DNA Threading Research Articles

Kinetics of DNA Threading Intercalation by a Rigid Ruthenium Complex Dimer

Compounds that can intercalate DNA by threading between melted bases serve as a model of therapeutic drugs for malignant tumors. An essential property of effective antitumor activity requires a drug candidate to exhibit a slow dissociation rate from the intercalation state of DNA-drug complex. In particular, previous bulk experiments reported a very slow dissociation rate for the rigid Ruthenium complex dimer ΔΔ-P (Δ,Δ-[µ-(11,11'-bidppz)(phen)4Ru2]4+). We examined the kinetics of ΔΔ-P threading intercalation into an individual λ-DNA molecule held between a micropipette and an optical trap over a concentration range of 2-100 nM. DNA extensions due to threading intercalation are measured at several concentrations for constant stretching forces of 10-60 pN. As a result, fractional binding is determined as a function of force and concentration from the time-dependent approach to equilibrium extension, which yields equilibrium binding affinity. We also obtain the force and concentration-dependent on and off rates for the threading reaction. Furthermore, these measurements allow us to obtain the force-dependent and zero force binding affinity. By fitting these rates to the expected exponential dependence on force, we are able to extract the equilibrium change in DNA extension due to a single intercalation event as well as the distance to the transition state from the bound and unbound equilibrium states. These results show that the DNA length must increase significantly both for association and dissociation of the ligand, which explains the extremely slow binding kinetics.

DNA Capture and Translocation through Nanoscale Pores—a Fine Balance of Electrophoresis and Electroosmosis

DNA Capture and Translocation through Nanoscale Pores—a Fine Balance of Electrophoresis and Electroosmosis

Bridging Ligand Length Controls AT Selectivity and Enantioselectivity of Binuclear Ruthenium Threading Intercalators

The slow dissociation of DNA threading intercalators makes them interesting as model compounds in the search for new DNA targeting drugs, as there appears to be a correlation between slow dissociation and biological activity. Thus, it would be of great value to understand the mechanisms controlling threading intercalation, and for this purpose we have investigated how the length of the bridging ligand of binuclear ruthenium threading intercalators affects their DNA binding properties. We have synthesised a new binuclear ruthenium threading intercalator with slower dissociation kinetics from ct-DNA than has ever been observed for any ruthenium complex with any type of DNA, a property that we attribute to the increased distance between the ruthenium centres of the new complex. By comparison with previously studied ruthenium complexes, we further conclude that elongation of the bridging ligand reduces the sensitivity of the threading interaction to DNA flexibility, resulting in a decreased AT selectivity for the new complex. We also find that the length of the bridging ligand affects the enantioselectivity with increasing preference for the ΔΔ enantiomer as the bridging ligand becomes longer.

Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening

SummaryYeast RNA polymerase (Pol) II general factor TFIIE and the TFIIH subunit Ssl2/XPB function in transition of the preinitiation complex (PIC) to the open complex. We find that the three TFIIE winged helix (WH) domains form a heterodimer, with the Tfa1/TFIIEα WH binding the Pol II clamp and the Tfa2/TFIIEβ tandem WH domain encircling promoter DNA that becomes single stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. In contrast to previous proposals, comparison of the PIC and open complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double stranded DNA translocase, feeding 15 bp of double stranded DNA into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, will lead to DNA unwinding and the open state.

Loading mechanisms of ring helicases at replication origins

Threading of DNA through the central channel of a replicative ring helicase is known as helicase loading, and is a pivotal event during replication initiation at replication origins. Once loaded, the helicase recruits the primase through a direct protein–protein interaction to complete the initial ‘priming step’ of DNA replication. Subsequent assembly of the polymerases and processivity factors completes the structure of the replisome. Two replisomes are assembled, one on each strand, and move in opposite directions to replicate the parental DNA during the ‘elongation step’ of DNA replication. Replicative helicases are the motor engines of replisomes powered by the conversion of chemical energy to mechanical energy through ATP binding and hydrolysis. Bidirectional loading of two ring helicases at a replication origin is achieved by strictly regulated and intricately choreographed mechanisms, often through the action of replication initiation and helicase-loader proteins. Current structural and biochemical data reveal a wide range of different helicase-loading mechanisms. Here we review advances in this area and discuss their implications.

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.

Influence of Biological Media on the Structure and Behavior of Ferrocene-Containing Cationic Lipid/DNA Complexes Used for DNA Delivery

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li(2)SO(4)) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately -35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about -20 mV in OptiMEM and -10 mV in Li(2)SO(4)), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.

The SV40 Large T-Antigen Origin Binding Domain Directly Participates in DNA Unwinding

The origin binding domain (OBD) of SV40 large T-ag serves a critical role during initiation of DNA replication to position T-ag on the origin. After origin recognition, T-ag forms a double hexamer over the origin. Within each hexamer, the OBD adopts a lock washer structure where the origin recognizing A1 and B2 loops face toward the helicase domain and likely become unavailable for binding DNA. In this study, we investigated the role of the central channel of the OBD hexamer in DNA replication by analyzing the effects of mutations of residues lining the channel. All mutants showed binding defects with origin DNA and ssDNA especially at low protein concentrations, but only half were defective at supporting DNA replication in vitro. All mutants were normal in unwinding linear origin DNA fragments. However, replication defective mutants failed to unwind a small origin containing circular DNA whereas replication competent mutants did so normally. The presence of RPA and/or pol/prim restored circular DNA unwinding activity of compromised mutants probably by interacting with the separated DNA strands on the T-ag surface. We interpret these results to indicate a role for the OBD central channel in binding and threading ssDNA during unwinding of circular SV40 DNA. Mixing experiments suggested that only one monomer in an OBD hexamer was necessary for DNA unwinding. We present a model of DNA threading through the T-ag complex illustrating how single-stranded DNA could pass close to a trough generated by key residues in one monomer of the OBD hexamer.

Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division

Sister chromatid cohesion is mediated by DNA catenation and proteinaceous cohesin complexes. The recent visualization of PICH (Plk1-interacting checkpoint helicase)-coated DNA threads in anaphase cells raises new questions as to the role of DNA catenation and its regulation in time and space. In the present study we show that persistent DNA catenation induced by inhibition of Topoisomerase-IIalpha can contribute to sister chromatid cohesion in the absence of cohesin complexes and that resolution of catenation is essential for abscission. Furthermore, we use an in vitro chromatid separation assay to investigate the temporal and functional relationship between cohesin removal and Topoisomerase-IIalpha-mediated decatenation. Our data suggest that centromere decatenation can occur only after separase activation and cohesin removal, providing a plausible explanation for the persistence of centromere threads after anaphase onset.

Nanopores as Biosensors: DNA Sequencing and Chiral Discrimination

Biosensors are stochastic sensors inspired by biology. They are potentially important for many applications in bionanotechnology, from DNA sequencing to single-molecule detection and even chiral discrimination. While the macromolecular properties of the individual components i.e. sensor and analyte are well-characterised, the intricacies of their interaction are less well understood. For full exploitation of biomolecules as stochastic sensors, detailed knowledge of their interactions with other biological and chemical species is desirable. Thus, we have performed a series of molecular dynamics simulations of the bacterial toxin, alpha hemolysin (aHL) and derivative model pores to address issues such as the mechanism of DNA transport through the pore, and the molecular basis of chiral discrimination when the protein is fitted with a molecular adapter (in this case the cyclic molecule, beta cyclodextrin (bCD)).We study the orientational discrimination of the DNA molecule by restraining the DNA at one end, inside the protein barrel, and applying an electric field. Simulations of the wildtype protein and mutants give good agreement with published experimental data and allow us to explore the molecular basis of discrimination.Our simulations of a model pore (the aHL barrel with only selected sidechains included), allow us to probe the mechanism of DNA threading into the pore once it has already entered the vestibule of the protein. Our results indicate that only key sidechains are required for the interaction with the DNA molecule, and thus have important implications for the future design of engineered protein pores.Our third set of simulations explores the ability of pores fitted with bCD to discriminate between the enantiomers of ibuprofen. We have used simplified models of pores with full atomistic representation of the bCD and ibuprofen molecules to capture the subtleties of their interaction under an applied external field.

Analysis of Short Tandem Repeats by Parallel DNA Threading

The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs.

Synthesis and DNA threading properties of quaternary ammonium [Ru(phen)2(dppz)]2+ derivatives

Ruthenium complexes with one dipyrido[3,2-a:2'-3'-c]phenazine (dppz) ligand, e.g. [Ru(phen)(2)(dppz)](2+) (phen=phenanthroline), shows strong binding to double helical DNA and are well-known DNA "light-switch" molecules. We have here investigated four new [Ru(phen)(2)(dppz)](2+) derivatives with different bulky quaternary ammonium substituents on the dppz ligand to find relationships between molecular structure and intercalation kinetics, which is considered to be of importance for antitumor applicability. Linear dichroism spectroscopy shows that the enantiomers of the new complexes exhibit very similar binding geometries (intercalation of dppz moiety between adjacent DNA base pairs) as the enantiomers of the parent [Ru(phen)(2)(dppz)](2+) complex. Absorption spectra and luminescence properties provide further evidence for a final intercalative binding mode which has to be reached by threading of a bulky moiety between the strands of the DNA. Delta-enantiomers of all the new complexes show much slower association and dissociation kinetics than that of a reference complex without a cationic substituent. Kinetics were not very different whether the bulky quaternary group was derived from hexamethylene tetramine or 1,4-diazabicyclo-(2,2,2)octane (DABCO) or whether it had one or two positive charges. However, a complex in which the hexamethylene tetramine substituent is attached via a phenyl group showed a lowered association rate, in addition to an improved quantum yield of luminescence. A second positive charge on the DABCO substituent resulted in a much slower dissociation rate, suggesting that the distance from the Ru-centre and the amount of charge are both important for threading intercalation kinetics.

When Segregation Hangs by a Thread

When Segregation Hangs by a Thread

Supercoil-Accelerated DNA Threading Intercalation

The effect of DNA supercoiling on a sterically very demanding threading intercalation process is investigated here. We find that the threading rate of a dimeric ruthenium complex into a negatively supercoiled plasmid at low binding density is 2 orders of magnitude higher than into the cleaved linear form. Further saturation is on the other hand kinetically hampered in comparison to the relaxed DNA. We also observe that threading kinetics correlates with the inhibition of luciferase expression from the plasmid construct. The results show how the target torsional strain can function as a control of DNA threading kinetics and gene expression efficiency.

Finding at-DNA - Kinetic Recognition of Long Adenine-Thymine Stretches by Metal-Ligand Complexes

High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.

Length of the hypermutation motif DGYW/WRCH in the focus of statistical limits. Implications for a double-motif or extended motif recognition models

The motif DGYW/WRCH (Mh) and its frequently discussed simplified derivative GYW/WRC (Mhs) are involved in immunoglobulin (Ig) hypermutation. Both these motifs appear to be markedly shorter than the corresponding conventionally predicted minima of valid sequence lengths (MVSL). The same conclusion concerning both Mh and Mhs can also be obtained in the combined case including a less strict semi-empirically defined w-value and one nucleotide length tolerance related to MVSL. Such disagreement indicates considerably low information content in Mh and Mhs when evaluating these motifs as alphabetical structures (words). This fact raises a question of actually recognized structures (presumably longer than Mh and Mhs). Interestingly, both Mh and Mhs dimers or pairs of closely located Mh or Mhs achieve confirmation of length validity in the case of w=0.05, suggesting thus double-motif recognition as one of statistically consistent explanations. This possibility is also in agreement with the results of our model sequence study of mRNA derived from variable Ig gene sequences (rIgV) with respect to the most frequently occurring structures formed by motif overlaps in all model sequence sets. On the other hand, additional superior occurrence of motif pairs at a structurally important distance of a single DNA thread was found in the conserved domain (cd00099) related sequences of Elasmobranchii origin and less markedly in the corresponding human rIgV, but not in a randomly selected human subset of rIgV. The data are discussed with respect to statistical evaluation and structural properties of hypermutation motifs or the competent enzyme, i.e. activation-induced cytidine deaminase.

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AUSTRALIA — Malaria Breakthrough in Australia. AUSTRALIA — Advances in Early Alzheimer's Diagnosis. AUSTRALIA — Genetic Test for Epilepsy Sufferers Developed By Victoria, Australia Scientists. AUSTRALIA — New R & D Facility Leads Way for Australian Biotech Sector. AUSTRALIA — Australia Launches New Center to Focus on HIV Prevention, Treatment Research. CHINA — Genomics Center Researchers Identify Key Mechanism in DNA Damage Response. CHINA — Hemophiliacs in Guangdong Suffer From Lack of Treatment. CHINA — Microbix to Build Flu Vaccine Plant in China. CHINA — Applied Biosystems, 454 Life Sciences and Illumina Join 1000 Genomes Project. INDIA — Indian Scientists Develop GM Mice Through Transgenic Sperm. JAPAN — Experts Identify Genes for Bird Flu Replication. JAPAN — Boosting Survival of Insulin-cell Transplants For Type 1 Diabetes. JAPAN — Medtronic Launches First Insertable Cardiac Monitor in Japan. JAPAN — Sewing DNA Thread with Lasers, Hooks and Microbobbins. KOREA — CSD Korea launches its Patient Longitudinal Database. NEW ZEALAND — NZ Signs Science Agreement with EU. SINGAPORE — Singapore Researchers Identify Viral Factor That Induces Deadly Fungal Infection. SINGAPORE — Singapore, Indonesia Enhance Collaboration to Fight Bird Flu. SINGAPORE — Singapore Scientists Beat More Than 140 Teams to Claim Top Award at 46th American Society of Neuroradiology 2008 Meeting. TAIWAN — Government Plans Development of Five Bio-Agricultural Parks. TAIWAN — Local Researchers Cast New Light on Genetic Mutations. TAIWAN — Enterovirus Cases Hit Record High. VIETNAM — New Cholera Outbreaks in Vietnam. VIETNAM — Vietnam, WB Join Hands to Improve Health Services in Remote Areas.

Tuning the DNA Conformational Perturbations Induced by Cytotoxic Platinum−Acridine Bisintercalators: Effect of Metal Cis/Trans Isomerism and DNA Threading Groups

Four highly charged, water soluble platinum-acridine bisintercalating agents have been synthesized. Depending on the cis/trans isomerism of the metal and the nature of the acridine side chains, bisintercalation induces/stabilizes the classical Watson-Crick B-form or a non-B-form. Circular dichroism spectra and chemical footprinting experiments suggest that 4, the most active derivative in HL-60 cells, produces a structurally severely perturbed DNA with features of a Hoogsteen base-paired biopolymer.

Mechanically Manipulating the DNA Threading Intercalation Rate

The dumbbell shaped binuclear ruthenium complex DeltaDelta-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur.

DNA threading bis(9-aminoacridine-4-carboxamides): Effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC 50 values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.