Nuclease substrate specificity

Microbial RNases with known substrate specificity are listed in Table XII. Nucleases with DNase activity are not included. It should be noted here that the lists of RNases in both animal and plant kingdoms are presented in the monograph by Privat de Garilhe (4) and in a chapter by E. A. Barnard in Annual Review of Biochemistry (1969) (135). 

Nucleases are broadly defined as the enzymes that degrade polynucleotides. They comprise a large family of enzymes that show diverse reaction and substrate specificities DNA-specific (DNase) and/or RNA-specific (RNase), nucleotide sequence-specific or nonspecific, double-strand-specific and/or single-strand-specific, exonucleolytic (5 3 and 3 5 ) and/or endonucleolytic, and generating... 

P or 5 -P products. Many polymerases, including reverse transcriptases, also exhibit intrinsic nuclease activities with widely varying reaction and substrate specificities, which makes the rational division of nucleases even more complex. 

This section describes two representative single-strand-specific endonucleases nuclease SI from a fungus Aspergillus oryzae and mung bean nuclease from mung bean sprouts. The two enzymes are very similar in many respects both are thermostable, zinc-dependent glycoproteins of similar size and they share most of the substrate specificities. The most pronounced difference that exists between... 

The nuclease from a marine bacteria Alteromonas espejiana (Bal31) is an extracellular nuclease with wide-ranging substrate specificities. The Alteromonas nuclease is commonly known as Bal31 nuclease, nuclease Bal31, or more simply Bal31. The enzyme catalyzes the degradation of ssDNA both endo- and exonucleo-lytically and of linear dsDNA exonucleolytically from 3 termini generating mostly blunt ends. Products are predominantly 5 -mononucleotides. 

Importantly, the ERCCl/XPF structure-specific nuclease has an additional role in the repair of cisplatin adducts besides its function in NER the recombinational repair of interstrand crosslinks (19). ERCCl- or XPF-deficient hamster mutant cell lines are hypersensitive to DNA crosslink agents, much more so than to ultraviolet-induced pyrimide dimers, the classical substrates for NER (20,21). Moreover, co-localization of ERCCl foci and RAD51 foci in response to cisplatin treatment has recently been found and may represent recruitment of ERCCl/XPF to sites of recombination repair (22). Previous studies have shown that BRCAl, involved in homologous recombination repair, also plays a major role in the repair of cisplatin DNA damage (23). 

Staphylococcal nuclease is a phosphodiesterase which can cleave either DNA or RNA to produce 3 -phosphomononucleosides (3-16, 20,25). The rates of hydrolysis of these substrates are dependent on the conformation of the substrate, Ca2+ concentration, and the ionic strength and the pH of the buffer (3, 54). Denatured DNA is hydrolzed more rapidly than native DNA (3, 12, 54-56), which reflects the important effect of substrate conformation on catalysis. In native DNA the Xp-dTp and Xp-dAp bonds are preferentially attached (7, 12, 14, 15, 55). With denatured DNA the order of cleavage appears to be nearly random (14, 15, 56, 57). The Xp-dCp and Xp-dGp linkages in the helical regions of DNA, which are more extensively stabilized, are more resistant to hydrolysis. The specific order of release of various mononucleotides from native compared to denatured DNA suggests that in the hydrolysis of DNA specificity toward the constitutent bases is less important than the substrate conformation (54-57). 

Fig. 2). The staphylococcal enzyme may appear to be more akin in its mode of action to the spleen enzyme because they both hydrolyze DNA and RNA to 3 -nucleotides, whereas the venom enzyme releases 5 -nucleotides. However, their mode of action and specificity are quite different, and the structural requirements of the staphylococcal enzyme substrates are perhaps more nearly similar to those of the venom enzyme. The principal difference is that the staphylococcal enzyme cleaves the diester bond between the phosphate and the 5 -carbon of the sugar, whereas the venom enzyme cleaves on the other side of the phosphate, that is, between the phosphate and the nonspecific hydroxylic component of the diester bond. In contrast to both spleen and venom diesterases, the primary product released by staphylococcal nuclease hydrolysis is a derivative bearing a hydroxyl group (on the 5 position) rather than a phosphoryl group. Therefore, the 3 -phosphoryl product formed from polynucleotide hydrolysis is a secondary consequence of such cleavage. 

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

Earlier work indicated that in the specific CAP-DNA complex the DNA was bent 90° away from the protein but was not distorted in the nonspecific complex. (CAP bound to its noncognate DNA site) [3, 36], This DNA bending phenomenon was exploited to construct a new CAP-OP nuclease capable of single site cleavage [37]. The crystal structure of the specific CAP-DNA complex revealed that amino acids 24-26 and 89-91 of CAP were close to the DNA substrate [38], With this in mind, residue 26 was mutated... 

Fig. 2.8. Nearest neighbour analysis and quantitative depurination analysis of a defined product from a primed synthesis reaction. When radioactive dATP (or dGTP) is used in the primed synthesis, depurination analysis will yield pyrimidine tracts each of which terminate in a radioactive 3 -phosphate. Thus only those depurination products which lie 5 -adjacent to the labelled nucleotide will be labelled. Each depurination product will be labelled to the same specific activity thus greatly simplifying the quantitation. Digestion of the labelled product with a mixture of micrococcal nuclease and bovine spleen phosphodiesterase yields the nucleoside 3 -monophosphates. Identification of the labelled products (by paper electrophoresis at pH 3.S) gives the nearest neighbours to the labelled substrate.

Three levels of protein-nucleic acid recognition have been observed. Nature provides three examples of protein-nucleic acid interactions which we shall consider. The nucleic acid component can be (1) a single nucleotide, e.g., a coenzyme or a substrate, (2) a single-stranded DNA or RNA as in ribonucleases A and T, or (3) a double-stranded DNA or RNA as in the highly specific complexes with repressors in the tRNA-synthetase complex, or in the unspecific nuclease DNase I. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

With the exception of enzymes such as proteases, nucleases, and amylases, which act on macromolecular substrates, enzyme molecules are considerably larger than the molecules of their substrates. Consideration of the structure of an enzyme s active site and its relationship to the structures of the enzyme s substrate(s) in its ground and transition states is necessary to understand the rate enhancement and specificity of the chemical reactions performed by the enzyme,... 

Phospholipase A2 (EC 3.1.1.4) " " is a member of a class of lypolytic enzymes that hydrolyze their lipid substrates at an organized lipid-water interface. This enzyme specifically catalyses the hydrolysis of the 2-acyl ester bond of 3-5 -phyosphoglycerides. It has an absolute requirement for Ca " and binds this ion in a 1 1 molar ratio to the enzyme, with a dissociation constant of 2-4 mM. The x-ray structure of the 124-residue bovine enzyme has been determined. It has about 50% a-helical and 10% j8-sheet structure. Ca " " is bound at the active site (Figure 3) and is coordinated to backbone carbonyl atoms of Tyr-28, Gly-30, Gly-32, the two carboxylate oxygens of Asp-49 and two HjO molecules, for a total coordination number of seven. As was the case for staphylococcal nuclease, the Ca " " ligands are supplied from noncontiguous regions of the polypeptide chain. 

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