Characteristics of Plasmonic Filters with a Notch Located Along Rectangular Resonators

We propose a plasmonic filter with a notch located along a rectangular resonator. The finite difference time domain method is utilized to investigate and analyze the transmission characteristics of the filter. Results reveal that the introduction of the notch affects the first and second resonant modes of the resonator in different manners due to different magnetic field distributions inside the resonator. The evolution of the transmission-peak wavelengths as a function of the notch position with the same total resonator length is given. Effects of geometrical parameters of the notch on peak wavelengths are also studied. The corresponding theoretical model of our proposal is discussed, which agrees well with simulation results.     

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

HMG-D complexed to a bulge DNA: an NMR model

An NMR model is presented for the structure of HMG-D, one of the DROSOPHILA: counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.     

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.     

Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor

Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic samples with a wide range of possible sensor designs such as interferometers and resonators ^1,2. Most of the existing RI sensing applications focus on biological materials in aqueous solutions in visible and IR frequencies, such as DNA hybridization and genome sequencing. At terahertz frequencies, applications include quality control, monitoring of industrial processes and sensing and detection applications involving nonpolar materials.Several potential designs for refractive index sensors in the terahertz regime exist, including photonic crystal waveguides ³, asymmetric split-ring resonators ⁴, and photonic band gap structures integrated into parallel-plate waveguides ⁵. Many of these designs are based on optical resonators such as rings or cavities. The resonant frequencies of these structures are dependent on the refractive index of the material in or around the resonator. By monitoring the shifts in resonant frequency the refractive index of a sample can be accurately measured and this in turn can be used to identify a material, monitor contamination or dilution, etc.The sensor design we use here is based on a simple parallel-plate waveguide ^6,7. A rectangular groove machined into one face acts as a resonant cavity (Figures 1 and 2). When terahertz radiation is coupled into the waveguide and propagates in the lowest-order transverse-electric (TE₁) mode, the result is a single strong resonant feature with a tunable resonant frequency that is dependent on the geometry of the groove ^6,8. This groove can be filled with nonpolar liquid microfluidic samples which cause a shift in the observed resonant frequency that depends on the amount of liquid in the groove and its refractive index ⁹.Our technique has an advantage over other terahertz techniques in its simplicity, both in fabrication and implementation, since the procedure can be accomplished with standard laboratory equipment without the need for a clean room or any special fabrication or experimental techniques. It can also be easily expanded to multichannel operation by the incorporation of multiple grooves ¹⁰. In this video we will describe our complete experimental procedure, from the design of the sensor to the data analysis and determination of the sample refractive index.     

Structural Basis for Promutagenicity of 8-Halogenated Guanine

8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.     

Structural insight into dynamic bypass of the major cisplatin‐DNA adduct by Y‐family polymerase Dpo4

Y‐family DNA polymerases bypass Pt‐GG, the cisplatin‐DNA double‐base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y‐family DNA polymerase, Dpo4, in complex with Pt‐GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3′G (first insertion) and 5′G (second insertion) of Pt‐GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation‐coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt‐GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4‐mediated Pt‐GG bypass was addressed by a dpo‐4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.     

Interaction between DNA polymerase λ and RPA during translesion synthesis

Replication of damaged DNA (translesion synthesis, TLS) is realized by specialized DNA polymerases. Additional protein factors such as replication protein A (RPA) play important roles in this process. However, details of the interaction are unknown. Here we analyzed the influence of the hRPA and its mutant hABCD lacking domains responsible for protein-protein interactions on ability of DNA polymerase lambda to catalyze TLS. The primer-template structures containing varying parts of extended strand (16 and 37 nt) were used as model systems imitating DNA intermediate of first stage of TLS. The 8-oxoguanine disposed in +1 position of the template strand in relation to 3 -end of primer was exploited as damage. It was shown that RPA stimulated TLS DNA synthesis catalyzed by DNA polymerase lambda in its globular but not in extended conformation. Moreover, this effect is dependent on the presence of p70N and p32C domains in RPA molecule.     

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ^−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.     

Response Line-Shapes in Compact Coupled Plasmonic Resonator Systems

A compact plasmonic coupled-resonator system, consisting of a stub resonator and baffles in the metal–insulator–metal waveguide, is numerically investigated with the finite element method. Simulations show that sharp and asymmetric response line-shapes can occur in the system. The asymmetric line-shapes in the transmission spectra depend on the relative positions of the resonant wavelengths between the single-stub resonator and the inner resonator constructed by the baffle and the stub resonator, while the other part of the transmission spectra (except the asymmetric part) maintains the spectral features of the structure constructed by the baffles. An analytic model and a relative phase analysis based on the scattering matrix theory are used to describe and explain this phenomenon. These sharp and asymmetric response line-shapes are important for improving the nano-plasmonic devices’ performances.     

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5′-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis.     

Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay

In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.     

DNA synthesis and endomitoses in the giant nuclei of the silkgland of Bombyx mori.

At the first symptoms of organisation of the silkgland in the embryo, mitoses stop and nuclei start to grow. Through autoradiographic studies, performed after sequenced labeling with [3H] and [14C] thymidine, the durations of the different phases of DNA synthesis cycles (T = 42 to 48 h, S = 22 to 25 h, G = 20 to 23 h) are established. These durations are in fact identical during the second and the third instar, and the same, whatever region is concerned : posterior, middle or anterior parts. A model has been established to account for the distribution of the S phases during the second and third instars. The DNA synthesis in all nuclei of a given region has been followed during the first four instars by labelling with [3H]thymidine. The activity goes through maxima and minima, depending on the percent of nuclei synthesizing DNA and their synchronism, both being characteristic of each region; long resting periods are observed during the molting stages of the first three instars in the middle and the anterior parts. The coincidence is obvious between the maxima and minima and respectively the S and G phases of the model. DNA assays agree with the distribution of cycles established by autoradiography if one admits that each cycle does lead to a doubling of the amount of DNA. The overall DNA synthesis from the diploid value is estimated to correspond to 18-19 endomitoic cycles in the nucleic of the posterior part, 19-20 cycles of those of the middle part and 13 in those of the anterior part. The analysis of chromosome structures, by squashing the nuclear content, shows that existence of a complete endomitotic cycle: the doubling of chromosomes is associated with condensed structures, alternating with a decondensed state of chromatin, responsible for the DNA synthesis. The female heterochromatin undergoes a restricted morphological cycle delayed with respect to bulk chromatin. It is characterized by a late DNA duplication and by non dispersed daughter chromosomes. Some of these aspects are, to a lesser extent, reproduced in groups of autosomic chromosomes.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

GOLD DNA-CONJUGATES: ION SPECIFIC SELF-ASSEMBLY OF GOLD NANOPARTICLES VIA THE DG-QUARTET

The Oxytricha telomere DNA hairpin 5′-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dG-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K⁺ is less efficient while Li⁺ and Cs⁺ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

Plasmonic Waveguide Filters Based on Tunneling and Cavity Effects

This work presents a bandstop plasmonic filter that comprises a metal–insulator–metal (MIM) waveguide and a few pairs of strip cavities that are embedded in the metal. The strip cavity acts as both a near-field antenna and an MIM resonator. The central frequency and the bandwidth of the forbidden band are inversely related to the cavity length and the cavity-to-waveguide distance, respectively. These results correlate with the predictions of the ring resonator model but only under the resonant condition that double the effective length of cavity is an integer multiple of the guiding wavelength in the cavity.     

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase λ (Pol λ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol λ. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol λ efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol λ could modulate the cytotoxic effect of AraC.     

A MIM Filter Based on a Side-Coupled Crossbeam Square-Ring Resonator

In this paper, a surface plasmon polarition filter based on a side-coupled crossbeam square-ring resonator is presented and the transmission characteristics of the filter are analyzed by using the finite difference time domain method. The simulation results indicate that the proposed resonator supports multiple resonant modes, and these resonant modes can be adjusted all together by varying the length and refractive index of the outer square ring or partially adjusted by changing the width and refractive index of the crossbeam. By adding two coupled waveguides to the structure, we further demonstrate that a multiple wavelength download filter can be achieved via different coupled waveguides. The proposed structure has potential applications in plasmonic integrated circuits.     

The Werner syndrome exonuclease facilitates DNA degradation and high fidelity DNA polymerization by human DNA polymerase δ

DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3' → 5' DNA exonuclease activities. Pol δ exonuclease removes 3'-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3'-terminal mismatches to enable primer extension by Pol δ. Consistent with our in vitro observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity in vivo. Cells expressing limiting amounts (～10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.