Nuclease

Nucleases are enzymes that degrade nucleic acids by cleaving phosphodiester linkages. They may be specific for DNA or RNA, or they may act on both. A nuclease specific for DNA is called a deoxyribonuclease (DNase) and for RNA, ribonuclease (RNase). 

Is there any specificity with respect to the location of the bond to be cleaved by a nuclease within the polynucleotide chain  

Nucleases are of two general types (1) exonucleases and (2) endonucleases. Exonucleases bind to a terminus (5 or 3 ) and remove nucleotides either one or a few at a time. Some require a 3 terminus and operate in the 5 — 3 direction (5 — 3 exonucleases) others (3 — 5 exonucleases) start at a 3 terminus and degrade in the opposite direction. There are some exonucleases that will work from either terminus. Exonucleases show no base or sequence specificity. 

Endonucleases do not require a terminus and will catalyze the cleavage of a polynucleotide chain at one or more sites. Frequently, they are specific for certain sites (specific base sequences) within the polynucleotide. 

Question Will nucleases cleave both single-stranded and double-stranded nucleic acids  

Nucleases.— Although the structures of ribonuclease A and ribonuclease 21,22 y, gJ.g solved at high resolution some years ago, there is still considerable interest in the structure and activity of these proteins. Carlisle and his colleagues (unpublished work) have completed an independent analysis of 

A crystallographic study of the binding of cupric ion with ribonuclease S has been carried out in order to investigate its inhibitory effects with this enzyme. Allewell and WyckofF have studied at 6 A resolution the binding of Cu to RNase S, to RNase E, and 41-DNP-RNase S, in the presence of 3M-(NH4)2S04 and acetate buffer. At pH 5.5, with 0.1 M acetate buffer, there are four intramolecular binding sites which are close to His-119, 

His-105, and Glu-86, and a sulphate ion bound at the active centre on both RNase S and RNase E. There are a further three intermolecular sites. In the 41-DNP-RNase there are minor changes and the site close to the sulphate is absent. At pH 7, in the presence of 0.67M acetate and 0.3M ethanolamine, the binding at all sites is decreased, although the occupancies at the nitrogen ligands decrease less than at oxygen ligands. 

Cotton et al. report the high-resolution structure of an inhibitor complex of the extracellular nuclease of Staphylococcus aureus. The 2 A resolution electron-density map was based on the analysis of two heavy-atom derivatives one involves the replacement of the calcium ion for a barium, while the other uses 5-iododeoxyuridine 3, S -diphosphate in place of thymidine 3 5 -diphosphate, a replac rait of Me by 1. 

The electron-density map allows a complete tracing of the polypeptide chain except for a few residues at the chain termini which are thought to be disordered as they project into solvent. About 30 of the 149 residues form three separate sections of helix, while 24 residues form a rather irregular, antiparallel pleated sheet. The inhibitor is bound in a large pocket which is predominantly hydrophobic, with the exception of the calcium and inhibitor ligands. 

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Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

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. 

Both phosphoramidate and phosphate triester derivatives have been used as linkers to attach reporter groups to oligonucleotides. These derivatives are not entirely resistant to nucleases and they possess a chiral center. They have not been widely iavestigated as antisense dmgs. 

The a-anomeric form of a 2 -deoxyribose, which has the base inverted with respect to the natural P-anomeric form, can be synthesized by using the phosphoramidite method sugar modification renders the derivatives nuclease-resistant. These analogues form parallel duplexes with complementary RNA... 

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. 

Composition and Methods of Manufacture. The vaccine consists of a mixture of purified capsular polysaccharides from 23 pneumococcal types that are responsible for over 90% of the serious pneumococcal disease in the world (47,48). Each of the polysaccharide types is produced separately and treated to remove impurities. The latter is commonly achieved by alcohol fractionation, centrifugation, treatment with cationic detergents, proteolytic en2ymes, nucleases or activated charcoal, diafiltration, and lyophili2ation (49,50). The vaccine contains 25 micrograms of each of the types of polysaccharide and a preservative such as phenol or thimerosal. 

Lipoteichoic acids (from gram-positive bacteria) [56411-57-5J. Extracted by hot phenol/water from disrupted cells. Nucleic acids that were also extracted were removed by treatment with nucleases. Nucleic resistant acids, proteins, polysaccharides and teichoic acids were separated from lipoteichoic acids by anion-exchange chromatography on DEAE-Sephacel or by hydrophobic interaction on octyl-Sepharose [Fischer et al. Ear J Biochem 133 523 1983]. 

Figure 6 Thermodynamic cycle for multi-substate free energy calculation. System A has n substates system B has m. The free energy difference between A and B is related to the substate free energy differences through Eq. (41). A numerical example is shown in the graph (from Ref. 39), where A and B are two isomers of a surface loop of staphylococcal nuclease, related by cis-trans isomerization of proline 117. The cis trans free energy calculation took into account 20 substates for each isomer only the six or seven most stable are included in the plot.

Figure 7 Experimental and theoretical inelastic neutron scattering spectrum from staphylococcal nuclease at 25 K. The experimental spectrum was obtained on the TFXA spectrometer at Oxford. The calculated spectrum was obtained from a normal mode analysis of the isolated molecule. (From Ref. 28.)...

Figure 2.15 The Greek key motif is found in antiparallel p sheets when four adjacent p strands are arranged in the pattern shown as a topology diagram in (a). The motif occurs in many p sheets and is exemplified here by the enzyme Staphylococcus nuclease (b). The four p strands that form this motif are colored red and blue.

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

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... 

Nuclease SI Both a Cleaves single-stranded but not double-stranded nucleic acids... 

FIGURE 11.32 All example of nuclease specificity The specificity of RNA hydrolysis by bovine pancreatic RNase. This RNase cleaves h at 3 -pyriinidines, yielding oligonncleoddes with pyrimidine 3 -P04 ends. 

C. B. Anfinsen (Bethesda) work on ribo-nuclease, especially concerning the connection between the amino-acid sequence and the biologically active conformation. 

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