DNA Topology (dna + topology)

Distribution by Scientific Domains

Selected Abstracts

Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing protein

FEBS JOURNAL, Issue 6 2010
Tokiha Masuda
The Mhr1 protein is necessary for mtDNA homologous recombination in Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction during homologous recombination, and is generally catalyzed by the RecA/Rad51 family of proteins in an ATP-dependent manner. Mhr1 catalyzes HP through a mechanism similar, at the DNA level, to that of the RecA/Rad51 proteins, but without utilizing ATP. However, it has no sequence homology with the RecA/Rad51 family proteins or with other ATP-independent HP proteins, and exhibits different requirements for DNA topology. We are interested in the structural features of the functional domains of Mhr1. In this study, we employed the native fluorescence of Mhr1's Trp residues to examine the energy transfer from the Trp residues to etheno-modified ssDNA bound to Mhr1. Our results showed that two of the seven Trp residues (Trp71 and Trp165) are spatially close to the bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg2+ -dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are involved in the DNA-binding activity of Mhr1. In addition, in vivo complementation analyses showed that a region around Trp165 is important for the maintenance of mtDNA. On the basis of these results, we discuss the function of the region surrounding Trp165. [source]

DNA tertiary structure and changes in DNA supercoiling upon interaction with ethidium bromide and gyrase monitored by UV resonance Raman spectroscopy

U. Neugebauer
Abstract The tertiary structure of DNA is important for many of its biological functions. In this work supercoiled and relaxed forms of purified plasmid DNA pBR322 in dilute aqueous solutions are investigated by means of UVRR spectroscopy to assess changes in B-DNA conformation. Spectral variation in the CO and exocyclic NH2 vibration above 1600 cm,1 indicate changes in hydrogen bonding. A minor shift of the CN stretching mode of adenosine and guanosine at 1487 cm,1 supports these findings. Changes in ribose conformation are visible in the spectral region 1320,1360 cm,1 by vibrational coupling of the ribose pucker to the vibrations of the purine and pyrimidine bases. The intercalating phenanthridinium drug ethidium bromide is known to reduce the negative supercoiling of DNA. This change in DNA topology is reflected in variations of the UVRR marker bands of DNA identified above. Principal component analysis helped to extract the features of interest from the complex spectra of the intercalation complex. Within the bacterial cells the change in DNA topology is achieved by the action of topoisomerases. In this work, the DNA-binding subunit GyrA of the enzyme gyrase was extracted from E. coli and applied to relaxed and supercoiled pBR322. The observed changes in the vibrational signature of the relaxed DNA in the presence of GyrA indicate a change of topology towards the supercoiled form. With already supercoiled DNA no further change in DNA topology is observed. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Untangling intracellular DNA topology

Olivier Espeli
Summary The biochemical steps by which bacterial topoisomerases alter the topology of DNA are well known. However, it has been a more vexing task to establish physiological roles and sites of action of the different topoisomerases within the context of the bacterial cell cycle. This difficulty can be attributed in part to the redundancy among the activities of the different enzymes. In this microreview, we will focus on recent progress in understanding the topological structure of the chromosome, analysis of topoisomerase mechanism in single-molecule assays and recent data on the regulation and integration of topoisomerase activity within the cell cycle that have all brought a new perspective to the action of topoisomerases in the bacterial cell. [source]

DNA topology and topoisomerases

Teaching a "knotty" subject
Abstract DNA is essentially an extremely long double-stranded rope in which the two strands are wound about one another. As a result, topological properties of the genetic material, including DNA underwinding and overwinding, knotting, and tangling profoundly influence virtually every major nucleic acid process. Despite the importance of DNA topology, it is a conceptionally difficult subject to teach because it requires students to visualize three-dimensional relationships. This article will familiarize the reader with the concept of DNA topology and offer practical approaches and demonstrations to teaching this "knotty" subject in the classroom. Furthermore, it will discuss topoisomerases, the enzymes that regulate the topological state of DNA in the cell. These ubiquitous enzymes perform a number of critical cellular functions by generating transient breaks in the double helix. During this catalytic event, topoisomerases maintain genomic stability by forming covalent phosphotyrosyl bonds between active site residues and the newly generated DNA termini. Topoisomerases are essential for cell survival. However, because they cleave the genetic material, these enzymes also have the potential to fragment the genome. This latter feature of topoisomerases is exploited by some of the most widely prescribed anticancer and antibacterial drugs currently in clinical use. Finally, in addition to curing cancer, topoisomerase action also has been linked to the induction of specific types of leukemia. [source]

Purification, crystallization and preliminary X-ray diffraction experiments on the breakage-reunion domain of the DNA gyrase from Mycobacterium tuberculosis

Jérémie Piton
Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57,kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7,Ĺ depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase. [source]

Super-Resolution Imaging of DNA Labelled with Intercalating Dyes

CHEMPHYSCHEM, Issue 13 2009
Cristina Flors Dr.
Imaging DNA: DNA intercalating cyanine dyes provide a simple and convenient method to image DNA topology by means of fluorescence microscopy with a spatial resolution better than the diffraction limit of light (see picture). [source]