CG Steps Research Articles

Advancing attenuation estimation through integration of the Hessian in multiparameter viscoacoustic full-waveform inversion

Accurate seismic attenuation models of subsurface structures not only enhance subsequent migration processes by improving fidelity, resolution, and facilitating amplitude-compliant angle gather generation but also provide valuable constraints on subsurface physical properties. Leveraging full-wavefield information, multiparameter viscoacoustic full-waveform inversion ( Q-FWI) simultaneously estimates seismic velocity and attenuation ( Q) models. However, a major challenge in Q-FWI is the contamination of crosstalk artifacts, where inaccuracies in the velocity model are mistakenly mapped to the inverted attenuation model. While incorporating the Hessian is expected to mitigate these artifacts, the explicit implementation is prohibitively expensive due to its formidable computational cost. In this study, we formulate and develop a Q-FWI algorithm via the Newton-conjugate gradient (CG) framework, where the search direction at each iteration is determined through an internal CG loop. In particular, the Hessian is integrated into each CG step in a matrix-free fashion using the second-order adjoint-state method. We find through synthetic experiments that our Newton-CG Q-FWI significantly mitigates crosstalk artifacts compared with the limited-memory Broyden-Fletcher-Goldfarb-Shanno method and the CG method, albeit with a notable computational cost. In the discussion of several key implementation details, we also determine the significance of the approximate Gauss-Newton Hessian, the second-order adjoint-state method, and the two-stage inversion strategy.

Environmental and cost analysis for polyhydroxyalkanoate production from glycerol byproduct: A case study from integrated soap and biodiesel plants

This work focuses on the sustainable integrated production of bioplastic, also known as Polyhydroxyalkanoate (PHA). Glycerol by-product generates from soap production and biodiesel production plant were utilized as the feedstock to overcome the current drawbacks in PHA production. Different purities of glycerol (i.e,16 wt%, 40 wt% and 60 wt%) were undergone pretreatment steps such as acidification, filtration, evaporation, hexane washing, methanol recovery and ion exchange to obtain 98 wt% of glycerol prior to PHA production. Capital of equipment purchase was estimated by using correlation and graphical method. It was found that, cost of chemical purchase for pretreatment has a high impact on the overall economy of the production. Economic feasibility of all proposed process routes was assessed by calculating the Payback period. Lastly, environmental impact of each pretreatment method was assessed by conducting an LCA by setting the annual target of 750,000 kg as the functional unit. Global warming potential, acidification potential and ecotoxicity of freshwater were chosen as the impact categories in this process. It was found that route 4 with feedstock purity of 60 wt% CG and pretreatment steps of methanol recovery, neutralization, centrifugation, water evaporation and distillation was found to be the most economically and environmentally sustainable among the assessed process routes.

Effect of the Thermal Fluctuations of the Photophysics of GC and CG DNA Steps: A Computational Dynamical Study

Here we refine and assess two computational procedures aimed to include the effect of thermal fluctuations on the electronic spectra and the ultrafast excited state dynamics of multichromophore systems, focusing on DNA duplexes. Our approach is based on a fragment diabatization procedure that, from a given Quantum Mechanical (QM) reference method, can provide the parameters (energy and coupling) of the reference diabatic states on the basis of the isolated fragments, either for a purely electronic excitonic Hamiltonian (FrDEx) or a linear vibronic coupling Hamiltonian (FrD-LVC). After having defined the most cost-effective procedure for DNA duplexes on two smaller fragments, FrDEx is used to simulate the absorption and Electronic Circular Dichroism (ECD) spectra of (GC)5 sequences, including the coupling with the Charge Transfer (CT) states, on a number of structures extracted from classical Molecular Dynamics (MD) simulations. The computed spectra are close to the reference TD-DFT calculations and fully consistent with the experimental ones. We then couple MD simulations and FrD-LVC to simulate the interplay between local excitations and CT transitions, both intrastrand and interstrand, in GC and CG steps when included in a oligoGC or in oligoAT DNA sequence. We predict that for both sequences a substantial part of the photoexcited population on G and C decays, within 50-100 fs, to the corresponding intrastrand CT states. This transfer is more effective for GC steps that, on average, are more closely stacked than CG ones.

Sequence dependent influence of an A…A mismatch in a DNA duplex: An insight into the recognition by hZαADAR1 protein

Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A…A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A…A mismatch in the midst of 5′CAG/3′GAC, 5′GAC/3′CAG and 5′CAA/3′GAT (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A…A mismatch in the midst of 5′GAA/3′CAT, 5′GAG/3′CAC, 5′AAC/3′TAG, 5′AAG/3′TAC, 5′TAA/3′AAT, 5′TAT/3′AAA and 5′AAT/3′TAA sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZαADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZαADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5′GAC/3′CAG upon titrating with hZαADAR1. Intriguingly, 5′AAC/3′TAG, 5′AAG/3′TAC and 5′GAA/3′CAT exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5′CAA/3′GAT. These indicate that although A…A mismatch could induce a local B-Z junction transiently, hZαADAR1 requires the presence of a G…C/C…G base pair adjacent to the A…A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZαADAR1 in the presence of the A…A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A…A mismatch towards the B-Z junction.

The effects of a neutral cosolute on the B to Z transition for DNA duplexes incorporating both CG and CA steps

ABSTRACTIn the cell, nearly 40% of the volume is occupied by macromolecular crowding agents and smaller osmolytes accumulate in response to environmental stresses. Of particular interest is the influence of osmolytes on the transition of the right-handed B-DNA to the left-handed Z-DNA. Due to the correlation between Z-DNA formation potential and regions of active transcription, Z-DNA is believed to serve a vital role in the transcription process, and changes in osmolyte concentration may influence transcription as a part of the stress response. We utilized circular dichroism spectroscopy to monitor changes in conformation of DNA duplexes containing a full-turn of Z-DNA in the presence and absence of PEG 200. We used PEG 200 as a model neutral cosolute. Sodium ion titrations revealed that PEG 200 influenced the folding of Z-DNA compared to dilute solution conditions by decreasing the free energy of folding, increasing folding cooperativity, and decreasing the in vitro [Na+] and Δn required for folding for all sequences tested, even those that included 40% CA steps instead of the classic CG repeats. Moreover, the presence of 40% PEG 200 induced the Z-form conformation in sequences that would not fully adopt the Z-form structure even in 5 M NaCl. These results suggest that osmolytes may play a significant role in supporting the transient formation of Z-DNA in vivo, and that sequences containing a significant amounts of CA instead of CG repeats may more favorably adopt the Z-conformation as a part of binding and regulatory processes than had been previously considered.

Improvement of Cardiorespiratory Fitness in Young men with Cardiovascular Risks Participating in Pedometer-based Workplace Programme

Lack of exercise among Malaysians and the increasing cardiovascular disease morbidity and mortality among young men are of concern. In response to the wide use of pedometers, daily targets of 10,000 steps/day have been recommended. In Malaysia, data on workplace exercise intervention is scarce. A programme was designed to enable subjects to increase their level of walking as part of their daily work routine and self-monitored by using pedometers. The aim of this study was to evaluate the improvement of cardiorespiratory fitness (CRF) through a pedometer based walking programme at the workplace. A total of 70 young men (20-40 years old) who were sedentary, achieving less than 5,000 steps/day in casual walking with 2 or more cardiovascular risk factors were recruited in IKBN Hulu Langat. Subjects were randomly assigned to a control (CG) (n=34; no change in walking) and pedometer group (PG) (n=36; minimum target: 8,000 steps/day). Blood lipid profile, anthropometric and CRF were measured at baseline and after 12 weeks. At post intervention, the CG step counts were similar (4983 ± 366 vs 5697 ± 407 steps/day). The PG increased step count from 4996 ± 805 to 10,128 ± 511 steps/day (P<0.001). Results for lipid and anthropometrics variables were significantly improved for time and group effect (p<0.001). In PG, the CRF was significantly increased for time and effect (p<0.01) for VO2 peak from 31.54 ± 9.66 to 40.15 ± 9.55(ml/kg/min but no change in CG (31.46 ± 6.15 vs 31.60 ± 8.99 ml/kg/min). The walking programme improved health status in terms of improving biophysical profiles and cardiorespiratory fitness.

Enhanced sampling simulations of DNA step parameters.

A novel approach for the selection of step parameters as reaction coordinates in enhanced sampling simulations of DNA is presented. The method uses three atoms per base and does not require coordinate overlays or idealized base pairs. This allowed for a highly efficient implementation of the calculation of all step parameters and their Cartesian derivatives in molecular dynamics simulations. Good correlation between the calculated and actual twist, roll, tilt, shift, and slide parameters is obtained, while the correlation with rise is modest. The method is illustrated by its application to the methylated and unmethylated 5'-CATGTGACGTCACATG-3' double stranded DNA sequence. One-dimensional umbrella simulations indicate that the flexibility of the central CG step is only marginally affected by methylation.

5-Methylation of Cytosine in CG:CG Base-Pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile "opening" mode and two shear "sliding" and "tearing" modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics.

Probing Sequence-Specific DNA Flexibility in A-Tracts and Pyrimidine-Purine Steps by Nuclear Magnetic Resonance 13C Relaxation and Molecular Dynamics Simulations

Sequence-specific DNA flexibility plays a key role in a variety of cellular interactions that are critical for gene packaging, expression, and regulation, yet few studies have experimentally explored the sequence dependence of DNA dynamics that occur on biologically relevant time scales. Here, we use nuclear magnetic resonance (NMR) carbon spin relaxation combined with molecular dynamics (MD) simulations to examine the picosecond to nanosecond dynamics in a variety of dinucleotide steps as well as in varying length homopolymeric A(n)·T(n) repeats (A(n)-tracts, where n = 2, 4, or 6) that exhibit unusual structural and mechanical properties. We extend the NMR spin relaxation time scale sensitivity deeper into the nanosecond regime by using glycerol and a longer DNA duplex to slow overall tumbling. Our studies reveal a structurally unique A-tract core (for n > 3) that is uniformly rigid, flanked by junction steps that show increasing sugar flexibility with A-tract length. High sugar mobility is observed at pyrimidine residues at the A-tract junctions, which is encoded at the dinucleotide level (CA, TG, and CG steps) and increases with A-tract length. The MD simulations reproduce many of these trends, particularly the overall rigidity of A-tract base and sugar sites, and suggest that the sugar-backbone dynamics could involve transitions in sugar pucker and phosphate backbone BI ↔ BII equilibria. Our results reinforce an emerging view that sequence-specific DNA flexibility can be imprinted in dynamics occurring deep within the nanosecond time regime that is difficult to characterize experimentally at the atomic level. Such large-amplitude sequence-dependent backbone fluctuations might flag the genome for specific DNA recognition.

DNA intercalation without flipping in the specific ThaI–DNA complex

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.

The hydration and unusual hydrogen bonding in the crystal structure of an RNA duplex containing alternating CG base pairs

The X-ray structures of two crystal forms of the RNA duplex [CGCG(5-FC)G]2 are the first crystal structures for an RNA duplex containing alternating CG base pairs with unmodified 2′-hydroxyl groups. The triclinic and the rhombohedral crystal forms (PDB ids 3jxq, 3jxr) differ in the molecular packing, the amount of ordered structure and in molecular details, especially the hydration. The P1 data extend to 1.45 Å resolution, while the R32 data reach 1.25 Å. In both structures the RNA oligomers take the form of a right-handed half-turn with conformational parameters close to the canonical A-RNA. The duplexes stack coaxially in the crystal lattice. The geometry of C:G vs. 5-FC:G pairing is similar in terms of H-bond geometry. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. Both structures contain ordered water molecules, forming characteristic hydration network in the major and the minor grooves. In addition to water, the triclinic structure contains four magnesium cations, one of them forming an inner complex with a phosphate. Two types of unusual interactions are observed. In the major groove, the 5-fluorocytidine residues form C–F⋯H–O–H hydrogen bonds with water molecules. In the minor groove, intermolecular contacts include C–H hydrogen bonds (C1′–H1⋯O2′ and C4′–H4′⋯O4′) between two ribose rings.

Four divergent Arabidopsis ethylene‐responsive element‐binding factor domains bind to a target DNA motif with a universal CG step core recognition and different flanking bases preference

The Arabidopsis ethylene-responsive element-binding factor (AtERF) family of transcription factors has approximately 120 members, all of which possess a highly conserved ERF domain. AtERF1, AtERF4, AtEBP and CBF1 are members from different phylogenetic subgroups within the family. Electrophoretic mobility shift assay analyses revealed that the ERF domains of these four proteins were capable of binding specifically to either GCC or dehydration-responsive element (DRE) motifs. In vitro and in vivo binding assays of the four AtERFs with the DRE motif showed that the recognition of the CG step was indispensable in all four of the specific binding reactions, implying that there may be a universal binding characteristic of various ERF domains binding to a given consensus (e.g. the DRE motif). In addition, the core DNA-binding motifs preferred by the four AtERFs were identified, and all of these motifs contained a conserved CG step core. Thus, conserved recognition of the CG step may be the foundation of the formation of the stable complex by the ERF domain with the DRE motif, which is probably determined by the highly conserved residues presented in the DNA contact surface among the whole AtERF family members. The different preferences at flanking bases of individual ERF domains, which appear to be attributed to the subfamily- or subgroup-specific residues, may be essential discrimination of the target binding motif from various similar sequences by divergent AtERF domains.

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6'-acylated kanamycin A (1) and 6'-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K(d)'s ranging from 235 to 1035 nM, with an average K(d) of 618 nM. For 2, the selected hairpins bind with K(d)'s ranging from 135 to 2300 nM, with an average K(d) of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5'GC and 5'CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.

X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)2 molecules by a bis(9-aminoacridine-4-carboxamide) derivative.

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 Å resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)2 molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.

Stabilization Energies of the Hydrogen-Bonded and Stacked Structures of Nucleic Acid Base Pairs in the Crystal Geometries of CG, AT, and AC DNA Steps and in the NMR Geometry of the 5‘-d(GCGAAGC)-3‘ Hairpin: Complete Basis Set Calculations at the MP2 and CCSD(T) Levels

Stabilization energies of the H-bonded and stacked structures of a DNA base pair were studied in the crystal structures of adenine-thymine, cytosine-guanine, and adenine-cytosine steps as well as in the 5'-d(GCGAAGC)-3' hairpin (utilizing the NMR geometry). Stabilization energies were determined as the sum of the complete basis set (CBS) limit of MP2 stabilization energies and the Delta E(CCSD(T)) - Delta E(MP2) correction term evaluated with the 6-31G*(0.25) basis set. The CBS limit was determined by a two-point extrapolation using the aug-cc-pVXZ basis sets for X = D and T. While the H-bonding energies are comparable to those of base pairs in a crystal and a vacuum, the stacking energies are considerably smaller in a crystal. Despite this, the stacking is still important and accounts for a significant part of the overall stabilization. It contributes equally to the stability of DNA as does H-bonding for AT-rich DNAs, while in the case of GC-rich DNAs it forms about one-third of the total stabilization. Interstrand stacking reaches surprisingly large values, well comparable to the intrastrand ones, and thus contributes significantly to the overall stabilization. The hairpin structure is characterized by significant stacking, and both guanine...cytosine pairs possess stacking energies larger than 11.5 kcal/mol. A high portion of stabilization in the studied hairpin comes from stacking (similar to that found for AT-rich DNAs) despite the fact that it contains two GC Watson-Crick pairs having very large H-bonding stabilization. The DFT/B3LYP/6-31G** method yields satisfactory values of interaction energies for H-bonded structures, while it fails completely for stacking.

Selective intercalation of charge neutral intercalators into GG and CG steps: implication of HOMO-LUMO interaction for sequence-selective drug intercalation into DNA.

We have synthesized naphthopyranone epoxide 4 from D-isoascorbic acid together with its three diastereoisomers. DNA alkylation of ODNs containing 5'XGT3' and 5'TGY3' by 4 (11R, 13R), where X and Y are any nucleotide bases, occurred at all G residues except at G of the 5'TGC3' sequence. In contrast, the three other diastereoisomers of 4 showed only weak G alkylation activity. Differential (1)H NMR NOE of the 4-G adduct confirmed the G-N7 alkylation at the epoxide carbon of 4 with concomitant S(N)2 ring opening of the epoxide. Quantitative HPLC analysis of G alkylation efficiency for 4 showed the order of G alkylation susceptibility as TGGT approximately CGT >> TGA > AGT > TGT >> TGC. The order was fully consistent with those reported for aflatoxin B(1) oxide and kapurimycin A(3), suggesting that the sequence selectivity observed for these DNA alkylating agents is not structure dependent but most likely due to the intrinsic property of DNA sequences. We found that the order of G alkylation susceptibility obtained for 4 completely matched the calculated HOMO energy level of G-containing sequences. These results underscore that 4 is a unique molecular probe for ranking the HOMO level of G-containing sequences by well-known G alkylation chemistry and suggests that the intercalation of charge neutral intercalators is a HOMO-controlled process.

An A-type double helix of DNA having B-type puckering of the deoxyribose rings

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(CnGn) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.

Recombinant Human DNA (Cytosine-5) Methyltransferase: II. STEADY-STATE KINETICS REVEAL ALLOSTERIC ACTIVATION BY METHYLATED DNA

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at </=20 microM AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V(max(app)) resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a role in vivo in the regulation of maintenance methylation.

Selective Binding of the TATA Box-Binding Protein to the TATA Box-Containing Promoter: Analysis of Structural and Energetic Factors

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding —NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.

A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA.

The 3D solution structure of the GCC-box binding domain of a protein from Arabidopsis thaliana in complex with its target DNA fragment has been determined by heteronuclear multidimensional NMR in combination with simulated annealing and restrained molecular dynamic calculation. The domain consists of a three-stranded anti-parallel beta-sheet and an alpha-helix packed approximately parallel to the beta-sheet. Arginine and tryptophan residues in the beta-sheet are identified to contact eight of the nine consecutive base pairs in the major groove, and at the same time bind to the sugar phosphate backbones. The target DNA bends slightly at the central CG step, thereby allowing the DNA to follow the curvature of the beta-sheet.