In-silicon studies on hydration in EcoRI-cognate DNA complex.

Restriction endonucleases (REs) cleave DNA at specific site in presence of Mg2+ ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg2+ and Ca2+ and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg2+ ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg2+ loaded EcoRI-DNA complex. We also perform steered MD simulations and observe that the rate of decrease of fraction of hydrogen bonds is slower in the mutated complex than the unmutated complex.

Effect of osmolytes on the EcoRI endonuclease: Insights into hydration and protein dynamics from molecular dynamics simulations.

Osmolytes play an important role in cellular physiology by modulating the properties of proteins, including their molecular specificity. EcoRI is a model restriction enzyme whose specificity to DNA is altered in the presence of osmolytes. Here, we investigate the effect of two different osmolytes, glycerol and DMSO, on the dynamics and hydration of the EcoRI enzyme using molecular dynamics simulations. Our results show that the osmolytes, alter the essential dynamics of EcoRI. Particularly, we observe that the dynamics of the arm region of EcoRI which is involved in DNA binding is significantly altered. In addition, conformational free energy analyses reveals that the osmolytes bring about a change in the landscape similar to that of EcoRI bound to cognate DNA. We further observe that the hydration of the enzyme for each of the osmolyte is different, indicating that the mechanism of action of each of these osmolytes could be different. Further analyses of interfacial water dynamics using rotational autocorrelation function reveals that while the protein surface contributes to a slower tumbling motion of water, osmolytes, additionally contribute to the slowing of the angular motion of the water molecules. Entropy analysis also corroborates with this finding. We also find that the slowed rotational motion of interfacial waters in the presence of osmolytes contributes to a slowed relaxation of the hydrogen bonds between the interfacial waters and the functionally important residues in the protein. Taken together, our results show that osmolytes alter the dynamics of the protein by altering the dynamics of water. This altered dynamics, mediated by the changes in the water dynamics and hydrogen bonds with functionally important residues, may contribute to the altered specificity of EcoRI in the presence of osmolytes.

Protein-DNA target search relies on quantum walk.

Protein-DNA interactions play a fundamental role in all life systems. A critical issue of such interactions is given by the strategy of protein search for specific targets on DNA. The mechanisms by which the protein are able to find relatively small cognate sequences, typically 15-20 base pairs (bps) for repressors, and 4-6 bps for restriction enzymes among the millions of bp of non-specific chromosomal DNA have hardly engaged researchers for decades. Recent experimental studies have generated new insights on the basic processes of protein-DNA interactions evidencing the underlying complex dynamic phenomena involved, which combine three-dimensional and one-dimensional motion along the DNA chain. It has been demonstrated that protein molecules have an extraordinary ability to find the target very quickly on the DNA chain, in some cases, with two orders of magnitude faster than the diffusion limit. This unique property of protein-DNA search mechanism is known as facilitated diffusion. Several theoretical mechanisms have been suggested to describe the origin of facilitated diffusion. However, none of such models currently has the ability to fully describe the protein search strategy. In this paper, we suggest that the ability of proteins to identify consensus sequences on DNA is based on the entanglement of π-π electrons between DNA nucleotides and protein amino acids. The π-π entanglement is based on Quantum Walk (QW), through Coin-position entanglement (CPE). First, the protein identifies a dimer belonging to the consensus sequence, and localize a π on such dimer, hence, the other π electron scans the DNA chain until the sequence is identified. Focusing on the example of recognition of consensus sequences of EcoRV or EcoRI, we will describe the quantum features of QW on protein-DNA complexes during the search strategy, such as walker quadratic spreading on a coherent superposition of different vertices and environment-supported long-time survival probability of the walker. We will employ both discrete- or continuous-time versions of QW. Biased and unbiased classical Random Walk (CRW) have been used for a long time to describe the Protein-DNA search strategy. QW, the quantum version of CRW, has been widely studied for its applications in quantum information applications. In our biological application, the walker (the protein) resides at a vertex in a graph (the DNA structural topology). Differently to CRW, where the walker moves randomly, the quantum walker can hop along the edges in the graph to reach other vertices entering coherently a superposition across different vertices spreading quadratically faster than CRW analogous evidencing the typical speed up features of the QW. When applied to a protein-DNA target search problem, QW gives the possibility to achieve the experimental diffusional motion of proteins over diffusion classical limits experienced along DNA chains exploiting quantum features such as CPE and long-time survival probability supported by the environment. In turn, we come to the conclusion that, under quantum picture, the protein search strategy does not distinguish between one-dimensional (1D) and three-dimensional (3D) cases.

Restriction enzymes use a 24 dimensional coding space to recognize 6 base long DNA sequences.

Restriction enzymes recognize and bind to specific sequences on invading bacteriophage DNA. Like a key in a lock, these proteins require many contacts to specify the correct DNA sequence. Using information theory we develop an equation that defines the number of independent contacts, which is the dimensionality of the binding. We show that EcoRI, which binds to the sequence GAATTC, functions in 24 dimensions. Information theory represents messages as spheres in high dimensional spaces. Better sphere packing leads to better communications systems. The densest known packing of hyperspheres occurs on the Leech lattice in 24 dimensions. We suggest that the single protein EcoRI molecule employs a Leech lattice in its operation. Optimizing density of sphere packing explains why 6 base restriction enzymes are so common.

The Role of Noncognate Sites in the 1D Search Mechanism of EcoRI.

A one-dimensional (1D) search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of 1D diffusion can help resolve the competing demands of a fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of a 1D search by quantum dot-labeled EcoRI. Our data supports the view that proteins search DNA via rotation-coupled sliding over a corrugated energy landscape. We observed that whereas EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations, its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites, which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence-independent energy landscape during fast search but rapidly interconverts with a "hemispecific" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding, which can then lead to target recognition.

Molecular and genetic characterization of the pOV plasmid from Pasteurella multocida and construction of an integration vector for Gallibacterium anatis.

BACKGROUND: The pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties in Pasteurellaceae family bacteria. METHODS: Antimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(-) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with EcoRI for cloning fragments into the pBluescript II KS(-) vector to obtain constructs and to determine the full DNA sequence of pOV. RESULTS: The pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(-), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing strA-strB into the G. anatis chromosome. CONCLUSION: The pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.

Experimental measurement of binding energy, selectivity, and allostery using fluctuation theorems.

Thermodynamic bulk measurements of binding reactions rely on the validity of the law of mass action and the assumption of a dilute solution. Yet, important biological systems such as allosteric ligand-receptor binding, macromolecular crowding, or misfolded molecules may not follow these assumptions and may require a particular reaction model. Here we introduce a fluctuation theorem for ligand binding and an experimental approach using single-molecule force spectroscopy to determine binding energies, selectivity, and allostery of nucleic acids and peptides in a model-independent fashion. A similar approach could be used for proteins. This work extends the use of fluctuation theorems beyond unimolecular folding reactions, bridging the thermodynamics of small systems and the basic laws of chemical equilibrium.

Investigating the effect of charged amino acids on DNA conformation in EcoRI-DNA complex: a molecular dynamics simulation study.

Sequence-specific binding of proteins to DNA is essential for almost all the cellular processes like transcription, translation, replication, etc. One among the various mechanisms that has been identified so far that contributes to the specificity in protein-DNA interaction is the DNA conformational change. Electrostatic neutralization of the phosphate groups by the positively charged amino acid residues in proteins is thought to bring about such conformational changes in DNA. Here, we employ molecular dynamics simulations to examine the effect of charge on amino acids Lys113, Arg145, and Asp91 which are attached to the scissile phosphate on the conformation of DNA in EcoRI-DNA complex. The results indicate that the charge of these amino acids is essential for maintaining the local conformation of DNA in the EcoRI-bound form. Interestingly, we observe that the positively charged amino acids Lys113 and Arg145 have a long-range influence on the DNA conformation, whereas the negatively charged amino acid Asp91 has only a localized effect on the DNA conformation. The charge on the amino acids also alters the collective dynamics of EcoRI. Collectively, the results shed light on the diversity of the effect of charges on DNA conformation as well as on protein dynamics.

Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity.

Water plays important but poorly understood roles in the functions of most biomolecules. We are interested in understanding how proteins use diverse search mechanisms to locate specific sites on DNA; here we present a study of the role of closely associated waters in diverse translocation mechanisms. The bacterial DNA adenine methyltransferase, Dam, moves across large segments of DNA using an intersegmental hopping mechanism, relying in part on movement through bulk water. In contrast, other proteins, such as the bacterial restriction endonuclease EcoRI, rely on a sliding mechanism, requiring the protein to stay closely associated with DNA. Here we probed how these two mechanistically distinct proteins respond to well-characterized osmolytes, dimethyl sulfoxide (DMSO), and glycerol. The ability of Dam to move over large segments of DNA is not impacted by either osmolyte, consistent with its minimal reliance on a sliding mechanism. In contrast, EcoRI endonuclease translocation is significantly enhanced by DMSO and inhibited by glycerol, providing further corroboration that these proteins rely on distinct translocation mechanisms. The well-established similar effects of these osmolytes on bulk water, and their differential effects on macromolecule-associated waters, support our results and provide further evidence of the importance of water in interactions between macromolecules and their ligands.

Extracting enzyme processivity from kinetic assays.

A steady-state analysis for the catalytic turnover of molecules containing two substrate sites is presented. A broad class of Markovian dynamic models, motivated by the action of DNA modifying enzymes and the rich variety of translocation mechanisms associated with these systems (e.g., sliding, hopping, intersegmental transfer, etc.), is considered. The modeling suggests an elementary and general method of data analysis, which enables the extraction of the enzyme's processivity directly and unambiguously from experimental data. This analysis is not limited to the initial velocity regime. The predictions are validated both against detailed numerical models and by revisiting published experimental data for EcoRI endonuclease acting on DNA.

Association between the APOB XbaI and EcoRI polymorphisms and lipids in Chinese: a meta-analysis.

BACKGROUND: No previous meta-analysis was to report the association between the apolipoprotein B (APOB) XbaI and EcoRI polymorphisms and serum lipids in Chinese. We performed the study to investigate their potentially association. METHODS AND RESULTS: Studies in English and Chinese were found via a systematic search of Pubmed, Embase, CNKI and Wanfang databases. The dominant genetic model and random-effects model were used to pool data from individual studies. As a result, a total of 30 articles with 5611 subjects for XbaI and 2653 subjects for EcoRI were included in the current study. For the XbaI polymorphism, overall, subjects carrying X+ allele were significantly associated with higher TC,TG and LDL compared with X-X- genotype (Pvalue = 0.0006, OR (95 %) = -0.55 (-0.86,-0.23); Pvalue = 0.0004, OR (95 %) = -0.30 (-0.47,-0.14); (Pvalue = 0.05, OR (95 %) = -0.23(-0.46,-0.00), respectively). Similar results were observed in the subgroups of Han, healthy individuals (HT), coronary heart disease (CHD), cerebral infarction (CI), and cholelithiasis. For HDL, positive association between X+ allele with Lower lipid value was found in CHD and CI subgroups. For EcoRI polymorphism, overall, the E- allele carriers were found to be obviously linked with elevated LDL and lower HDL compared with E + E+ genotype (Pvalue = 0.02,OR (95 %) = -0.27 (-0.49,-0.05); Pvalue = 0.01, OR (95 %) = 0.17 (0.03, 0.30), respectively). TC was significantly high in subjects carrying E- allele in the subgroup of hyperlipidemia. No evidence of publication bias was observed. CONCLUSIONS: The two genetic variants of APOB may be associated with serum lipids in Chinese.

DNA electrochemical sensor for detection of PRSS1 point mutation based on restriction endonuclease technique.

To construct a restriction endonuclease based biosensor technology for PRSS1 genotyping. We designed a thiol-modified hairpin probe where the neck has EcoRI endonuclease recognition sites according to the PRSS1 gene c.410 C>T (p.T137 M) mutation and it was fixed on the gold electrode. Different charge generated by the binding of MB to phosphate groups of DNA before and after hybridization was used for distinguishing the different genotypes and quantity. This showed that the novel sensor can better distinguish the complementary sequence, single-base mismatches, and completely noncomplementary sequences, and the linear range for the logarithm was Y=-0.0242 X+0.1574, R=0.9912(Y=current, X=log target DNA concentration); the detection limit for DNA detection is estimated to be 50 fM.

An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets.

Effect of particle size on conformation and enzymatic activity of EcoRI adsorbed on CdS nanoparticles.

Better understanding of the interaction between nanoparticles (NPs) and protein is the basis for biological and biomedical applications of NPs. Water-soluble fluorescent CdS NPs have been widely used in the biological and biomedical fields and the study on effect of CdS NPs size on conformation and enzymatic activity of protein might be very important in its application. In this work, the interaction of CdS NPs with different size with type II restriction endonuclease (EcoRI) were investigated by atomic force microscopy, transmission electron microscopy, UV-vis spectroscopy, fluorescence quenching method, CD spectra, laser scanning confocal microscopy and gel electrophoresis. It was found that the equilibrium constant (kD) as well as the cooperativity degree of CdS NPs-EcoRI binding (Hill constant, n) strongly depended on the CdS NPs size. The different curvature of CdS NPs surface could result in different changes of EcoRI conformation. The gel electrophoresis indicated that the decrease in α-helix content more or less affected the activity of EcoRI.

Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

Sensitive Cu2+-Cu2+ distance measurements in a protein-DNA complex by double-quantum coherence ESR.

Double quantum coherence (DQC) ESR spectroscopy is applied to measure the Cu(2+)-Cu(2+) distance in the EcoRI-DNA complex. A simple method is proposed to reduce the contribution of nuclear hyperfine and quadrupole interactions to such data. The effects of such interactions between the electron spin of Cu(2+) and neighboring nuclei on the DQC data make it difficult to measure the nanometer range interspin distance. The DQC data is in good agreement with results obtained by double electron electron resonance (DEER) spectroscopy. At the same time, the signal-to-noise ratio per shot in DQC is high. Taken together, these results provide impetus for further development of paramagnetic metal ion-based DQC techniques.

Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model.

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side-chain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein-DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems.

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

Spectroscopic studies on the interaction between EcoRI and CdS QDs and conformation of EcoRI in EcoRI-CdS QDs bioconjugates.

Water-soluble fluorescent quantum dots (QDs) have been widely used in biological and biomedical fields, and the interaction between QDs and proteins and the conformational structure of the protein in the bioconjugate has attracted increasing attention. In this study, UV-Vis spectroscopy, fluorescence quenching, CD spectra, gel electrophoresis and Fourier transform infrared (FTIR) spectroscopic techniques were used to systematically investigate the interaction between type II restriction endonuclease (EcoRI) and CdS QDs and the conformational structure of EcoRI in the EcoRI-CdS QDs bioconjugates. The results indicated that electrostatic interactions played a major role in the binding reaction at pH 6.0, and the nature of quenching was static, resulting in forming CdS QDs-EcoRI bioconjugates. FTIR and CD spectra studies indicated a decrease of α-helical and turn structures accompanied by the increase of ß-sheet structures of EcoRI in the bioconjugates. This study showed that the interaction between EcoRI and CdS QDs resulted in a change in the secondary structure of EcoRI after it was conjugated with CdS QDs, but the enzyme activity was maintained.