Identification of Arginine Finger as the Starter of the Biomimetic Motor in Driving Double-Stranded DNA

Discrimination of monomer from multimer DNA disk-shaped toruses by freezeetch TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100111161 ◽

1984 ◽

Vol 42 ◽

pp. 210-211

Author(s):

George C. Ruben ◽

Kenneth A. Marx

Keyword(s):

Biochemical Data ◽

Dna Structures ◽

Ice Surface ◽

Double Stranded Dna ◽

Data Support ◽

Dna Organization ◽

Trivalent Cations

In vitro collapse of DNA by trivalent cations like spermidine produces torus (donut) shaped DNA structures thought to have a DNA organization similar to certain double stranded DNA bacteriophage and viruses. This has prompted our studies of these structures using freeze-etch low Pt-C metal (9Å) replica TEM. With a variety of DNAs the TEM and biochemical data support a circumferential DNA winding model for hydrated DNA torus organization. Since toruses are almost invariably oriented nearly horizontal to the ice surface one of the most accessible parameters of a torus population is annulus (ring) thickness. We have tabulated this parameter for populations of both nicked, circular (Fig. 1: n=63) and linear (n=40: data not shown) ϕX-174 DNA toruses. In both cases, as can be noted in Fig. 1, there appears to be a compact grouping of toruses possessing smaller dimensions separated from a dispersed population possessing considerably larger dimensions.

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Synchrony of Digestion of SV40 DNA by Exonuclease III Determined by EM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116991 ◽

1975 ◽

Vol 33 ◽

pp. 674-675

Author(s):

Ray Wu ◽

G. Ruben ◽

B. Siegel ◽

P. Spielman ◽

E. Jay

Keyword(s):

Dark Field ◽

Uranyl Acetate ◽

Enzyme Digestion ◽

Dna Molecules ◽

Exonuclease Iii ◽

Aluminum Films ◽

Double Stranded Dna ◽

Before And After ◽

Exo Iii ◽

Sv40 Dna

A method for determining long nucleotide sequences of double-stranded DNA is being developed. It involves (a) the synchronous digestion of the DNA from the 3' ends with EL coli exonuclease III (Exo III) followed by (b) resynthesis with labeled nucleotides and DNA polymerase. A crucial factor in the success of this method is the degree to which the enzyme digestion proceeds synchronously under proper conditions of incubation (step a). Dark field EM is used to obtain accurate measurements on the lengths and distribution of the DNA molecules before and after digestion with Exo III, while gel electrophoresis is used in parallel to obtain a mean length for these molecules. It is the measurements on a large enough sample of individual molecules by EM that provides the information on how synchronously the digestion proceeds. For length measurements, the DNA molecules were picked up on 20-30 Å thick carbon-aluminum films, using the aqueous Kleinschmidt technique and stained with 7.5 x 10-5M uranyl acetate in 90% ethanol for 3 minutes.

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Torus Circumference and Thickness Parameters From a Linear DNA Size Series Suggest How Toruses Self-Assemble

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119430 ◽

1985 ◽

Vol 43 ◽

pp. 522-523

Author(s):

George C. Ruben ◽

Kenneth A. Marx

Keyword(s):

Tertiary Structure ◽

Dna Complexes ◽

Tertiary Structures ◽

Self Assembling ◽

Double Stranded Dna ◽

Calf Thymus ◽

Dna Organization ◽

Condensed Dna ◽

Dna Size

Certain double stranded DNA bacteriophage and viruses are thought to have their DNA organized into large torus shaped structures. Morphologically, these poorly understood biological DNA tertiary structures resemble spermidine-condensed DNA complexes formed in vitro in the total absence of other macromolecules normally synthesized by the pathogens for the purpose of their own DNA packaging. Therefore, we have studied the tertiary structure of these self-assembling torus shaped spermidine- DNA complexes in a series of reports. Using freeze-etch, low Pt-C metal (10-15Å) replicas, we have visualized the microscopic DNA organization of both calf Thymus( CT) and linear 0X-174 RFII DNA toruses. In these structures DNA is circumferentially wound, continuously, around the torus into a semi-crystalline, hexagonal packed array of parallel DNA helix sections.

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