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1.
Base dynamics, heretofore observed only at TpA steps in DNA, were investigated as a function of sequence context by NMR spectroscopy. The large amplitude conformational dynamics have been previously observed in TnAn segments where n > or = 2. In order to determine whether the dynamic characteristics occur in more general sequence contexts, we examined four self-complementary DNA sequences, [d(CTTTA-NATNTAAAG)2] (where N = A, C, T, G and N = complement of N). The anomalous broadening of the TpA adenine H2 resonance which is indicative of large amplitude base motion was observed in all nine unique four nucleotide contexts. Furthermore, all the adenine H2 resonances experienced a linewidth maximum as a function of temperature, which is a characteristic of the dynamic process. Interestingly, the temperature of the linewidth maximum varied with sequence indicating that the thermodynamics of TpA base dynamics are also sequence dependent. In one example, neither a T preceding nor an A trailing the TpA step was required for base dynamics. These results show that base dynamics, heretofore observed in only a few isolated sequences, occurs at all TpA steps which are either preceded or followed by a thymine or adenine, respectively, and may be characteristic of all TpA steps in DNA notwithstanding sequence context.  相似文献   

2.
Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA. Calculations were carried out on the 136 cases embedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All simulations were carried out using a well-defined state-of-the-art MD protocol, the AMBER suite of programs, and the parm94 force field. In a previous article (Beveridge et al. 2004. Biophysical Journal. 87:3799-3813), the research design, details of the simulation protocol, and informatics issues were described. Preliminary results from 15 ns MD trajectories were presented for the d(CpG) step in all 10 unique sequence contexts. The results indicated the sequence context effects to be small for this step, but revealed that MD on DNA at this length of trajectory is subject to surprisingly persistent cooperative transitions of the sugar-phosphate backbone torsion angles alpha and gamma. In this article, we report detailed analysis of the entire trajectory database and occurrence of various conformational substates and its impact on studies of context effects. The analysis reveals a possible direct correspondence between the sequence-dependent dynamical tendencies of DNA structure and the tendency to undergo transitions that "trap" them in nonstandard conformational substates. The difference in mean of the observed basepair step helicoidal parameter distribution with different flanking sequence sometimes differs by as much as one standard deviation, indicating that the extent of sequence effects could be significant. The observations reveal that the impact of a flexible dinucleotide such as CpG could extend beyond the immediate basepair neighbors. The results in general provide new insight into MD on DNA and the sequence-dependent dynamical structural characteristics of DNA.  相似文献   

3.
In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.  相似文献   

4.
The structurally correlated dihedral angles epsilon and zeta are known for their large variability within the B-DNA backbone. We have used molecular modelling to study both energetic and mechanical features of these variations which can produce BI/BII transitions. Calculations were carried out on DNA oligomers containing either YpR or RpY dinucleotides steps within various sequence environments. The results indicate that CpA and CpG steps favour the BI/BII transition more than TpA or any RpY step. The stacking energy and its intra- and inter-strand components explain these effects. Analysis of neighbouring base pairs reveals that BI/BII transitions of CpG and CpA are easiest within (Y)n(R)n sequences. These can also induce a large vibrational amplitude for TpA steps within the BI conformation.  相似文献   

5.
A combination of high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy was used to analyze the products of X-irradiated aqueous solutions of the dinucleoside monophosphate thymidylyl(3'-5')-2'-deoxyadenosine, d(TpA), and its sequence isomer 2'-deoxyadenylyl(3'-5')thymidine, d(ApT). The products of d(TpA) include both bases and nucleotides and a variety of thymine modifications of d(TpA) including the two cis and two trans glycol stereoisomers, two cis monohydroxy derivatives, an N-formamide derivative, and the hydroxymethyl derivative. Attention is focused on using NMR spectral features to distinguish among the various stereoisomers. The radiation chemistry of d(ApT) is also explored and differences in product formation compared with d(TpA) are described, particularly the formation of two products involving modification of adenine base. The potential of the HPLC-NMR approach to the study of radiation chemistry in DNA model compounds is discussed.  相似文献   

6.
In a previous NMR study we detected the presence of particular motions and hydration properties within the DNA fragment d(CTACTGCTTTAG).d(CTAAAGCAGTAG). Now, we report on an NMR and molecular modelling analysis of this sequence focusing our attention on the biologically important TpA steps. NOe and coupling constant restraints were introduced in three different modelling protocols: X-PLOR and JUMNA used with Flex and AMBER94 as force-fields. Despite their differences the protocols produce similar mean B-DNA structures (r.m.s.d. <1 A). The new information confirms our previous experimental results on the narrowing of the minor groove along the T8T9T10/A17A16A15 run and the sudden widening at the T10pA11 step ending this run. It is further shown that this step displays a large positive roll with its T10:A15 and A11:T14 base-pairs likely stabilised by amino-amino and amino-carbonyl interactions in the major groove. A relationship between roll values and amino-amino and amino-carbonyl distances strongly suggests that electrostatics or bifurcated hydrogen-bonds could be responsible for induction of positive rolls in TpA steps. Such edge-to-edge interactions could explain the slower motions shown by the adenine A15. The influence of these interactions on the stabilisation of particular DNA conformers is discussed using our data and those provided by the recent literature.  相似文献   

7.
The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.  相似文献   

8.
NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.  相似文献   

9.
Molecular dynamics simulations were performed on the duplex DNA dodecamers d(CGCGAA TT CGCG): d(CGCGAATTCGCG) and d(GCACGAA TT AAG): d(CTTAATTCGTGC), where TT denotes a cis, syn cyclobutane thymine dimer. The constant temperature and pressure algorithm of the AMBER 4.1 molecular-modeling package was used with explicit water and counterions, periodic boundary conditions and electrostatic interactions evaluated by the particle-mesh Ewald method. Results were analyzed by the CURVES algorithm and its implementation in DIALS and WINDOWS. Calculated distortions of DNA structure by the thymine dimer were qualitatively and quantitatively similar for the two sequences. Despite the enhanced flexibility of the native TpA dinucleotide step, major deviations from the B-DNA values of helicoidal parameters were found only at the Ap and p dinucleotide steps in both sequences. Only the AT base pairs of the two sequences that contain the 5' thymine of the dimers exhibited weakened Watson-Crick hydrogen bonds and anomalous stretching. Hence, we conclude that the pattern of structural perturbations responsible for recognition of cis, syn thymine dimers by repair enzymes is not sensitive to their sequence context.  相似文献   

10.
We report here the release of a web-based tool (MDDNA) to study and model the fine structural details of DNA on the basis of data extracted from a set of molecular dynamics (MD) trajectories of DNA sequences involving all the unique tetranucleotides. The dynamic web interface can be employed to analyze the first neighbor sequence context effects on the 10 unique dinucleotide steps of DNA. Functionality is included to build all atom models of any user-defined sequence based on the MD results. The backend of this interface is a relational database storing the conformational details of DNA obtained in 39 different MD simulation trajectories comprising all the 136 unique tetranucleotide steps. Examples of the use of this data to predict DNA structures are included. Availability: http://humphry.chem.wesleyan.edu:8080/MDDNA. Supplementary information: Supplementary data including color figures are available at Bioinformatics online.  相似文献   

11.
We calculated the interatomic distances between all couples of non-hydrogen atoms belonging to the neighboring Watson-Crick base pairs in the available crystal structures of DNA. Their standard deviations revealed remarkably large differences in the variability of the base stacking geometries of the particular steps. In line with experimental studies in solution, (CpA)-(TpG) and (TpA).(TpA) were identified as the most variable or flexible steps in the crystal structures of B-DNA. On the other hand, base stacking geometries of the (ApT).(ApT) steps were the most invariant, which was very surprising because all three steps composed only of C and G were much more flexible. This finding suggests that conformational stability of DNA and the rigidity have different origins. Furthermore, the nucleotide sequence dependence of the flexibility was almost reversed in A-DNA because the most flexible steps in B-DNA were the least flexible in A-DNA. The most invariant steps of B-DNA were variable in A-DNA. The (ApT).(ApT) step was a notable exception to this rule because it belonged to the most rigid steps in both B-DNA and A-DNA. The present results are fully consistent with the properties that poly(dA-dT).poly(dA-dT), poly(dA).poly(dT), poly(dAdC).poly(dG-dT) and poly(dA-dG).poly(dC-dT) exhibit in solution.  相似文献   

12.
Topoisomerase II enzymes are essential enzymes that modulate DNA topology and play a role in chromatin compaction. While these enzymes appear to recognize and cleave the DNA in a nonrandom fashion, factors that underlie enzyme specificity remain an enigma. To gain new insights on these topics, we undertake, using NMR and molecular dynamics methods, studies of the structural and dynamic features of a 21 bp DNA segment preferentially cleaved by topoisomerases II. The large size of the oligonucleotide did not hamper the determination of structures of sufficient quality, and numerous interesting correlations between helicoidal parameters already depicted in crystals and molecular dynamics simulations are recovered here. The main feature of the sequence is the occurrence of a large opening of the base pairs in a four-residue AT-rich region located immediately at the 5′ end of one of the cleaved sites. This opening seems to be largely dependent on sequence context, since a similar opening is not found in the other AT base pairs of the sequence. Furthermore, two adenine nucleotides of the same portion of the oligonucleotide present slow internal motions at the NMR timescale, revealing particular base dynamics. In conclusion, this AT-rich region presents the most salient character in the sequence and could be involved in the preferential cleavage by topoisomerase II. The examination of preferred sites in the literature pointed out the frequent occurrence of AT-rich sequences, namely matrix attachment region and scaffold attachment region sequences, at the sites cleaved by topoisomerase II. We could infer that the particular flexibility of these sequences plays an important role in enabling the formation of a competent cleavage complex. The sequences could then be selected based on their facility to undertake conformational change during the complex formation, rather than purely based on binding affinity.  相似文献   

13.
A database of X-ray crystal structures of double helical DNA oligomers has been used to analyse the role of the sugar-phosphate backbone in coupling the conformational properties of neighbouring dinucleotide steps. The base step parameters which are most strongly coupled to the backbone degrees of freedom are slide and shift, and these are the two dinucleotide step parameters which show strong correlations along a sequence: the value of slide follows the values in the neighbouring steps, whereas shift tends to alternate. This conformational coupling is mediated by the shared furanose rings at the step junctions: a change in the value of slide causes a change in the mean value of the same strand 3' and 5'-chi torsion angle, and a change in the mean value of the 3' and 5' sugar pseudo-rotation phase angle, P; a change in the value of shift causes a difference between the same strand 3' and 5'-chi in A-DNA and a difference between the 3' and 5'-P in B-DNA. We have used a database of tetranucleotide X-ray crystal structures to parameterise a simple model for the coupling of slide and shift. Using this junction model together with our dinucleotide step potential energy maps described previously, we can in principle calculate the structure of any DNA oligomer. The parameterisation indicates that the rotational step parameters are accurate to within 5 degrees, and the translational step parameters are accurate to within 0.5 A. The model has been used to study the potential energy surfaces of all possible tetranucleotide sequences, and the calculations agree well with the experimental data from X-ray crystal structures. Some dinucleotide steps are context independent (AA/TT, AT and TA), because the conformational properties of all possible neighbouring steps are compatible. When the conformational properties of the neighbours are not compatible, the behaviour of a step cannot be understood at the dinucleotide level. Thus the conformations of CG, GC and GG/CC are all strongly context dependent. The remaining mixed sequence steps show weakly context-dependent behaviour. The approach allows the calculation of the relative stability and flexibility of tetranucleotide sequences, and the results indicate why TATA is used as an origin of replication. Clear predictions are made about sequences which have not yet been characterised crystallographically. In particular, poly(CCA).poly(TGG) is predicted to have an unusual structure which lies between the C and D-DNA polymorphs.  相似文献   

14.
Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.  相似文献   

15.
One and two dimensional NMR techniques have been used together with molecular modelling to obtain the solution structure for the photoproduct d(TpA)*. The NMR data confirm that the cyclobutane linkage is formed between the bonds thymine C6-C5 and adenine C5-C6. The 2D NOE data are used as constraints in a distance geometry calculation. The structures obtained show a trans-syn cyclobutane linkage and the glycosidic angles are SYN and ANTI for thymidine and deoxyadenosine, respectively. The coupling constant data are used to check the backbone torsion angles of the obtained structures. Typical torsion angles are a gamma+ and beta t for the deoxyadenosine residue. A free molecular dynamics simulation of a trans-syn d(TpA) photoproduct confirmed all these structural characteristics.  相似文献   

16.
Circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques have been used to characterize the structural properties of the two self-complementary DNA octamers d(TGACGTCA) (I) and d(ACTGCAGT) (II). These display as distinctive features reverse sequences and central steps CpG and GpC, respectively. CD experiments lead to B-form DNA spectra characterized by larger magnitude signals in the case of octamer (I). NMR COSY spectra indicate that in the two octamers all the residues are predominantly south, S, (2'-endo) sugar conformation. NMR NOESY spectra show most of the glycosidic angles confined in the range predicted for B-form DNA although important heterogeneity is noticed along the chains, more pronounced in the case of octamer (I). Both the increase of north, N, (3'-endo) sugar conformation and P (pseudorotation phase angle) deviation from its standard B-form DNA value (162 degrees) express local sequence dependent structure distortions, remarkably visible in CpG step of octamer (I) and agreeing with NOESY cross-peaks intensities. Results interpreted according to Calladine's rules indicate higher cross-chain strains in octamer (I) than in octamer (II). All together, we find evidence to support for octamer (I) in solution local structures with A-DNA properties likely dictated by the central CpG step. Such structures could be involved in the DNA recognition by proteins and anticancerous drugs.  相似文献   

17.
The short-time (submicrosecond) bending dynamics of duplex DNA were measured to determine the effect of sequence on dynamics. All measurements were obtained from a single site on duplex DNA, using a single, site-specific modified base containing a rigidly tethered, electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of single-step sequence-dependent bending force constants, determined from the mean squared amplitude of bending relative to the end-to-end vector using the modified weakly bending rod model. The bending dynamics at a single site are a function of the sequence of the nucleotides constituting the duplex DNA. We developed and examined several dinucleotide-based models for flexibility. The models indicate that the dominant feature of the dynamics is best explained in terms of purine- and pyrimidine-type steps, although distinction is made among all 10 unique steps: It was found that purine-purine steps (which are the same as pyrimidine-pyrimidine steps) were near average in flexibility, but the pyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereas purine-pyrimidine steps were more than half as flexible as average DNA. Therefore, the range of stepwise flexibility is approximately fourfold and is characterized by both the type of base pair step (pyrimidine/purine combination) and the identity of the bases within the pair (G, A, T, or C). All of the four models considered here underscore the complexity of the dependence of dynamics on DNA sequence with certain sequences not satisfactorily explainable in terms of any dinucleotide model. These findings provide a quantitative basis for interpreting the dynamics and kinetics of DNA-sequence-dependent biological processes, including protein recognition and chromatin packaging.  相似文献   

18.
We have prepared novel DNA footprinting substrates that contain all 64 symmetrical hexanucleotide sequences. These were contained in two restriction fragments that were cloned into the pUC19 polylinker site; each fragment was also obtained in both orientations. These fragments were used to assess the sequence binding preferences of the synthetic quinoxaline antibiotic TANDEM. We found that, although the ligand binds to most TpA steps, the affinity is affected by the flanking sequences. The best binding sites contain the tetranucleotide sequence ATAT, although YATATR is a better site than RATATY. TTAA always is a poor binding site, especially TTTAAA. The binding to GTAC is strongly dependent on the flanking bases, with good binding to GGTACC but none at all to CGTACG.  相似文献   

19.
Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.  相似文献   

20.
The interaction of the antibiotic drug norfloxacin with double-stranded DNA containing interior 5'-CpG-3', 5'-GpC-3', and 5'-GpG-3' steps was studied by 1H NMR. The drug is in fast exchange on the NMR timescale. A highly selective broadening of the imino proton resonances assigned to central CpG steps was observed after addition of drug, indicating an intercalation-like interaction. DNA sequences with central CpG steps also displayed broadening of non-hydrogen-bonded cytosine amino protons in the major groove upon addition of norfloxacin. Furthermore, a sequence-independent selective broadening of the adenine H2 resonance and an upfield shift of the guanine amino proton resonance, both protons located in the minor groove, was observed. Two-dimensional-NOESY spectra showed that no significant structural changes were induced in the DNA by the drug. The results suggest that the planar two-ring system of norfloxacin partially intercalates into CpG steps and that the drug also exhibits non-specific groove binding.  相似文献   

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