DNA Binding Orientation Research Articles

Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB).

The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding proteins.

Multiple Lac-Mediated Loops Revealed by Bayesian Statistics and Tethered Particle Motion

The bacterial transcription factor LacI loops DNA by binding to two distinct binding sites on DNA. Despite being one of the most well-studied model systems for transcriptional regulation, the number of possible loops, and their structures, are unclear. To clarify this question, we have developed a new analysis method for tethered particle motion (TPM), a versatile single-molecule technique commonly used to study Lac-mediated looping and other DNA-protein interactions in vitro. The method, called vbTPM, is based on a variational Bayes treatment of hidden Markov models. It learns the number of distinct states (e.g., DNA-protein conformations) in a TPM trajectory directly from the data with better resolution than existing methods, and also corrects for common experimental artifacts. Studying short (about 100 bp) LacI-mediated loops, we are able to resolve states and interconversion patterns that are best explained in terms of at least three distinct loop structures. These results indicate that changes in Lac conformation and DNA binding orientation both contribute to the repertoire of LacI-mediated loops, and provides new input for quantitative models of looping and transcriptional regulation.

DNA-fiber EPR spectroscopy as a tool to study DNA–metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II)·Gly-Gly-His-derived metallopeptides

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II)·Gly-Gly-His-derived metallopeptides containing d- vs. l-amino acid substitutions in the first peptide position. This examination included studies of Cu(II)· d-Arg-Gly-His and Cu(II)· d-Lys-Gly-His for comparison to metallopeptides containing l-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II)· d/ l-Pro-Gly-His and Cu(II)· d/ l-Pro-Lys-His. Results indicated that l-Arg/Lys to d-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA, whereas the replacement of l-Pro by d-Pro in Cu(II)· l-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness observed between the d- vs. l-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the ε-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II)·Xaa-Xaa′-His metallopeptides with DNA are also discussed.

Effect of a conjugated acridine moiety on the binding and reactivity of Cu(II)[9-acridinylmethyl-1,4,7-triazacyclononane] with DNA

The DNA binding orientation and dynamic behavior of Cu(II) complexes of 1,4,7-triazacyclononane ([9]aneN 3), 1, and an acridine conjugate, 2, were investigated by DNA fiber EPR (EPR = electron paramagnetic resonance) spectroscopy. Crystal and molecular structure of 2 were determined by X-ray diffraction. It has been shown that 1 binds to DNA in two different modes at room temperature; one species is rapidly rotating and the other is immobilized randomly on the DNA. The introduction of acridine to [9]aneN 3 fixed the [Cu([9]aneN 3)] 2+ moiety of 2 in two different environments on the DNA: the g ∣∣ axis of one species ( g ∥ = 2.26) is aligned perpendicularly to the DNA fiber axis whereas that of the other ( g ∥ = 2.24) aligns < 90° with the DNA fiber axis. The different DNA binding structures of 1 and 2 are reflected also in their different efficiencies of DNA cleavage; 2 was found to be more effective both in oxidative and hydrolytic cleavage reactions.

DNA-binding Orientation and Domain Conformation of the E. coli Rep Helicase Monomer Bound to a Partial Duplex Junction: Single-molecule Studies of Fluorescently Labeled Enzymes

The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3′-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the “closed” conformation when bound to a 3′-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3′-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3′-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.

Synthesis, Properties and Functions of Peptide Nucleic Acids (PNA) and Their Derivatives

AbstractFor Abstract see ChemInform Abstract in Full Text.

ペプチド核酸（ＰＮＡ）およびその類縁体からなる機能性人工核酸の合成と性質

In 1991 Nielsen et al. first described peptide nucleic acid (PNA), which is a DNA mimic with a pseudopeptide backbone composed of N-(2-aminoethyl) glycine units with the nucleobases attached to the glycine nitrogen via carbonyl methylene linkers. PNA is a very attractive molecule as a DNA mimic because it shows high biological stability with strong binding-affinity to complementary DNA and RNA. The limitations of PNA include low aqueous solubility, ambiguity in DNA binding orientation and poor membrane permeability. Various PNA derivatives were reported to improve physicochemical and biological properties of PNA. Chirality was introduced into the achiral PNA backbone to influence the selectivity in DNA binding orientation. PNA backbone was rigidified by introduction of ring structure to preorganize PNA for duplex formation. This review focuses on the chemistry and biological activity of PNA, and gives an overview of backbone modifications of PNA.

MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily.

The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

Cytosine Methylation Enhances DNA Damage Produced by Groove Binding and Intercalating Enediynes: Studies with Esperamicins A1 and C

Methylation of the C5 position of cytosine in CG dinucleotides represents an important element in the control of gene expression in eukaryotic cells. This major groove modification of DNA causes changes in DNA conformation that are recognized by DNA-binding proteins and DNA-damaging chemicals. We have observed that CG methylation affects the DNA damage produced by the enediyne esperamicin A1 and its analog lacking the intercalating anthranilate, esperamicin C. Fragments of the human phosphoglycerate kinase gene (PGK1) and the plasmid pUC19 were methylated with SssI methylase and subjected to damage by esperamicins A1 and C. Damage produced by esperamicin A1 was enhanced 1.5-2-fold near a single CG sequence at position -101 in PGK1 and in a region containing several methylated CG dinucleotides between positions -120 and -131. Esperamicin C-induced damage was enhanced to a similar degree in PGK1 but only at the site that contained multiple CG dinucleotides. There was enhancement of damage for both drugs in the pUC19 fragment at several sites near CG sequences. Analysis of the chemistry of esperamicin-induced DNA damage suggests that cytosine methylation does not affect the identity of drug-abstracted hydrogen atoms. The damage chemistry was also used to identify the DNA binding orientation of the esperamicins. The anthranilate of esperamicin A1 is predicted to intercalate in a CT step four base pairs in a 3'-direction to the CG sequence at -101 in PGK1 and in a CG dinucleotide at the site containing multiple CGs (positions -120 to -131). These observations are consistent with other studies that indicate a long range effect of cytosine methylation on DNA conformation.

Differential effects of DNA supercoiling on radical-mediated DNA strand breaks.

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA structure and dynamics that accompany superhelical tension, we have investigated the effect of torsional tension on DNA damage produced by five oxidizing agents: gamma-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positively supercoiled plasmid DNA prepared by a recently developed technique, we compared the quantity of strand breaks produced by the five agents in negatively and positively supercoiled pUC19. It was observed that strand breaks produced by gamma-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly reactive intermediates (e.g., hydroxyl radical), but do not interact directly with DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. In the case of Fe2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O2 in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, a sigmoidal Cu2+/H2O2 damage response was observed in the negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu2+ suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigation into the role of supercoiling in base damage.