M.TaqI facilitates the base flipping via an unusual DNA backbone conformation

MD simulations have been carried out to understand the dynamical behavior of the DNA substrate of the Thermus aquaticus DNA methyltransferase (M.TaqI) in the methylation process at N6 of adenine. As starting structures, an x-ray structure of M.TaqI in complex with DNA and cofactor analogue (PDB code: 1G 38) and free decamer d(GTTCGATGTC)(2) were taken. The x-ray structure shows two consecutive BII substates that are not observed in the free decamer. These consecutive BII substates are also observed during our simulation. Additionally, their facing backbones adopt the same conformations. These double facing BII substates are stable during the last 9 ns of the trajectories and result in a stretched DNA structure. On the other hand, protein-DNA contacts on 5' and 3' phosphodiester groups of the partner thymine of flipped adenine have changed. The sugar and phosphate parts of thymine have moved further into the empty space left by the flipping base without the influence of protein. Furthermore, readily high populated BII substates at the GpA step of palindromic tetrad TCGA rather than CpG step are observed in the free decamer. On the contrary, the BI substate at the GpA step is observed on the flipped adenine strand. A restrained MD simulation, reproducing the BI/BII pattern in the complex, demonstrated the influence of the unusual backbone conformation on the dynamical behavior of the target base. This finding along with the increased nearby interstrand phosphate distance is supportive to the N6-methylation mechanism.     

Influence of a cis,syn‐cyclobutane pyrimidine dimer damage on DNA conformation studied by molecular dynamics simulations

The photo‐induced formation of cis‐syn‐cyclobutane pyrimidine dimers (CPD) is a highly mutagenic and cancerogenic DNA lesion. In bacteria photolyases can efficiently reverse the dimer formation employing a light‐driven reaction after looping out the CPD damaged bases into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The CPD damage may alter the DNA structure and dynamics already in the absence of the repair enzyme which can facilitate the initial binding of a photolyase repair enzyme. To characterize the effect of a CPD damage, extensive comparative molecular dynamics (MD) simulations on duplex DNA with central regular or CPD damaged nucleotides were performed supplemented with simulations of the DNA‐photolyase complex. Although no spontaneous flipping out transitions of the damaged bases were observed, the simulations showed significant differences in the conformational states of regular and CPD damage DNA. The isolated damaged DNA adopted transient conformations which resembled the global shape of the repair enzyme bound conformation more closely compared to regular B‐DNA. In particular, these conformational changes were observed in most of helical and structural parameters where the protein bound DNA differs drastically from regular B‐DNA. It is likely that the transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out process of the damaged DNA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 215–222, 2015.     

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5'-AACNNNNNNGTGC-3', where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.     

DNA bending by EcoRI DNA methyltransferase accelerates base flipping but compromises specificity.

EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52 degrees and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (KDDNA) was reduced 10-fold, and kcat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme-DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.     

Molecular dynamics simulations and free energy calculations of base flipping in dsRNA

The family of adenosine deaminases acting on RNA (ADARs) targets adenosines in RNA that is mainly double stranded. Some substrates are promiscuously deaminated whereas others, such as the mammalian glutamate receptor B (gluR-B) pre-mRNA, are more selectively deaminated. Many DNA/RNA-base modification enzymes use a base flipping mechanism to be able to reach their target base and it is believed that ADARs function in a similar way. In this study we used molecular dynamics (MD) simulations to describe two sites on the gluR-B pre-mRNA, the selectively targeted R/G site and the nontargeted 46 site, in an attempt to explain the substrate specificity. We used regular MD and also a forced base flipping method with umbrella sampling to calculate the free energy of base opening. Spontaneous opening of the mismatched adenosine was observed for the R/G site but not for the 46 site.     

Bending and flexibility of methylated and unmethylated EcoRI DNA

We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the EcoRI restriction site. Our findings agree well with recent crystal and NMR structures of the EcoRI dodecamer, where an overall bend of seven degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a "generic" DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or direction, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides. We demonstrate that our approach can augment the understanding of DNA structure and dynamics by adding information about the global structure and flexibility of the sequence. We also show that cyclization kinetics can be used to study the properties of modified nucleotides.     

Role of base flipping in specific recognition of damaged DNA by repair enzymes

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G.U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol(-1) lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol(-1) lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol(-1), making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.     

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.     

Structure and axis curvature in two dA6 x dT6 DNA oligonucleotides: comparison of molecular dynamics simulations with results from crystallography and NMR spectroscopy

Molecular dynamics (MD) simulations have been performed on the A6 containing DNA dodecamers d(GGCAAAAAACGG) solved by NMR and d(CGCAAAAAAGCG) solved by crystallography. The experimental structures differ in the direction of axis bending and in other small but important aspects relevant to the DNA curvature problem. Five nanosecond MD simulations of each sequence have been performed, beginning with both the NMR and crystal forms as well as canonical B-form DNA. The results show that all simulations converge to a common form in close proximity to the observed NMR structure, indicating that the structure obtained in the crystal is likely a strained form due to packing effects. A-tracts in the MD model are essentially straight. The origin of axis curvature is found at pyrimidine-purine steps in the flanking sequences.     

Axis Curvature and Ligand Induced Bending in the CAP-DNA Oligomers

The origin of DNA axis curvature in complexes of the catabolite activator protein with DNA is studied using multiple molecular dynamics (MD) simulations of the free and protein-bound forms of the DNA. The results are compared to available solution and crystal structure data. The MD simulations reproduce the experimentally observed bend in DNA and indicate that ∼40% of the bending observed in the complex is intrinsic to the DNA sequence, whereas ∼60% is induced on protein binding. The MD provides a model for the dynamical structure of the DNA free in solution and for ligand-induced bending.     

The effects of sequence context on base dynamics at TpA steps in DNA studied by NMR.

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.     

Molecular dynamics studies of sequence-directed curvature in bending locus of trypanosome kinetoplast DNA.

The macroscopic curvature induced in the double helical B-DNA by regularly repeated adenine tracts (A-tracts) plays an exceptional role in structural studies of DNA because this effect presents the most well documented example of sequence specific conformational modulations. Recently, a new hypothesis of its physical origin has been put forward. According to it, the intrinsic bends in B-DNA may represent one of the consequences of the compressed frustrated state of its backbone. The compressed backbone hypothesis agrees with many data and explains some controversial experimental observations. The original arguments of this theory came out from MD simulations of a DNA fragment with a strong bending propensity. Its sequence, however, was not experimental. It was constructed empirically so as to maximize the magnitude of bending in calculations. To make sure that our computations reproduce the experimental effect we carried out similar simulations with an A-tract repeat of a natural base pair sequence found in a bent locus of a minicircle DNA. We demonstrate spontaneous development of static curvature in the course of MD simulations excluding any initial bias except the base pair sequence. Its direction and magnitude agree with experimental estimates. The results confirm earlier qualitative conclusions and agree with the hypothesis of a compressed backbone as the origin of static bending in B-DNA.     

Markov state models elucidate the stability of DNA influenced by the chiral 5S-Tg base

The static and dynamic structures of DNA duplexes affected by 5S-Tg (Tg, Thymine glycol) epimers were studied using MD simulations and Markov State Models (MSMs) analysis. The results show that the 5S,6S-Tg base caused little perturbation to the helix, and the base-flipping barrier was determined to be 4.4 kcal mol⁻¹ through the use of enhanced sampling meta-eABF calculations, comparable to 5.4 kcal mol⁻¹ of the corresponding thymine flipping. Two conformations with the different hydrogen bond structures between 5S,6R-Tg and A19 were identified in several independent MD trajectories. The 5S,6R-Tg:O6H_O6•••N1:A19 hydrogen bond is present in the high-energy conformation displaying a clear helical distortion, and near barrier-free Tg base flipping. The low-energy conformation always maintains Watson–Crick base pairing between 5S,6R-Tg and A19, and 5S-Tg base flipping is accompanied by a small barrier of ca. 2.0 K_BT (T = 298 K). The same conformations are observed in the MSMs analysis. Moreover, the transition path and metastable structures of the damaged base flipping are for the first time verified through MSMs analysis. The data clearly show that the epimers have completely different influence on the stability of the DNA duplex, thus implying different enzymatic mechanisms for DNA repair.     

Analysis of the stability of looped-out and stacked-in conformations of an adenine bulge in DNA using a continuum model for solvent and ions

A combination of conformational search, energy minimization, and energetic evaluation using a continuum solvent treatment has been employed to study the stability of various conformations of the DNA fragment d(CGCAGAA)/d(TTCGCG) containing a single adenine bulge. The extra-helical (looped-out) bulge conformation derived from a published x-ray structure and intra-helical (stacked bulge base) model structures partially based on nuclear magnetic resonance (NMR) data were used as start structures for the conformational search. Solvent-dependent contributions to the stability of the conformations were calculated from the solvent exposed molecular surface area and by using the finite difference Poisson-Boltzmann approach. Three classes (I-III) of bulge conformations with calculated low energies can be distinguished. The lowest-energy conformations were found in class I, corresponding to structures with the bulge base stacked between flanking helices, and class II, composed of structures forming a triplet of the bulge base and a flanking base pair. All extra-helical bulge structures, forming class III, were found to be less stable compared with the lowest energy structures of class I and II. The results are consistent with NMR data on an adenine bulge in the same sequence context indicating an intra-helical or triplet bulge conformation in solution. Although the total energies and total electrostatic energies of the low-energy conformations show only relatively modest variations, the energetic contributions to the stability were found to vary significantly among the classes of bulge structures. All intra-helical bulge structures are stabilized by a more favorable Coulomb charge-charge interaction but destabilized by a larger electrostatic reaction field contribution compared with all extra-helical and most triplet bulge structures. Van der Waals packing interactions and nonpolar surface-area-dependent contributions appear to favor triplet class II structures and to a lesser degree also the intra-helical stacked bulge conformations. The large conformational variation found for class III conformers might add a favorable entropic contribution to the stability of the extra-helical bulge form.     

Molecular dynamics simulation of hydrated d(CGGGTACCCG)4 as a four-way DNA Holliday junction and comparison with the crystallographic structure

Molecular dynamics (MD) simulation of decamer sequence (CGGGTACCCG)₄ as a four-way Holliday junction is reported here for 15.0 ns at three different temperatures 100, 200 and 300 K, respectively, using AMBER force field. Particle mesh Ewald method has been utilised to deal long-range interaction potentials. After MD simulation, various parameters of the junction model including backbone and helical parameters have been worked out and the dynamical pathway is discussed. Structural analysis and geometrical calculations were carried out through X3DNA. The computational results obtained are compared with the previously reported crystallographic outcomes. The width and depth of the major and minor grooves of the duplex of the four arms of the DNA junction have been calculated. The variations in the C1′–C1′ distances between the two complementary strands are discussed in detail. A close observation of the results reveals that the conformation of the average simulated structure at low temperature is of ‘B’ form and the structural integrity of the DNA junction having a twofold sequence symmetry is temperature dependent. It also seems that besides the other parameters (i.e. presence of ions, solvents, etc.), temperature may be playing a key role in preserving the structural integrity of the DNA junction.     

Flipping duplex DNA inside out: a double base-flipping reaction mechanism by Escherichia coli MutY adenine glycosylase

The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058-1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s(-1) followed by the adenine at 16 s(-1). A protein isomerization step following base flipping at 1.9 s(-1) was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision.     

Opening dynamics of 8‐oxoguanine in DNA

8‐oxoguanine is a major lesion of genomic DNA that results from oxidation of guanine by reactive oxygen species. The repair of this lesion is initiated by 8‐oxoguanine glycosylases, which excise the damaged base by “flipping” it outside the DNA double helix. The molecular mechanisms involved in the specific recognition of the damaged base by the enzyme are not yet fully understood. Several models have proposed that, in DNA, the base pair between 8‐oxoguanine and cytosine may possess altered dynamic properties that could help the enzyme locate the lesion and could favor the selective extra‐helical flipping of the damaged base. To test this proposal, we have characterized the spontaneous opening of the base pair between 8‐oxoguanine and cytosine in a DNA double helix using NMR spectroscopy and proton exchange. The results show that the rate of spontaneous opening of 8‐oxoguanine and the lifetime of the base in the extra‐helical state are the same as those of a canonical guanine‐cytosine base pair, in the same base sequence context. This finding suggests that the opening dynamics of 8‐oxoguanine, when paired with cytosine in DNA, does not play a significant role in the recognition of the lesion by glycosylases. Copyright © 2013 John Wiley & Sons, Ltd.     

Sequence specific DNA binding of Ets-1 transcription factor: molecular dynamics study on the Ets domain--DNA complexes

Molecular dynamics (MD) simulations for Ets-1 ETS domain-DNA complexes were performed to investigate the mechanism of sequence-specific recognition of the GGAA DNA core by the ETS domain. Employing the crystal structure of the Ets-1 ETS domain-DNA complex as a starting structure we carried out MD simulations of: (i). the complex between Ets-1 ETS domain and a 14 base-pair DNA containing GGAA core sequence (ETS-GGAA); (ii). the complex between the ETS domain and a DNA having single base-pair mutation, GGAG sequence (ETS-GGAG); and (iii). the 14 base-pair DNA alone (GGAA). Comparative analyses of the MD structures of ETS-GGAA and ETS-GGAG reveal that the DNA bending angles and the ETS domain-DNA phosphate interactions are similar in these complexes. These results support that the GGAA core sequence is distinguished from the mutated GGAG sequence by a direct readout mechanism in the Ets-1 ETS domain-DNA complex. Further analyses of the direct contacts in the interface between the helix-3 region of Ets-1 and the major groove of the core DNA sequence clearly show that the highly conserved arginine residues, Arg391 and Arg394, play a critical role in binding to the GGAA core sequence. These arginine residues make bidentate contacts with the nucleobases of GG dinucleotides in GGAA core sequence. In ETS-GGAA, the hydroxyl group of Tyr395 is hydrogen bonded to N7 nitrogen of A(3) (the third adenosine in the GGAA core), while the hydroxyl group makes a contact with N4 nitrogen of C(4') (the complementary nucleotide of the fourth guanosine G(4) in the GGAG sequence) in the ETS-GGAG complex. We have found that this difference in behavior of Tyr395 results in the relatively large motion of helix-3 in the ETS-GGAG complex, causing the collapse of bidentate contacts between Arg391/Arg394 and the GG dinucleotides in the GGAG sequence.