Formation of Nucleation Center in Single Double-Stranded DNA Chain

Abstract Faithful DNA replication is required for transmission of the genetic material across generations. The basic mechanisms underlying this process are shared among all organisms: progressive unwinding of the long double-stranded DNA; synthesis of RNA primers; and synthesis of a new DNA chain. These activities are invariably performed by a multi-component machine called the replisome. A detailed description of this molecular machine has been achieved in prokaryotes and phages, with the replication processes in eukaryotes being comparatively less known. However, recent breakthroughs in the in vitro reconstitution of eukaryotic replisomes have resulted in valuable insight into their functions and mechanisms. In conjunction with the developments in eukaryotic replication, an emerging overall view of replisomes as dynamic protein ensembles is coming into fruition. The purpose of this review is to provide an overview of the recent insights into the dynamic nature of the bacterial replisome, revealed through single-molecule techniques, and to describe some aspects of the eukaryotic replisome under this framework. We primarily focus on Escherichia coli and Saccharomyces cerevisiae (budding yeast), since a significant amount of literature is available for these two model organisms. We end with a description of the methods of live-cell fluorescence microscopy for the characterization of replisome dynamics.

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Genetic information ‘created’ by archaebacterial DNA polymerase

Biochemical Journal ◽

10.1042/bj3240667 ◽

1997 ◽

Vol 324 (2) ◽

pp. 667-671 ◽

Cited By ~ 22

Author(s):

Norio OGATA ◽

Takanori MIURA

Keyword(s):

Dna Polymerase ◽

Genetic Information ◽

Repetitive Sequences ◽

Chromatographic Purification ◽

Related Sequence ◽

Thermococcus Litoralis ◽

Double Stranded Dna ◽

Dna Chain ◽

Cellular Dna ◽

Template Complex

DNA polymerase catalyses replication of cellular DNA. The reaction requires a primer–template complex, and a new DNA chain grows from the 3′ end of the primer along the template; no genetic information is created in this reaction. We demonstrate that DNA polymerase from Thermococcus litoralis, a hyperthermophilic marine Archaea, can synthesize up to 50000 bp of linear double-stranded DNA in the complete absence of a primer–template complex, indicating that genetic information is ‘created.’ The possibility of DNA contamination in the reaction mixture, which may serve as a primer and/or template, was vigorously excluded; for example, pretreatment of DNA polymerase with DNase I or extensive chromatographic purification of the substrate, deoxyribonucleoside 5′-triphosphates, did not abolish the primer–template-independent DNA synthesis. The DNA synthesized was (CTAGATAT)n, (TAGATATCTATC)n or a related sequence. Similar repetitive sequences are found in centromeric satellite DNA of many organisms. The significance of this ab initioDNA synthesis is that genetic information can flow from protein to DNA.

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New coarse-grained molecular dynamics model of double stranded DNA chain for DNA origami

2016 IEEE 11th Annual International Conference on Nano/Micro Engineered and Molecular Systems (NEMS) ◽

10.1109/nems.2016.7758291 ◽

2016 ◽

Author(s):

Hiromasa Yagyu ◽

Do-Nyun Kim ◽

Osamu Tabata

Keyword(s):

Molecular Dynamics ◽

Dna Origami ◽

Coarse Grained ◽

Dynamics Model ◽

Double Stranded Dna ◽

Dna Chain ◽

Coarse Grained Molecular Dynamics

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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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