Duplex DNA Molecules Research Articles

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5′-phosphoryl and 3′-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown.Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells.Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids.Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA.

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Natural Products as Anti-Cancerous Therapeutic Molecules Targeted towards Topoisomerases.

Topoisomerases are reported to resolve the topological problems of DNA during several cellular processes, such as DNA replication, transcription, recombination, and chromatin remodeling. Two types of topoisomerases (Topo I and II) accomplish their designated tasks by introducing single- or double-strand breaks within the duplex DNA molecules, and thus maintain the proper structural conditions of DNA to release the topological torsions, which is generated by unwinding of DNA to access coded information, in the course of replication, transcription, and other processes. Both the topoisomerases have been looked at as crucial targets against various types of cancers such as lung, melanoma, breast, and prostate cancers. Conceptually, targeting topoisomerases will disrupt both DNA replication and transcription, thereby leading to inhibition of cell division and consequently stopping the growth of actively dividing cancerous cells. Since the discovery of camptothecin (an alkaloid) as an inhibitor of Topo I in 1958, a number of derivatives of camptothecin were developed as potent inhibitors of Topo I. Two such derivatives of camptothecin, namely, topotecan and irinotecan, have been commonly used as US Food and Drug Administration (FDA) approved drugs against Topo I. Similarly, the first Topo II inhibitor, namely, etoposide, an analogue of podophyllotoxin, was developed in 1966 and got FDA approval as an anti-cancer drug in 1983. Subsequently, several other inhibitors of Topo II, such as doxorubicin, mitoxantrone, and teniposide, were developed. These drugs have been reported to cause accumulation of cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Thus, the present review describes the anticancer potential of plant-derived secondary metabolites belonging to alkaloids, flavonoids and terpenoids directed against topoisomerases. Furthermore, in view of the recent advances made in the field of computer-aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of these potent anti-cancerous therapeutic molecules.

Convergence in LINE-1 nucleotide variations can benefit redundantly forming triplexes with lncRNA in mammalian X-chromosome inactivation

BackgroundAssociations between X-inactive transcript (Xist)–long noncoding RNA (lncRNA) and chromatin are critical intermolecular interactions in the X-chromosome inactivation (XCI) process. Despite high-resolution analyses of the Xist RNA-binding sites, specific interaction sequences are yet to be identified. Based on elusive features of the association between Xist RNA and chromatin and the possible existence of multiple low-affinity binding sites in Xist RNA, we defined short motifs (≥5 nucleotides), termed as redundant UC/TC (r-UC/TC) or AG (r-AG) motifs, which may help in the mediation of triplex formation between the lncRNAs and duplex DNA.ResultsThe study showed that r-UC motifs are densely dispersed throughout mouse and human Xist/XIST RNAs, whereas r-AG motifs are even more densely dispersed along opossum RNA-on-the-silent X (Rsx) RNA, and also along both full-length and truncated long interspersed nuclear elements (LINE-1s, L1s) of the three species. Predicted secondary structures of the lncRNAs showed that the length range of these sequence motifs available for forming triplexes was even shorter, mainly 5- to 9-nucleotides long. Quartz crystal microbalance (QCM) measurements and Monte Carlo (MC) simulations indicated that minimum-length motifs can reinforce the binding state by increasing the copy number of the motifs in the same RNA or DNA molecule. Further, r-AG motifs in L1s had a similar length-distribution pattern, regardless of the similarities in the length or sequence of L1s across the three species; this also applies to high-frequency mutations in r-AG motifs, which suggests convergence in L1 sequence variations.ConclusionsMultiple short motifs in both RNA and duplex DNA molecules could be brought together to form triplexes with either Hoogsteen or reverse Hoogsteen hydrogen bonding, by which their associations are cooperatively enhanced. This novel triplex interaction could be involved in associations between lncRNA and chromatin in XCI, particularly at the sites of L1s. Potential binding of Xist/XIST/Rsx RNAs specifically at L1s is most likely preserved through the r-AG motifs conserved in mammalian L1s through convergence in L1 nucleotide variations and by maintaining a particular r-UC/r-AG motif ratio in each of these lncRNAs, irrespective of their poorly conserved sequences.

Abstract 422: Duplex sequencing for MRD detection in acute myeloid leukemia

Abstract The purpose of this study is to develop a universally applicable Duplex Sequencing (DS) assay for extremely sensitive detection of minimal residual disease (MRD) in acute myeloid leukemia (AML). The presence of MRD plays a critical role in AML recurrence and mortality, and is an important prognostic marker to guide clinical care and clinical trial risk stratification. Unfortunately, current MRD detection methods, including light microscopy, cytogenetics, qPCR, and flow cytometry have shortcomings in terms of sensitivity, general applicability, and/or inter-laboratory reproducibility. Duplex Sequencing eliminates technical artefacts observed with other sequencing technologies and delivers an unprecedented level of accuracy and sensitivity, on widely available NGS platforms. Here, we report validation of our DS MRD mutation detection assay, with a background mutation level more than 100-fold below single-stranded tag-based error correction (SSCS). Our complete DS MRD probe panel targets mutation hotspots or full coding sequences in 29 genes recurrently mutated in AML, representing mutations found in approximately 90% of patients. Supplementing with a targeted RNA-seq panel for recurrent gene fusions would approach 100%. To simulate low frequency mutations in MRD, we generated 2 DNA dilution series: either AML patient DNA or cell line DNA with known AML mutations that were serially diluted into normal human DNA. Replicate libraries were prepared with custom targeted probe panels and sequenced to over 50,000x or over 1,000,000x total Duplex depth (original Duplex DNA molecules sequenced per site), respectively. Expected mutations were detected down to MAFs of 1.9x10-5 and 6.0x10-6. Background mutation levels of 5.7x10-7 and 5.8x10-7, were respectively seen in each series, and likely reflect the true age-associated biological background, rather than technical noise. SSCS artefacts in these samples were approximately 100x higher than the frequency of DS mutations, and obscured expected mutations in the lower dilutions. We are further validating our method in both retrospective and prospective studies. In retrospective samples from a completed clinical trial, we are comparing the sensitivity and accuracy of DS vs. multicolor flow cytometry for MRD with known clinical outcomes. The relative prognostic value of DNA from blood vs. bone marrow will be quantified with DS as well. We will also perform DS on prospective samples to examine the prognostic value of specific genes and MAFs to eliminate uninformative variants, which will also allow accurate quantification of low level mutations in non-leukemic clones that accumulate with aging and can confound detection of MRD. Elimination of sequencing artefacts is critical for the application of NGS assays to MRD detection, especially when expected mutations are unknown, and DS is an important new tool in high sensitivity cancer diagnostics. Citation Format: Jacob E. Higgins, Lindsey N. Williams, Christopher S. Hourigan, Jerald P. Radich, Jesse J. Salk. Duplex sequencing for MRD detection in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 422.

Development of a graphene oxide-based assay for the sequence-specific detection of double-stranded DNA molecules.

Graphene oxide (GO) is a promising material for the development of cost-effective detection systems. In this work, we have devised a simple and rapid GO-based method for the sequence-specific identification of DNA molecules generated by PCR amplification. The csp genes of Escherichia coli, which share a high degree of sequence identity, were selected as paradigm DNA templates. All tested csp genes were amplified with unlabelled primers, which can be rapidly removed at the end of the PCR taking advantage of the preferential binding to GO of single-stranded versus duplex DNA molecules. The amplified DNAs (targets) were heat-denatured and hybridized to a fluorescently-labelled single strand oligonucleotide (probe), which recognizes a region of the target DNAs displaying sequence variability. This interaction is extremely specific, taking place with high efficiency only when target and probe show perfect or near perfect matching. Upon GO addition, the unbound fraction of the probe was captured and its fluorescence quenched by the GO’s molecular properties. On the other hand, the probe-target complexes remained in solution and emitted a fluorescent signal whose intensity was related to their degree of complementarity.

Abstract 2395: Direct assessment of sequence heterogeneity in human cancers by Duplex Sequencing

Abstract The extent of heterogeneity in DNA sequence within human cancers has been difficult to fully assess, due to the absence of methods with sufficient sensitivity to detect minority variants. For example, next-generation sequencing could in principle detect sub-clonal mutations at any genomic position; however due to amplification errors and inaccuracies in the sequencing platform itself, such approaches are limited to detection of mutations present in >1% of cells. We have overcome this limitation by developing an approach, Duplex Sequencing, which improves the accuracy of next-generation sequencing by >100,000 fold. Duplex Sequencing is based upon separately tagging and sequencing the two strands of single molecules of duplex DNA. True mutations are present at the same position in both DNA strands and are complementary, whereas artifacts arising from amplification or sequencing errors are seen in only one strand. The calculated error rate of our approach is less than one artifactual mutation per billion nucleotides sequenced (ref. 1). We have also developed an enrichment approach based on sequential rounds of hybridization to biotinylated probes which enables efficient sequencing of targeted regions of the genome (ref. 2). Duplex Sequencing thereby enables exceptionally sensitive detection of sequence heterogeneity within any set of genes. We have now applied Duplex Sequencing to study of sequence heterogeneity in both normal and malignant samples. In acute myeloid leukemia (AML), we find multiple sub-clonal mutations which fall below the detection limit of conventional approaches, some of which encode mutations that can drive chemotherapy resistance and cancer progression. Our results indicate that prior studies have under-estimated the burden of sub-clonal mutations in AML by more than 1,000-fold. We have additionally studied chronic myeloid leukemia (CML); treatment of CML with inhibitors directed against the Abl kinase represents the prototypical targeted cancer therapy. With Duplex Sequencing, we find that Abl mutations are extremely uncommon at the time of CML diagnosis. In contrast, refractory patients who fail therapy frequently possess multiple sub-clonal mutations within the Abl kinase, most of which are below the resolution of conventional approaches but are readily detected with our method. In ongoing work, we have found multiple sub-clonal mutations within human prostate cancer and colon cancer, suggestive of widespread intratumor heterogeneity that may limit the efficacy of single-agent therapy in these diseases.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature.

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the trans-cis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

The complete mitochondrial genome of the orb-weaving spider Neoscona theisi (Walckenaer) (Araneae: Araneidae)

The complete mitochondrial genome of an orb-weaving spider Neoscona theisi (Walckenaer) is determined in this article. It is a typical circular duplex DNA molecule with a length of 14,156 bp, which encodes the same 37 genes as all metazoan mitogenomes and an A + T-rich region (D-loop). The overall A + T content is 75.2% (A: 35.5%; T: 39.8%; G: 15.6%; C: 9.3%). All of the protein-coding genes were initiated by ATN, with the exceptions in three genes. COI has a TTA start codon, COII, COIII and ND6 use TTG as initiation codon. Three genes (ND3, COII and ND5) end with incomplete stop codons (T or TA), while all other genes terminated with canonical stop codons (TAA or TAG). Among 22 transfer RNAs genes, six of them (tRNAAla, tRNASer(AGN), tRNASer(UCN), tRNAGly, tRNAArg and tRNAHis) lack the potential to form the cloverleaf-shaped secondary structure. The A + T-rich region is 559 bp with an A + T content of 79.6%.

Synthesis and characterisation of nickel Schiff base complexes containing the meso-1,2-diphenylethylenediamine moiety: selective interactions with a tetramolecular DNA quadruplex.

As part of a program of preparing metal complexes which exhibit unique affinities towards different DNA structures, we have synthesised the novel Schiff base complex N,N'-bis-4-(hydroxysalicylidine)meso-diphenylethylenediaminenickel(ii) (), via the reaction of meso-1,2-diphenylethylenediamine and 2,4-dihydroxybenzaldehyde. This compound was subsequently reacted with 1-(2-chloroethyl)piperidine or 1-(2-chloropropyl)piperidine, to afford the alkylated complexes N,N'-bis-(4-((1-(2-ethyl)piperidine)oxy)salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) () and N,N'-bis-(4-((1-(3-propyl)piperidine)oxy)-salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) (), respectively. These complexes were characterised by microanalysis and X-ray crystallography in the solid state, and in solution by (1)H and (13)C NMR spectroscopy. Electrospray ionisation mass spectrometry (ESI-MS) was used to confirm the identity of () and (). The affinities of () and () towards a discrete 16 mer duplex DNA molecule, and examples of both tetramolecular and unimolecular DNA quadruplexes, was explored using a variety of techniques. In addition, the affinity of two other complexes () and (), towards the same DNA molecules was examined. Complexes () and () were prepared by methods analogous to those which afforded () and (), however 1,2-phenylenediamine was used instead of meso-1,2-diphenylethylenediamine in the initial step of the synthetic procedure. The results of ESI-MS and DNA melting temperature measurements suggest that () and () exhibit a lower affinity than () and () towards the 16 mer duplex DNA molecule, while circular dichroism (CD) spectroscopy suggested that none of the four complexes had a major effect on the conformation of the nucleic acid. In contrast, ESI-MS and CD spectroscopy suggested that both () and () show significant binding to a tetramolecular DNA quadruplex. The results of ESI-MS and Fluorescence Resonance Energy Transfer (FRET) assays indicated that () and () did not bind as tightly to a unimolecular DNA quadruplex, although both complexes had a major effect on the CD spectrum of the latter. These results highlight that the presence of the meso-1,2-diphenylethylenediamine moiety in metal complexes of this type may provide a general method for instilling selectivity for some DNA quadruplexes over dsDNA.

Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

This protocol uses quantitative polymerase chain reaction (qPCR) to measure the number of DNA molecules containing a specific contiguous sequence in a sample of interest (e.g., genomic DNA or cDNA generated by reverse transcription). The sample is subjected to fluorescence-based PCR amplification and, theoretically, during each cycle, two new duplex DNA molecules are produced for each duplex DNA molecule present in the sample. The progress of the reaction during PCR is evaluated by measuring the fluorescence of dsDNA-dye complexes in real time. In the early cycles, DNA duplication is not detected because inadequate amounts of DNA are made. At a certain threshold cycle, DNA-dye complexes double each cycle for 8-10 cycles, until the DNA concentration becomes so high and the primer concentration so low that the reassociation of the product strands blocks efficient synthesis of new DNA and the reaction plateaus. There are two types of measurements: (1) the relative change of the target sequence compared to a reference sequence and (2) the determination of molecule number in the starting sample. The first requires a reference sequence, and the second requires a sample of the target sequence with known numbers of the molecules of sequence to generate a standard curve. By identifying the threshold cycle at which a sample first begins to accumulate DNA-dye complexes exponentially, an estimation of the numbers of starting molecules in the sample can be extrapolated.

DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA.

The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.

Sorting Out the Structure of Single-Stranded DNA

The mechanical properties of single stranded nucleic acids play a vital role in such dynamic processes as DNA replication and RNA folding. While experiments have demonstrated that duplex DNA molecules behave as ideal worm-like chains with a persistence length that can be measured accurately, the unfolded single-stranded form of DNA (ssDNA) has been surprisingly recalcitrant: different biophysical techniques lead to different conclusions. Making matters worse, all-atom molecular dynamics simulations of ssDNA do not agree with experiment. This has motivated new experimental and theoretical work. In recent force spectroscopy and computational studies, McIntosh, Stevens and Saleh propose models where excluded volume influences long-ranged structure, while specific ion interactions induce wrinkles at short length scales (Single-stranded DNA is not a wormlike-chain, Biophys. J. 104, 28). However, direct structural evidence for these models is lacking. Thus, we perform detailed investigations of ssDNA homopolymers using x-ray scattering, single molecule FRET, and quantitative characterization of the ion atmosphere in mono- and di-valent salt. We develop a novel data-driven ensemble optimization technique that allows us to visualize the conformational adaptation of the DNA backbone to changes in the ion atmosphere, including the distinct effects of base stacking and ion valence. Correlation functions obtained from the structure ensembles, without reference to polymer or electrostatic theories, nonetheless show signatures of electrostatic excluded volume and strong ion effects at short distances. We discuss the implications for new polyelectrolyte theories of ssDNA and the biological role of electrostatics in these systems.

Elucidating Restriction Endonucleases Reaction Mechanisms via Dwell-Time Distribution Analysis

We have developed a Total Internal Reflection Fluorescence (TIRF) microscopy based assay that allows us to simultaneously measure the length of the catalytic cycle for hundreds of restriction endonuclease molecules in one experiment. We stably attach thousands of short duplex DNA molecules, each labeled with a single quantum dot semiconductor nanocrystal, to a passivated glass surface within a flow channel. The disappearance of a quantum dot indicates that its DNA tether has been cleaved. We introduce restriction endonuclease molecules into the channel in the absence of magnesium, which permits binding to, but not cleavage of the surface immobilized DNA substrate. When buffer containing magnesium is introduced into the flow channel, DNA cleavage by the pre-bound restriction endonuclease molecules is initiated. This synchronization allows us to measure the lag time between the introduction of magnesium and the completion of DNA cleavage for the entire population of enzymes. Analysis of the dwell-time distributions can provide insights into the DNA cleavage mechanism. Our observations suggest that EcoRV, a dimeric Type II restriction endonuclease that cleaves the palindromic sequence GAT↓ATC (where ↓ is the cut site), requires two kinetic steps to complete duplex cleavage after prebinding. However, dwell-time distributions suggest that BcnI, which is active as a monomer and cleaves the pseudopalindromic sequence 5'-CC↓SGG-5' (where S stands for C or G), requires more than four kinetic steps to complete duplex cleavage. Furthermore, experiments performed with strand-specific DNA substrates suggest that the number of steps indicated by the dwell-time distribution depends on which strand of the recognition site must be cleaved to result in quantum dot release. By designing additional substrates that mimic the various intermediate states, we plan to dissect the mechanism by which BcnI cleaves each strand of the intact restriction site.

N-Terminal Disordered Domain of Saccharomyces cerevisiae Hop1 Protein Is Dispensable for DNA Binding, Bridging, and Synapsis of Double-Stranded DNA Molecules But Is Necessary for Spore Formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein-protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Δhop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Structural Analysis of Activated SgrAI–DNA Oligomers Using Ion Mobility Mass Spectrometry

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.

Salt‐Dependence of Homology Searching Step by RecA Nucleoprotein Filaments

AbstractRecA recombinase binds onto single‐stranded DNA (ssDNA) and searches for its homologous sequence along double‐stranded DNA (dsDNA). This homologous recombination process is required for repairing double‐stranded DNA breaks. Here, we applied lateral magnetic tweezers to stretch duplex DNA molecules (Lambda DNA, 48,502 bp) in order to monitor the homology searching step by RecA nucleoprotein filaments at the single‐molecule level. From individual searching events, the diffusion coefficient of the RecA nucleoprotein filament is determined to be ∼ 0.5‐1 μm2/s. The nearly salt‐independence of the diffusion coefficients of RecA nucleoprotein filaments implies that our observed RecA searching events were mainly carried out by the one‐dimensional sliding mode.

An Exonuclease III Protection-Based Electrochemical Method for Estrogen Receptor Assay

Estrogen receptor (ER), expressed in approximately 80% of primary breast cancer cells, has proven to be a valuable predictive factor of the disease. Herein, by making use of the specific binding of ER to its DNA response elements, we propose an Exonuclease III (Exo III) protection-based electrochemical method for detecting ER proteins. In this assay, the presence of ER can protect the duplex DNA molecules immobilized on an electrode surface from Exo III-catalyzed digestion, resulting in an increased electrochemical signal. Experimental results have revealed that the proposed method can allow the quantification of ER in the range of 0.5 to 100 nM with a satisfactory detection limit of 0.38 nM. Furthermore, since this approach can also be employed to detect ER directly in nuclear extracts, it may be of great use in biomedical applications in the future.

Unzipping Kinetics of Duplex DNA Containing Oxidized Lesions in an α-Hemolysin Nanopore

The unzipping kinetics for lesion-containing DNA duplexes was studied in an α-hemolysin (α-HL) nanopore. The lesion of focus was the guanine two-electron oxidation product, 8-oxo-7,8-dihydroguanine (OG), and its further oxidation products, the hydantoins guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). The voltage-driven unzipping of individual duplex DNA molecules with symmetrical overhangs was carried out by pulling one strand of the duplex through the α-HL channel using an electrical field. Entry from the 3' or 5' end produced distinct current blockages, allowing directional effects on unzipping kinetics to be investigated. We find that the strand dissociation of complementary duplexes or duplexes containing the slightly destabilizing lesion OG follows a first-order kinetic model, while opening of duplexes that contain the highly destabilizing lesions Gh or Sp is described by two sequential first-order reactions, in which the intermediate state is proposed to correspond to the duplex unzipped to the lesion site within the channel. The rate constants for strand separation of the duplexes containing single lesions were obtained from kinetic model fits to histograms of unzipping duration. For all duplexes, the rate constants for strand separation displayed a significant dependence on the direction of entry into the nanopore. For duplexes containing Gh, truncated duplexes were used to assign the measured rate constants for the first and second unzipping steps of symmetrically designed duplexes.

KMnO4 Footprinting

The KMnO4 footprinting method offers a rapid and easy way to detect and localize single-stranded regions within a duplex DNA molecule, such as it occurs for instance within an actively transcribing RNA polymerase-DNA complex or during R-loop formation in DNA-RNA hybrid structures. The method is based on the selective oxidation of single-stranded thymines in DNA. The modified nucleotides react with strong bases by ring opening and subsequent phosphodiester cleavage. Because the modified nucleotides will not be recognized by DNA polymerase sites of modification can also be analyzed by primer extension with Klenow DNA polymerase, which stops elongation one residue before the modification. Hence, localization of the modified base positions can be performed on denaturing polyacrylamide gels either after piperidine catalyzed phosphodiester cleavage of 3'- or 5'-32P-end-labeled DNA or by primer extension with non-labeled DNA employing 32P-labeled oligonucleotide primers. Due to the fact that KMnO4 can penetrate through membranes the footprinting method can also be used for footprint analyses within living cells.