New evidences on Tau-DNA interactions and relevance to neurodegeneration

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau.We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor,aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool `PromPredict'. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

CNT loading into cationic cholesterol suspensions show improved DNA binding and serum stability and ability to internalize into cancer cells

Methods which disperse single-walled carbon nanotubes (SWNTs) in water as `debundled', while maintaining their unique physical properties are highly useful. We present here a family of cationic cholesterol compounds (Chol(+)) {Cholest-5en-3 beta-oxyethyl pyridinium bromide (Chol-PB+), Cholest-5en-3 beta-oxyethyl N-methyl pyrrolidinium bromide (Chol-MPB+), Cholest-5en-3 beta-oxyethyl N-methyl morpholinium bromide (Chol-MMB+) and Cholest-5en-3 beta-oxyethyl diazabicyclo octanium bromide (Chol-DOB+)}. Each of these could be easily dispersed in water. The resulting cationic cholesterol (Chol(+)) suspensions solubilized single-walled carbon nanotubes (SWCNTs) by the non-specific physical adsorption of Chol(+) to form stable, transparent, dark aqueous suspensions at room temperature. Electron microscopy reveals the existence of highly segregated CNTs in these samples. Zeta potential measurements showed an increase in potential of cationic cholesterol aggregates on addition of CNTs. The CNT-Chol(+) suspensions were capable of forming stable complexes with genes (DNA) efficiently. The release of double-helical DNA from such CNT-Chol(+) complexes could be induced upon the addition of anionic micellar solution of SDS. Furthermore, the CNT-based DNA complexes containing cationic cholesterol aggregates showed higher stability in fetal bovine serum media at physiological conditions. Confocal studies confirm that CNT-Chol(+) formulations adhere to HeLa cell surfaces and get internalized more efficiently than the cationic cholesterol suspensions alone (devoid of any CNTs). These cationic cholesterol-CNT suspensions therefore appear to be a promising system for further use in biological applications.

Role of Specific Cations and Water Entropy on the Stability of Branched DNA Motif Structures

DNA three-way junctions (TWJs) are important intermediates in various cellular processes and are the simplest of a family of branched nucleic acids being considered as scaffolds for biomolecular nanotechnology. Branched nucleic acids are stabilized by divalent cations such as Mg2+, presumably due to condensation and neutralization of the negatively charged DNA backbone. However, electrostatic screening effects point to more complex solvation dynamics and a large role of interfacial waters in thermodynamic stability. Here, we report extensive computer simulations in explicit water and salt on a model TWJ and use free energy calculations to quantify the role of ionic character and strength on stability. We find that enthalpic stabilization of the first and second hydration shells by Mg2+ accounts for 1/3 and all of the free energy gain in 50% and pure MgCl2 solutions, respectively. The more distorted DNA molecule is actually destabilized in pure MgCl2 compared to pure NaCl. Notably, the first shell, interfacial waters have very low translational and rotational entropy (i.e., mobility) compared to the bulk, an entropic loss that is overcompensated by increased enthalpy from additional electrostatic interactions with Mg2+. In contrast, the second hydration shell has anomalously high entropy as it is trapped between an immobile and bulklike layer. The nonmonotonic entropic signature and long-range perturbations of the hydration shells to Mg2+ may have implications in the molecular recognition of these motifs. For example, we find that low salt stabilizes the parallel configuration of the three-way junction, whereas at normal salt we find antiparallel configurations deduced from the NMR. We use the 2PT analysis to follow the thermodynamics of this transition and find that the free energy barrier is dominated by entropic effects that result from the decreased surface area of the antiparallel form which has a smaller number of low entropy waters in the first monolayer.

Structure, stability and elasticity of DNA nanotubes

DNA nanotubes are tubular structures composed of DNA crossover molecules. We present a bottom up approach for the construction and characterization of these structures. Various possible topologies of nanotubes are constructed such as 6-helix, 8-helix and tri-tubes with different sequences and lengths. We have used fully atomistic molecular dynamics simulations to study the structure, stability and elasticity of these structures. Several nanosecond long MD simulations give the microscopic details about DNA nanotubes. Based on the structural analysis of simulation data, we show that 6-helix nanotubes are stable and maintain their tubular structure; while 8-helix nanotubes are flattened to stabilize themselves. We also comment on the sequence dependence and the effect of overhangs. These structures are approximately four times more rigid having a stretch modulus of similar to 4000 pN compared to the stretch modulus of 1000 pN of a DNA double helix molecule of the same length and sequence. The stretch moduli of these nanotubes are also three times larger than those of PX/JX crossover DNA molecules which have stretch moduli in the range of 1500-2000 pN. The calculated persistence length is in the range of a few microns which is close to the reported experimental results on certain classes of DNA nanotubes.

Use of S1 nuclease and formamide in combination for the reassociation studies on GC-rich DNA

The optimal parameters in the use of nuclease S1 in DNA reassociation kinetics in the presence of formamide have been determined. The conditions are especially suitable for the study of DNA rich in mole percent GC. A 10-fold dilution of the reassociation samples leading to a decrease in both NaCl and formamide concentrations, consequently resulting in a lowering of Tm by only 1.5°C, and the S1 digestion at temperatures identical to the reassociation assay in order to retain the stability of the duplex, are two important aspects of this system. Under these conditions, the kinetics of reassociation followed the theoretically predicted pattern, while the earlier reported methods have shown lower values.

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIalpha

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIalpha to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIalpha. The results indicate that topo IIalpha binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIalpha displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIalpha to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIalpha. These results implicate a dual role for topo IIalpha in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.

Interaction between the Z-type DNA duplex and 1,3-propanediamine:crystal structure of d(CACGTG)(2) at 1.2 angstrom resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 angstrom resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C3H10N2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 A resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

CA\TG Sequence at the 5'end of Oligo(A)-tracts Strongly Modulates DNA Curvature

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

CAITG sequence at the 5' end of Oligo(A)-tracts strongly modulates DNA curvature

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Structure and stability of human telomeric sequence

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)(n). We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T(2)AG(3))(4), d(G(3)T(2)A)(3)G(3), and d(G(3)T(2)AG(3)) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G(3)T(2)A)(3)G(3) and d(T(2)AG(3))(4) assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G(3)T(2)AG(3)) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide D(G(3)T(2)AG(3)) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T(2)AG(3))(4) suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.