Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site

The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins

Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

Scanning tunneling microscopy study of a DNA fragment of known size and sequence

Assays to visualize a DNA fragment that may serve as DNA test for further experiments were performed by scanning tunneling microscopy (STM). We present observations on a 350 bp restriction fragment from pBR322 deposited on natural graphite single crystal surfaces and imaged in vacuum. Measurement of the length and of helical parameters of the molecule in conjunction with the fact that no features of the substrate or electrical characteristics were suggestive of a graphite artifact, allowed unambiguous identification of the molecule. Imaging of the entire DNA molecule has been achieved and high, resolution details are shown. Their correspondence to precise DNA sequences may be deduced from their localization on the image since the DNA sequence and orientation of the molecule is known. Therefore, both global and local sequence-directed variations of structural features along a DNA molecule are now accessible to analysis with STM imaging. The ability of STM to investigate DNA structures involved in gene activity appears thus to be very promising.Des essais pour visualiser un fragment d'ADN qui puisse servir de test pour des expériences ultérieures ont été réalisés en microscopie par effet tunnel (STM). Nous présentons des observations faites sur un fragment de restriction de 350 pb provenant de pBR322, déposé sur la surface d'un monocristal naturel de graphite et imagé sous vide. La mesure de la longueur et des paramètres hélicoïdaux de la molécule s'ajoutant au fait qu'aucun aspect du substrat ou des caractéristiques électriques ne suggérait un artefact du graphite, ont permis une identification non ambiguë de la molécule. L'image de la molécule complète d'ADN a été obtenue et des détails en haute résolution sont montrés. Leur correspondance avec des séquences d'ADN précises peut être déduite de leur localisation sur l'image puisque la séquence et l'orientation de la molécule sont connues. Les variations générales de même que les variations locales des caractéristiques structurales le long de la molécule d'ADN sont donc maintenant accessibles à l'analyse par imagerie STM. La capacité du STM à analyser les structures d'ADN impliquées dans l'activité génique apparaît ainsi très prometteuse