Groove Atoms Research Articles

A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases

In A-family DNA polymerases (dPols), a functional 3′-5′ exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the −2 and − 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the −2 and − 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at −2 and − 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

Water spines and networks in G-quadruplex structures.

Quadruplex DNAs can fold into a variety of distinct topologies, depending in part on loop types and orientations of individual strands, as shown by high-resolution crystal and NMR structures. Crystal structures also show associated water molecules. We report here on an analysis of the hydration arrangements around selected folded quadruplex DNAs, which has revealed several prominent features that re-occur in related structures. Many of the primary-sphere water molecules are found in the grooves and loop regions of these structures. At least one groove in anti-parallel and hybrid quadruplex structures is long and narrow and contains an extensive spine of linked primary-sphere water molecules. This spine is analogous to but fundamentally distinct from the well-characterized spine observed in the minor groove of A/T-rich duplex DNA, in that every water molecule in the continuous quadruplex spines makes a direct hydrogen bond contact with groove atoms, principally phosphate oxygen atoms lining groove walls and guanine base nitrogen atoms on the groove floor. By contrast, parallel quadruplexes do not have extended grooves, but primary-sphere water molecules still cluster in them and are especially associated with the loops, helping to stabilize loop conformations.

Partial Base Flipping Is Sufficient for Strand Slippage near DNA Duplex Termini

Strand slippage is a structural mechanism by which insertion-deletion (indel) mutations are introduced during replication by polymerases. Three-dimensional atomic-resolution structural pathways are still not known for the decades-old template slippage description. The dynamic nature of the process and the higher energy intermediates involved increase the difficulty of studying these processes experimentally. In the present study, restrained and unrestrained molecular dynamics simulations, carried out using multiple nucleic acid force fields, are used to demonstrate that partial base-flipping can be sufficient for strand slippage at DNA duplex termini. Such strand slippage can occur in either strand, i.e. near either the 3' or the 5' terminus of a DNA strand, which suggests that similar structural flipping mechanisms can cause both primer and template slippage. In the repetitive mutation hot-spot sequence studied, non-canonical base-pairing with exposed DNA groove atoms of a neighboring G:C base-pair stabilizes a partially flipped state of the cytosine. For its base-pair partner guanine, a similar partially flipped metastable intermediate was not detected, and the propensity for sustained slippage was also found to be lower. This illustrates that a relatively small metastable DNA structural distortion in polymerase active sites could allow single base insertion or deletion mutations to occur, and stringent DNA groove molecular recognition may be required to maintain intrinsic DNA polymerase fidelity. The implications of a close relationship between base-pair dissociation, base unstacking, and strand slippage are discussed in the context of sequence dependence of indel mutations.

Influence of divalent magnesium ion on DNA: molecular dynamics simulation studies

A large amount of experimental evidence is available on the effect of magnesium ions on the structure and stability of DNA double helix. Less is known, however, on how these ions affect the stability and dynamics of the molecule. The static time average pictures from X-ray structures or the quantum chemical energy minimized structures lack understanding of the dynamic DNA–ion interaction. The present work addresses these questions by molecular dynamics simulation studies on two DNA duplexes and their interaction with magnesium ions. Results show typical B-DNA character with occasional excursions to deviated states. We detected expected stability of the duplexes in terms of backbone conformations and base pair parameter by the CHARMM-27 force field. Ion environment analysis shows that Mg2+ retains the coordination sphere throughout the simulation with a preference for major groove over minor. An extensive analysis of the influence of the Mg2+ ion shows no evidence of the popular predictions of groove width narrowing by dipositive metal ion. The major groove atoms show higher occupancy and residence time compared to minor groove for magnesium, where no such distinction is found for the charge neutralizing Na+ ions. The determining factor of Mg2+ ion’s choice in DNA binding site evolves as the steric hindrance faced by the bulky hexahydrated cation where wider major groove gets the preference. We have shown that in case of binding of Mg2+ to DNA non electrostatic contributions play a major role. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:5

Conserved structural features of ETS domain-DNA complexes

ETS proteins comprise a family of widespread transcription factors regulating expression of many animal genes. Structurally ETS proteins are characterized by conserved DNA-binding ETS domain, recognizing DNA sequences with trinucleotide GGA. Comparative analysis of structural features of ETS domain-DNA complexes was carried out and conserved contacts, important in terms of interaction stability and specificity were identified. The analysis revealed 9 conserved hydrogen bonds with DNA backbone phosphates and 2 conserved bidentate hydrogen bonds with DNA major groove atoms, one conserved hydrophobic cluster on protein-DNA interface, important for binding site recognition, and 12 conserved water molecules, possibly mediating ETS domain-DNA interaction. The results are represented in specialized data bank of protein-DNA complexes NPIDB.

Prediction of mono- and di-nucleotide-specific DNA-binding sites in proteins using neural networks

BackgroundDNA recognition by proteins is one of the most important processes in living systems. Therefore, understanding the recognition process in general, and identifying mutual recognition sites in proteins and DNA in particular, carries great significance. The sequence and structural dependence of DNA-binding sites in proteins has led to the development of successful machine learning methods for their prediction. However, all existing machine learning methods predict DNA-binding sites, irrespective of their target sequence and hence, none of them is helpful in identifying specific protein-DNA contacts. In this work, we formulate the problem of predicting specific DNA-binding sites in terms of contacts between the residue environments of proteins and the identity of a mononucleotide or a dinucleotide step in DNA. The aim of this work is to take a protein sequence or structural features as inputs and predict for each amino acid residue if it binds to DNA at locations identified by one of the four possible mononucleotides or one of the 10 unique dinucleotide steps. Contact predictions are made at various levels of resolution viz. in terms of side chain, backbone and major or minor groove atoms of DNA.ResultsSignificant differences in residue preferences for specific contacts are observed, which combined with other features, lead to promising levels of prediction. In general, PSSM-based predictions, supported by secondary structure and solvent accessibility, achieve a good predictability of ~70–80%, measured by the area under the curve (AUC) of ROC graphs. The major and minor groove contact predictions stood out in terms of their poor predictability from sequences or PSSM, which was very strongly (>20 percentage points) compensated by the addition of secondary structure and solvent accessibility information, revealing a predominant role of local protein structure in the major/minor groove DNA-recognition. Following a detailed analysis of results, a web server to predict mononucleotide and dinucleotide-step contacts using PSSM was developed and made available at or .ConclusionMost residue-nucleotide contacts can be predicted with high accuracy using only sequence and evolutionary information. Major and minor groove contacts, however, depend profoundly on the local structure. Overall, this study takes us a step closer to the ultimate goal of predicting mutual recognition sites in protein and DNA sequences.

Exploring DNA groove water dynamics through hydrogen bond lifetime and orientational relaxation

Dynamics of water molecules in the grooves of DNA are of great interest both for practical (functionality of DNA) and fundamental (as examples of confined systems) interest. Here the authors employ atomistic molecular dynamics simulations to understand varying water dynamics at the minor and the major grooves of a 38 base-pair long DNA duplex in water. In order to understand and quantify the diversity in the nature of hydrogen bond due to many hydrogen bond donors and acceptors present in the four bases, they have undertaken study of hydrogen bond lifetime (HBLT) correlation functions of all the specific hydrogen bonds between the base atoms and water molecules. They find that the HBLT correlation functions are in general multiexponential, with the average lifetime depending significantly on the specificity and may thus be biologically relevant. The average hydrogen bond lifetime is longer in the minor groove than that in the major groove by almost a factor of 2. Analysis further shows that water hydrogen bonds with phosphate oxygen have substantially shorter lifetimes than those with the groove atoms. They also compute two different orientational time correlation functions (OTCFs) of the water molecules present at the major and the minor grooves and attempt to correlate OTCF with HBLT correlation function. The OTCFs in the minor groove exhibit three time scales, with the time constant of the slowest component one to two orders of magnitude longer than what is observed for bulk water. A slow component is also present for the major groove water but with shorter time constant. Interestingly, correlation between reformations allowed HBLT correlation function [C(HB)(t)] and the OTCF markedly deviates from each other in the grooves, indicating enhanced rigidity of water molecules in the grooves.

Water and ion binding around r(UpA) 12and d(TpA) 12Oligomers - comparison with RNA and DNA (CpG) 12 duplexes

The structural and dynamic properties of the water and ion first coordination shell of the r(A-U) and d(A-T) base-pairs embedded within the r(UpA) 12and d(TpA) 12duplexes are described on the basis of two 2.4 ns molecular dynamics simulations performed in a neutralizing aqueous environment with 0.25 M added KCl. The results are compared to previous molecular dynamics simulations of the r(CpG) 12and d(CpG) 12structures performed under similar conditions. It can be concluded that: (i) RNA helices are more rigid than DNA helices of identical sequence, as reflected by the fact that RNA duplexes keep their initial A-form shape while DNA duplexes adopt more sequence-specific shapes. (ii) Around these base-pairs, the water molecules occupy 21 to 22 well-defined hydration sites, some of which are partially occupied by potassium ions. (iii) These hydration sites are occupied by an average of 21.9, 21.0, 20.1, and 19.8 solvent molecules (water and ions) around the r(G=C), r(A-U), d(G=C), and d(A-T) pairs, respectively. (iv) From a dynamic point of view, the stability of the hydration shell is the strongest for the r(G=C) pairs and the weakest for the d(A-T) pairs. (v) For RNA, the observed long-lived hydration patterns are essentially non-sequence dependent and involve water bridges located in the deep groove and linking OR atoms of adjacent phosphate groups. Maximum lifetimes are close to 400 ps. (vi) In contrast, for DNA, long-lived hydration patterns are sequence dependent and located in the minor groove. For d(CpG) 12, water bridges linking the (G)N3 and (C)O2 with the O4′ atoms of adjacent nucleotides with 400 ps maximum lifetimes are characterized while no such bridges are observed for d(TpA) 12. (vii) Potassium ions are observed to bind preferentially to deep/major groove atoms at RpY steps, essentially d(GpC), r(GpC), and r(ApU), by forming ion-bridges between electronegative atoms of adjacent base-pairs. On average, about half an ion is observed per base-pair. Positive ion-binding determinants are related to the proximity of two or more electronegative atoms. Negative binding determinants are associated with the electrostatic and steric hindrance due to the proximity of electropositive amino groups and neutral methyl groups. Potassium ions form only transient contacts with phosphate groups.

Ribozyme Catalysis from the Major Groove of Group II Intron Domain 5

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

A Single 2′-Hydroxyl Group Converts B-DNA to A-DNA : Crystal Structure of the DNA-RNA Chimeric Decamer Duplex d(CCGGC)r(G)d(CCGG) with a Novel Intermolecular G·C Base-paired Quadruplet

We have found that the introduction of a single 2′-hydroxyl group on the sugar-phosphate backbone of the B-DNA decamer d(CCGGCGCCGG) transforms it to A-DNA. Thus, for the first time the X-ray structures of the same sequence have been observed in both the A and B-DNA conformations, permitting a comparison. Crystals of the DNA-RNA chimeric decamer d(CCGGC)r(G)d(CCGG) belong to the orthorhombic space group P 2 12 12 1 with unit cell dimensions a = 25·63 Å, b = 45·24 Å and c = 47·99 Å, and one decamer duplex in the asymmetric unit. The structure was solved by a rigid body search using the coordinates of the isomorphous structure d(CCCGGCCGGG) and refined to an R value of 0·136 using 2753 unique reflections at 1·9 Å resolution. The final model contains 406 nucleotide atoms and 61 water molecules. The chimeric duplex exhibits typical A-DNA geometry, with all the sugars in the C (3′)- endo puckering and the base-pairs inclined and displaced from the helix axis. The 2′-hydroxyl groups on rG6 and rG16 protrude into the minor groove surface and form different types of hydrogen bonds; that on strand 1 forms an intermolecular hydrogen bond with the furanose ring O (4′) of a symmetry-related C1 residue, while that on strand 2 is involved in two water bridges. Crystal packing forces the G4·C17 base-pair in the top half of the duplex to slide significantly into the minor groove compared to the corresponding C7·G14 base-pair in the bottom half, resulting in these base-pairs exhibiting different base stacking and intermolecular interactions. The base G4 of the G4·C17 base-pair forms an unorthodox base "triple", G4 *(G10·C11), hydrogen-bonding through its minor groove sites N (2) and N (3) to the minor groove atoms N (2) and O (2) of both bases of the G10·C11 base-pair of a symmetry-related molecule. The base G10 of this triple in turn forms a second similar unorthodox base triple, G10 *(G3 *C18), with the adjacent base-pair G3·C18 of the duplex, thus G10 is involved in a double triple. On the other hand, in the bottom half of the duplex, the C7·G14 base pair is involved only in a single similar unorthodox base triple with G20, (C7·G14) *G20, while the adjacent base-pair rG6·C15 is involved in a novel quadruple with C1·G20, (rG6·C15) * (C1·G20), where the latter base-pairs are hydrogen-bonded to each other via the minor groove sites G(N (2))…C(O (2)). The double base triple and quadruple are features observed for the first time.

DNA B to D transition can be explained in terms of hydration economy of the minor groove atoms

Adjacent phosphate oxygen atoms in A and Z-DNA are located much closer together than in the B form and can be hydrated more economically due to the formation of water bridges between them, whereas in the B form phosphates are hydrated individually. This principle of hydration economy of phosphate groups discovered by Saenger and colleagues could not be applied to the B- D transition, which, like the B- A and B- Z transitions, occurs in a situation of water deficiency, because the distances between adjacent phosphates of individual polynucleotide chains in the D form are not much different from B-DNA. It follows from our calculations of B and D-DNA accessibility to solvent performed by the method of Lee & Richards, and from a simulation of solvent structure near DNA, that there is an economy of hydration only for the minor groove atoms. This feature and some experimental data can explain why only a limited range of sequences consisting of A · T or I · C pairs undergo the transition to the D form. The conformational transition in DNAs with such sequences to a poly[d(A)] · poly[d(T)]-like conformation ( B h -DNA), which is accompanied by a narrowing of the minor groove, can be explained in the same way. Calculations suggest that in the D-form minor groove of different A-T or I-C DNAs there is a double-layer hydration spine similar to that observed by Drew & Dickerson in the A-T tract of the d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer. The B- D and B- B h transitions in A + T-rich DNAs can have biological implications, e.g. they can facilitate DNA bending upon the interaction with proteins.