Single-molecule studies of repressor-DNA interactions show long-range interactions.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang YM
PROVIDER: S-EPMC1168954 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:DNA functions only in aqueous environments and adopts different conformations depending on the hydration level. The dynamics of hydration water and hydrated DNA leads to rotating and oscillating dipoles that, in turn, give rise to a strong megahertz to terahertz absorption. Investigating the impact of hydration on DNA dynamics and the spectral features of water molecules influenced by DNA, however, is extremely challenging because of the strong absorption of water in the megahertz to terahertz frequency range. In response, we have employed a high-precision megahertz to terahertz dielectric spectrometer, assisted by molecular dynamics simulations, to investigate the dynamics of water molecules within the hydration shells of DNA as well as the collective vibrational motions of hydrated DNA, which are vital to DNA conformation and functionality. Our results reveal that the dynamics of water molecules in a DNA solution is heterogeneous, exhibiting a hierarchy of four distinct relaxation times ranging from ∼8 ps to 1 ns, and the hydration structure of a DNA chain can extend to as far as ∼18 Å from its surface. The low-frequency collective vibrational modes of hydrated DNA have been identified and found to be sensitive to environmental conditions including temperature and hydration level. The results reveal critical information on hydrated DNA dynamics and DNA-water interfaces, which impact the biochemical functions and reactivity of DNA.
| S-EPMC8633837 | biostudies-literature
Project description:Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.
| S-EPMC1853322 | biostudies-literature
Project description:Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.
| S-EPMC7141782 | biostudies-literature
Project description:The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact "closed" form with a V-shaped repressor and also a more "open" form with an extended hinge. The "9C14" construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.
| S-EPMC1366758 | biostudies-literature
Project description:At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.
| S-EPMC2553838 | biostudies-literature
Project description:Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N6-methyladenosine (m6A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.
| S-EPMC7968351 | biostudies-literature
Project description:Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.
| S-EPMC8390797 | biostudies-literature
Project description:DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.
| S-EPMC4310750 | biostudies-literature
Project description:Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.
| S-EPMC6451117 | biostudies-literature
Project description:Recently, it was proposed that DNA looping by the lambda repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the lambda repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to -0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.
| S-EPMC2685085 | biostudies-literature