Torunn Berge

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy

Direct observation of DNA translocation and cleavage by the Eco KI endonuclease using atomic force microscopy

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 +/- 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Translocation-Independent Dimerization of the EcoKI Endonuclease Visualized by Atomic Force Microscopy

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA.

The binding mode of the DNA bisintercalator luzopeptin investigated using atomic force microscopy.

The luzopeptins are DNA bisintercalating antibiotics that contain a decadepsipeptide to which are attached two quinoline chromophores. We have used atomic force microscopy (AFM) to investigate the interaction between luzopeptin B and DNA in an attempt to shed light on the binding mode of this antibiotic. AFM images provided contour lengths which were used as a direct measure of bisintercalation. Binding of luzopeptin B was investigated using two different DNA sequences, one having a GC content of 42% and the other 59%, which revealed a higher degree of bisintercalation into the DNA sequences having the lower GC content. The measured increment in contour length was found to plateau at values corresponding to binding of one drug molecule every 40 and 72 bp to the 42 and 59% GC sequences, respectively. In addition to the length increase, a higher proportion of DNA molecules displaying complex morphology was observed as the concentration of luzopeptin was increased. Such molecules were not included in the measurements of contour length. We propose that the various manifestations of complex morphology arise from both inter- and intramolecular cross-linking of the DNA caused by binding of luzopeptin, providing direct evidence of cross-linked species by AFM imaging.

Imaging the surface of Staphylococcus aureus by atomic force microscopy

The surfaces of four strains of Staphylococcus aureus, which differed in their expression of capsular polysaccharides, were examined using atomic force microscopy. The images show that it is possible to get information about surface characteristics of S. aureus using atomic force microscopy (AFM) following simple preparation. Strains Smith Diffuse (serotype 2), Reynolds (serotype 5), Wood‐46 (capsule negative) and JL243 (capsule negative) were grown on medium known to promote the expression of capsular polysaccharides. The bacteria were air‐dried prior to being imaged using tapping‐mode AFM. Differences in the appearance of the bacterial surfaces were evident between the strains. The two capsule‐negative strains exhibited a smooth regular surface, as opposed to the mucoid appearance of the two strains having polysaccharide capsules. Moreover, comparison of images of the heavily encapsulated serotype 2 strain and the serotype 5 strain indicates that a type 2 capsule can be distinguished from a type 5 microcapsule.

Investigation of protein partnerships using atomic force microscopy.

The origin of contrast in atomic force microscopy (AFM) lies in the probes response to forces between itself and the sample. These forces most commonly result from changes in height as the tip is scanned over the surface, but can also originate in properties inherent in the sample. These have been exploited as further means of contrast and have spawned an array of similar imaging techniques, such as chemical force microscopy, magnetic force microscopy, and frictional force microscopy. All of these techniques use AFM as an extremely sensitive gauge to map forces at discrete sites on the surface. A natural extension of this approach is to map forces in an array, in order to create a force map. AFM can be used in aqueous or fluid environments, thus allowing the exploration of forces in biological systems under physiologically relevant conditions. By immobilizing one half of an interacting pair of proteins onto the tip and the other half onto the substrate, it is possible to investigate the electrostatic and hydrophobic interactions between them. We employed these techniques to examine the interaction between a pair of proteins of known affinity that are involved in exocytosis (NSF and α‐SNAP) and separately to demonstrate how two‐dimensional force mapping can be applied to the nuclear envelope to identify nuclear pore complexes. Microsc. Res. Tech. 44:368–377, 1999.

Selective area regrowth of n-GaAs with reduced interface carrier depletion using arsenic passivation

Selective area molecular beam epitaxy (MBE) of n-GaAs through a Si 3 N 4 mask has been explored. An arsenic capping layer was deposited in the MBE growth chamber prior to mask definition, in order to protect the epilayer surface during ex situ processing. After in situ thermal decapping and high-temperature (680°C) regrowth at a low growth rate (0.5 μm/h), the samples were examined with scanning electron and atomic force microscopy. The data unveil selective regrowth with lateral definition on a micrometer length scale and GaAs surfaces with a smooth mirror-like finish. Electrolytic carrier profiling and secondary ion mass spectrometry measurements of the homoepitaxial regrowth interface show a significant reduction of the interface potential barrier, from V bi = 0.45 V to V bi = 0.07 V, and reduced carbon impurity concentrations, compared to regrowth on non-passivated GaAs epilayers.

Patterned regrowth of n-GaAs by molecular beam epitaxy using arsenic passivation

Patterned regrowth of n-GaAs through a Si3N4 mask prepared on molecular beam epitaxy (MBE) grown n-GaAs layers has been explored. An arsenic capping layer was deposited in the MBE growth chamber prior to mask definition, in order to protect the GaAs surface during ex situ processing. Removal of this cap from the lithographically defined mask area was accomplished by hydrogen plasma etching prior to Si3N4 deposition. After in situ thermal desorption of the As cap and phase selective regrowth, the samples were inspected with scanning electron and atomic force microscopy to establish the growth selectivity and examine the surface morphology. Electrolytic carrier profiling and secondary ion mass spectrometry measurements were carried out to characterize the growth-interrupted GaAs–GaAs interface. We find that the surface morphology and defect density of regrown GaAs epilayers are comparable to those of continuously grown epilayers of the same overall thickness. Structuring of the arsenic cap with hydrogen pla...