Nucleases and DNA

The bacterial nucleases are enzymes which cut DNA specifically, and they are of intrinsic physiological importance as well as being indispensable in recombinant DNA research [38]. 

The endonucleases are a class of restriction enzymes which cleave doubly stranded DNA by recognition of specific (symmetric) nucleotide sequences [38, 39]. The enzymes are of considerable utility in studying sequence-specific interactions of proteins and small molecules with DNA. 

For metal complexes care should be taken that no inhibition of free enzyme occurs, which could affect the interpretation of the results. 

Many of the physical properties of polynucleotides are changed upon alteration of their structure this allows for the application of a wide range of techniques in such studies. Hydrodynamic properties such as viscosity, sedimentation coefficients, and buoyant density can all be readily monitored. Light and low-angle X-ray scattering are also well understood. Changes in spectroscopic properties are also conveniently studied. These have all been summarized in Physical Chemistry of Nucleic Acids [28], and the major effects which are discussed in this book are briefly reviewed below. 

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Mn may also be important in the regulation of enzyme activity, e.g. nonadenylylated glutamine synthetase requires Mg(II), but the adenylylated form binds Mn(II). Also the specificity of nucleases and DNA polymerases is changed when Mg(II) is replaced by Mn(II). The physiological significance of these differences is not clear. 

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

Fig. 6.3 Molecular model of the domains of the chimeric nuclease (constituted by an hybrid between a non-specific DNA cleavage domain and a zinc finger recognition domain) and DNA. The cleavage domain sits behind...

Piece of human DNA cut with same restriction nuclease and containing same sticky ends... 

The size of plasmids used for transfection can vary considerably, but most plasmids are 4,000 to 10,000 base pairs in size. Despite their differences all plasmids face the same barriers when transfected. Transfected DNA has to cross the cell membrane or the endosomal membrane, it has to be transported into the nucleus, and it has to be protected against cellular nucleases and degradation... 

FRET probes based on DNA are strong potential tools to investigate cellular processes where nucleases and a set of enzymes acting on DNA play a role. Most probes of this class are not particularly... 

Nuclease PI is another trizinc enzyme which cleaves the phosphodiester bond in single-stranded RNA and DNA. Protein crystallography has revealed that the structure of the three zinc site is very similar to that... 

Scheme 14 Schematic representations of the assay for nucleases and protease detection based on the complex of ACP, DNA-TR and peptide-H...

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

Conserved catalytic (adenylation) domain in NAD-dependent (bacterial) and ATP-dependent (archaeal-eukaryotic) DNA ligases (Aravind and Koonin, 1999) Conserved nucleotide joining-cleaving domain in type I and II topoisomerases, DnaG-type primases, OLD nucleases, and RecR (Toprim domain) (Aravind et al, 1998b)... 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

The weakly immunogenic protamine sulfate USP (1) condenses DNA to form a toroid structure of super-coiled DNA about 50 nm in diameter (2). The DNA in this form or in the preformed LPDI complex cannot be displaced from the protamine by polycations such as spermidine and histones or by other nucleic acids like genomic DNA (2). DNA in this toroid structure is transcriptionally inactive and this conformation allows for protection of DNA from enzymatic degradation by nucleases and other environmental assaults such as mechanical stress (1,2). After the liposome surrounds the toroid, the resulting homogenous LPDI nanoparticles are slightly less than... 

LPDI nanoparticles are homogenous, self-forming spheres between 100 and 200 nm in diameter that are formed from the spontaneous rearrangement of a lipid bilayer around a polycation condensed DNA core. The LPDI particles (lipopolyplexes) have benefits over lipoplexes, which are composed of liposomes and DNA. Homogenous particles are formed during preparation and thus allow a more consistent production of particles, as required by the FDA for clinical use. The LPDI particles also have a lower toxicity associated with them as opposed to lipoplexes, which can generate severe systemic inflammatory responses, most likely to the increased DNA content on the surface of the particles. The internalization of DNA inside the LPDI also has a benefit of DNA protection. The DNA is not nearly as accessible to nuclease attack and mechanical stress. Therefore, a lower quantity of DNA is used because it is protected inside of the LPDI for delivery. 

In common with the digestion of other macromolecules, nucleic acids are hydrolysed in a stepwise manner, by pancreatic nuclease (diesterase enzymes) which hydrolyse the bonds between two adjacent phosphate groups in RNA and DNA. The resultant oligoribonucleotides and oligode-oxy ribonucleotides are hydrolysed to form nucleoside monophosphates, which lose their phosphate to form nucleosides, by the action of pancreatic phosphatase. In brief, the process is ... 

One of the very early research tools that were used to study the nucleosomal state of active genes were the nucleases, DNase I and Micrococcal nuclease. With the development of protocols for the isolation of nuclei from cells, it was possible to add these reagents to probe the accessibility of DNA. DNase I makes single nicks in double stranded DNA and when the DNA is associated with histones within the nucleosome, the DNA is extensively protected. Those nicks that are observed are found to occur only after extensive digestion and are limited to the outside surface of the DNA in 10 base increments [7,8]. Weintraub and Groudine in 1976 [9] first used this nuclease and observed that when nuclei from chicken erythrocytes were treated with DNase I, the active /1-globin gene was preferentially... 

SINGLE-STEP REACTION Single-stranded RNA and DNA substrates, NUCLEASE SI Singlet dioxygen,... 

A multitude of nucleases cleave DNA, single- or double-stranded. They range from the pancreatic digestive enzyme DNase I through specialized nucleases that function during DNA repair and the hundreds of restriction endonucleases that have become so valuable in modern laboratory work. Some nucleases leave a 3 -phosphate ester at a cut end in a DNA chain, while others leave a 5 -phosphate end.824 Many nucleases are dealt with in later chapters. Only a few will be mentioned here. 

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