DNases nuclease

Saponaria Saporin 6 Saponaria officinalis PAG (rRNA) (DNA nuclease or... 

Large DNA Nuclease Small DNA fragments Plasmid Billions of copies... 

A major technical consideration in DNA-DNA hybridization is choice of a method for determining the amount of DNA which is single-stranded and double-stranded at various temperatures. Two methods have been most often employed the binding of double-stranded DNA to HAP (hydroxylapatite) and digestion with the enzyme SI DNA nuclease which cleaves single-stranded DNA into mono-nucleotides. We detail our procedures for the latter method in another chapter in this volume (Caccone and Powell). 

Katsarou M, Efthimiadou E, Psomas G, Karaliota A, Vourloumis D (2008) Novel copper(II) complex of N-propyl-norfloxacin and 1,10-phenanthroline with enhanced antileukemic and DNA nuclease activities. J Med Chem 51 470-478... 

When compared with nuclease SI under comparable conditions with a given substrate (e.g., 12-nt single-stranded termini of X DNA), nuclease MB is less active and less single strand specific than nuclease SI (10). 

The methylphosphonates differ from the phosphodiesters and phosphorothiolates in that the methyl derivatives are uncharged and are thus less water soluble. Moreover, compared to the naturally occurring phosphodiesters, the methylphosphonates form slightly less stable duplexes with complementary DNA and RNA sequences. This effect has been ascribed to the iaevitable chiraUty problem that is, if one isomer biads less well, the overall binding is decreased. Methylphosphonates can enter cell membranes by a passive mechanism and are completely resistant to nucleases. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

Fike most enzymes (see Chapter 14), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), while others are specific for RNA (the RNases). Still... 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

The urocanic-acid-modified chitosan showed good DNA binding abihty, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coup-Hng with urocanic acid [96]. 

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. 

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