DNA Binding Events Research Articles

In VitroDiagnostic Device with a Bio-Coupled Gate Field Effect Transistor

In this study, we report a simple and rapid biosensing method for the analysis andin vitromonitoring of biological processes, including DNAbinding events, antigen-antibody interactions, and cellular functions, using a semiconductor device. Most biological phenomena involve cell-cell communication processes that are mediated by the transport of sodium or potassium ions and other charged biomolecules, such as DNA, across ion channels in the cell membrane. Therefore, our approach focused on the direct detection of changes in ion concentrations by utilizing a semiconductor-based biosensor device. Our results demonstrated that our semiconductor-based biosensor platform achieves label-free and noninvasive biosensing that is suitable forin vitrodiagnosis.

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. Cancer Res; 77(18); 4934-46. ©2017 AACR.

Extensive and functional overlap of the STAT6 and RXR cistromes in the active enhancer repertoire of human CD14+ monocyte derived differentiating macrophages.

Macrophages are able to differentiate into classically polarized (M1) or alternatively polarized (M2) states upon encountering pro-inflammatory cytokines such as interferon (IFN) γ or anti-inflammatory cytokines such as interleukin (IL) -4/IL-13, respectively. Moreover, macrophages are known to regulate lipid metabolism via multiple members of the nuclear hormone receptor family, including the retinoid X receptors (RXR). It has been also documented that cytokines are able to modulate macrophage responses to lipid signals but the nature of these interactions and the underlying mechanisms of these processes especially at the level of the chromatinized genome are not well understood. Previous work from our laboratory suggested that STAT6 is a facilitator of nuclear receptor mediated transcriptional activity acting at the genome level. This prompted us to investigate genome-wide DNA binding events and the development of cistromes in human CD14+ monocyte-derived macrophages upon exposure to IL-4. We determined the impact of IL-4 on the PU.1, RXR and STAT6 cistromes within the active enhancer regions marked by H3K27-acetylation using chromatin immunoprecipitation followed by deep sequencing and integrated bioinformatics analyses. We found that about 2/3rd of the IL-4 induced STAT6 peaks co-localized with RXR peaks. These STAT6/RXR co-peaks differed at least in part from the non-overlapping RXR peaks regarding the most enriched de novo transcription factor binding motifs. Interestingly, RXR-binding was not regulated at the STAT6/RXR co-bound enhancers following IL-4 stimulation, but differential enhancer interactions were observed between the IL-4/STAT6 and RXR signaling pathways acting in a gene selective manner. Our results suggest that there is a novel, so far uncharacterized cistromic crosstalk between RXR and STAT6 that is likely to contribute to the formation of the active enhancer repertoire, transcriptome and differential signal-specific gene regulation of polarized macrophages.

UbaLAI is a monomeric Type IIE restriction enzyme.

Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5′-CC/WGG-3′ (where W = A/T, and ‘/’ marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5′-CCWGG-3′ sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5′-CCWGG-3′ sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.

Abstract 481: GRK5 as a Novel DNA-Binding Protein in Cardiomyocyte Hypertrophic Stress

Introduction: The pathogenesis and progression of heart failure (HF) encompasses aberrations in gene regulation, leading to ventricular remodeling and maladaptive cardiac hypertrophy. A common maladaptation in HF is an increase in the expression level of the G protein-coupled receptor kinase, GRK5. Recently, non-canonical roles for GRK5 have been uncovered that are crucial for the development of maladaptive hypertrophy and these are due to nuclear translocation and accumulation of GRK5. We have shown that GRK5 is a histone deacetylase kinase promoting de-repression of hypertrophic gene transcription through MEF2 and can also facilitate transcriptional activity of NFAT through DNA-binding. The latter is consistent with studies revealing that GRK5 contains a DNA-binding domain. Hypothesis: We hypothesize that the DNA-dependent roles of GRK5 are a mechanism by which GRK5 contributes to gene reprogramming in myocardial hypertrophic stress. Methods: To further study this, neonatal rat ventricular cardiomyocytes overexpressing GRK5 were subjected to α-adrenergic stress with 50μM phenylephrine (PE) or vehicle for 30 minutes to induce nuclear accumulation of GRK5. Chromatin was isolated and GRK5 immunoprecipitation (ChIP) employed to pull-down DNA associated with GRK5 (n=3 PE, n=4 vehicle). Next generation sequencing of this DNA provides an unbiased view of GRK5 DNA binding. Results: The GRK5 ChIP-Seq revealed that GRK5 translocation upon hypertrophic stress leads to multiple promoter, exon, and enhancer DNA-binding events. Gene ontology revealed enrichment of pathways known to be involved in hypertrophy, including the NFAT and Ras pathways. Promoters for genes relevant to cardiac hypertrophy, such as bone natriuretic peptide and early growth response 1 were also found to be enriched by GRK5 immunoprecipitation with PE treatment. In conclusion, we show that during hypertrophic stress, GRK5 mobilizes to the nucleus of myocytes and localizes around diverse genes involved in cardiomyocyte hypertrophy. Further studies in vivo will reveal whether GRK5 has a causal DNA-dependent role in gene reprogramming in HF.

Toward Direct Observation of the DNA Binding Dynamics of Monomeric Type IIP Restriction Endonucleases

Restriction endonucleases (REases) are enzymes that cleave duplex DNA at a recognition site specified by a particular nucleotide sequence. These enzymes are found in a wide range of bacterial species, and serve as a defense against infection by phage. Type IIP REases are typically antiparallel homodimers that recognize a palindromic DNA sequence and cleave within or close to the recognition site. However, some Type IIP REases cleave pseudopalindromic sequences, and some of these may be active as monomers. It is hypothesized that these monomeric REases sequentially cleave the two complementary strands of duplex DNA during a single DNA binding event. Since the two strands of DNA are antiparallel, after cleaving one strand, a monomeric REase would be required to rotate about the axis perpendicular to the DNA to be properly oriented to cleave the second strand. This type of reorientation is known a “flipping”. Although only a small number of proteins have been observed reorienting in this way, protein flipping may play an important role in binding to a specific DNA sequence. Direct observation of flipping during DNA cleavage will greatly improve our understanding of how monomeric REases mediate double strand breaks. We are currently using total internal reflection fluorescence (TIRF) microscopy to observe REase-mediated cleavage of quantum dot-labeled DNA at the single-molecule level. In our highly multiplexed assay, the disappearance of a quantum dot indicates cleavage of the DNA that tethers it to a functionalized glass coverslip. We plan to utilize single-molecule Forster resonance energy transfer (smFRET) to trace individual monomeric REases in real time through the entire process of DNA cleavage from substrate binding to product release. Our observations should provide significant insight into the mechanism by which these enzymes cleave duplex DNA.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s⁻¹). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (&gt;1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. <b>SIGNIFICANCE STATEMENT</b> The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.

Fabrication of a Horizontal and a Vertical Large Surface Area Nanogap Electrochemical Sensor.

Nanogap sensors have a wide range of applications as they can provide accurate direct detection of biomolecules through impedimetric or amperometric signals. Signal response from nanogap sensors is dependent on both the electrode spacing and surface area. However, creating large surface area nanogap sensors presents several challenges during fabrication. We show two different approaches to achieve both horizontal and vertical coplanar nanogap geometries. In the first method we use electron-beam lithography (EBL) to pattern an 11 mm long serpentine nanogap (215 nm) between two electrodes. For the second method we use inductively-coupled plasma (ICP) reactive ion etching (RIE) to create a channel in a silicon substrate, optically pattern a buried 1.0 mm × 1.5 mm electrode before anodically bonding a second identical electrode, patterned on glass, directly above. The devices have a wide range of applicability in different sensing techniques with the large area nanogaps presenting advantages over other devices of the same family. As a case study we explore the detection of peptide nucleic acid (PNA)−DNA binding events using dielectric spectroscopy with the horizontal coplanar device.

Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability.

The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species.

SsDNA-Functionalized Nanoceria: A Redox-Active Aptaswitch for Biomolecular Recognition.

Quantification of biomolecular binding events is a critical step for the development of biorecognition assays for diagnostics and therapeutic applications. This paper reports the design of redox-active switches based on aptamer conjugated nanoceria for detection and quantification of biomolecular recognition. It is shown that the conformational transition state of the aptamer on nanoceria, combined with the redox properties of these particles can be used to create surface based structure switchable aptasensing platforms. Changes in the redox properties at the nanoceria surface upon binding of the ssDNA and its target analyte enables rapid and highly sensitive measurement of biomolecular interactions. This concept is demonstrated as a general applicable method to the colorimetric detection of DNA binding events. An example of a nanoceria aptaswitch for the colorimetric sensing of Ochratoxin A (OTA) and applicability to other targets is provided. The system can sensitively and selectivity detect as low as 0.15 × 10(-9) m OTA. This novel assay is simple in design and does not involve oligonucleotide labeling or elaborate nanoparticle modification steps. The proposed mechanism discovered here opens up a new way of designing optical sensing methods based on aptamer recognition. This approach can be broadly applicable to many bimolecular recognition processes and related applications.

Abstract A43: Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX)

Abstract Introduction: Progesterone (P4) and progesterone receptors (PR) play elusive roles in breast cancer. Synthetic progestins in hormone replacement therapy increase breast tumorigenesis, yet these same progestins at high doses can be an effective therapy for some advanced breast tumors. The literature supports that progestins can either stimulate or inhibit breast cancer cell growth, and an increasing body of evidence supports P4 promotes expansion of breast cancer stem cells. Interplay between PR and estrogen receptors (ER) is emerging as important in dictating growth and response to treatment. There is historical precedence for PR interference of ER dependent gene transcription; recent reports identify that PR modulate ER DNA binding events. Given the importance of PR as a biomarker and potential therapeutic target, and the uncertainty surrounding its context-dependent actions, we aimed resolve this using breast cancer PDX models. The purpose of this study was to evaluate the effects of natural and synthetic progestins on growth and estrogen-dependent gene expression in luminal breast tumors, and to define contexts that dictate their divergent effects. Experimental procedures: Six luminal breast cancer PDX developed at UCD-AMC were used for these studies. Most profiled as Luminal B subtype. Tumors generally fell into two types: a pure luminal phenotype and a mixed luminal/basal phenotype based on cytokeratin expression. Tumors varied in expression of ER, PR, and androgen receptors (AR) from low (&amp;lt;10% positive cells), moderate (30-50%), to high (&amp;gt;90%). Tumors were grown in vivo under conditions of continuous placebo, 17β-estradiol (estrogen), estrogen plus progesterone (P4), or estrogen plus the synthetic progestin medroxyprogesterone acetate (MPA) for several months and growth parameters monitored. Receptor content was measure by immunohistochemistry and western blots. Gene expression profiles of select tumors were determined using Affymetrix® GeneChip human gene microarrays and/or RNAseq and results analyzed using Partek Genomics Suite™ software, and Metacore™ pathway analysis. Summary: In two pure luminal PDX with high receptor levels (ER, PR, AR) both P4 and MPA significantly inhibited estrogen-dependent growth (growth-inhibited, GI). Two PDX (one pure luminal, one mixed) with moderate ER and low PR and AR showed no significant effect of P4 or MPA on estrogen-dependent growth (growth neutral, GN). One mixed luminal/basal PDX with low ER and PR and moderate AR showed no effect with P4, whereas MPA simulated tumor growth above that with estrogen alone (growth stimulated, GS). Study of one PDX is ongoing. In the GI tumors both P4 and MPA reversed expression of ~30% of estrogen regulated genes. In particular these tumors had high estrogen-dependent expression of the Notch pathway, which was blocked by both P4 and MPA. Progestins did not alter estrogen-dependent genes in the GN or GS tumors, which did not include the Notch pathway. MPA stimulated a unique set of genes in the GS tumor, and was able to mobilize AR to the nucleus whereas P4 did not. Conclusion: Progestins have mixed effects on breast cancer PDX tumor growth. Tumors that have high ER and PR and are highly estrogen-dependent are more likely to exhibit strong growth inhibition by progestins (both P4 and MPA). Tumors with mixed luminal/basal phenotype that have less ER and PR and are moderately estrogen-dependent are less likely to be progestin-inhibited. In tumors with low ER and PR but high AR content MPA can act as an AR agonist to stimulate growth. Tumor-specific downstream estrogen-dependent pathways such as Notch may indicate whether progestins are therapeutic. Additional tumors are needed to confirm these results with the long term goal of developing a molecular assay to define those tumors that may benefit from progestin treatment. Citation Format: Jessica Finlay-Schultz, Heather M. Brechbuhl, Rachel B. Rosen, Austin E. Gillen, Britta M. Jacobsen, Peter Kabos, Carol A. Sartorius. Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A43.

Synthesis of 19F nucleic acid–polymer conjugates as real-time MRI probes of biorecognition

The efficacy of novel 19F nucleic acid–polymer conjugates as sensitive and selective in vitro reporters of DNA binding events is demonstrated through a number of rapid-acquisition MR sequences.

Construction of Plasmonic Core-Satellite Nanostructures on Substrates Based on DNA-Directed Self-Assembly as a Sensitive and Reproducible Biosensor.

We report the successful construction of plasmonic core-satellite nanostructured assemblies on two-dimensional substrates, based on a strategy of combining DNA-functionalized plasmonic nanoparticles (NPs) with the specific recognition ability toward target to enable satellite NPs to self-assemble around the core immobilized on substrates. A strongly coupled plasmonic resonance band was observed because of the close proximity between core and satellite NPs, which presented significant red-shift and enhanced extinction with respect to the local surface plasmon resonance (LSPR) band of individual core NPs on the substrate. The functionality of this core-satellite nanostructured assembly as a biosensor was further explored, and the changes in extinction intensity and the peak shift of the plasmonic coupling resonance band arising from the probe-target DNA binding event all proved to be useful criteria for target DNA detection. Moreover, high selectivity down to single-base mismatched DNA was achieved using this strongly coupled plasmonic core-satellite nanostructured assembly on a substrate. Such substrate-based detection was advantageous, and its reusability and high cycle stability were demonstrated after five cycles of disassembly and reassembly. Our work demonstrates the biosensing capacity of this DNA-functionalized plasmonic nanoassembly model system on two-dimensional substrate, which is also applicable to the detection of numerous DNA-recognized biomolecules. Likewise, the presented construction method can be extended to fabricate other compositional core-satellite nanoassemblies.

New approaches to uncertainty in chemical risk assessment – The example of bisphenol A

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91−266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91−266–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91−266–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.

"Hit-and-run": Transcription factors get caught in the act.

A key challenge for understanding transcriptional regulation is being able to measure transcription factor (TF)-DNA binding events with sufficient spatial and temporal resolution; that is, when and where TFs occupy their cognate sites. A recent study by Para et al. has highlighted the dynamics underlying the activation of gene expression by a master regulator TF. This study provides concrete evidence for a long-standing hypothesis in biology, the "hit-and-run" mechanism, which was first proposed decades ago. That is, gene expression is dynamically controlled by a TF that transiently binds and activates a target gene, which might stay in a transcriptionally active state after the initial binding event has ended. Importantly, the experimental procedure introduced, TARGET, provides a useful way for identifying multiple target genes transiently bound by their regulators, which can be used in conjunction with other well-established methods to improve our understanding of transcriptional regulatory dynamics.

Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.

Ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA utilizing ion-mediated cascade surface-enhanced Raman spectroscopy amplification.

Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring "ions-mediated cascade amplification (IMCA)", was proposed by utilizing the dissolved silver ions (Ag(+)) from silver nanoparticles (AgNPs). We found that using Ag(+) as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form "hot spots" and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag(+). Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag(+) from AgNPs using IMCA, low to 3.0 × 10(-5) fm/μL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic β-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis.

The DNA-binding network of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation.

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.