic data. The implications of the structural data for the mechanism by which cis-syn dimers are recognized by repair enzymes and bypassed by DNA polymerases are also discussed.     

A theoretical study oh the effect of "bound" water on the proton chemical shifts of the nucleic acid bases

Computations are performed on the proton chemical shifts due to hydrogen bonding between the purine and pyrimidine bases of the nucleic acids and water molecules of their first hydration shell. The water molecules should produce measurable shifts essentially for protons of the bases located close to the site of interaction. For the imino protons of the bases G-N1H and U-N3H participating in hydrogen bonding, the calculated delta delta is larger for the interaction of a base with a complementary base than for its interaction with water. Base pairing will thus produce a downfield shift in water but the measured delta delta due to pairing in this solvent will be smaller than in an inert solvent. Also, the chemical shift difference between G-N1H and U-N3H in water will be larger if the molecules are engaged in pairs than if they are not.     

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C?1H? NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.