Hydrolysis of Polynucleotides

Polynucleotides are split up in aqueous solution by heating or by extremes of pH (Chapter 10.4). DNA is more resistant to alkaline hydrolysis than RNA because of the involvement of 2 OH groups. Certain cations, particularly Ce +, catalyse the rapid hydrolysis of polynucleotide chains. 

During digestive processes, nucleoprotein is split into nucleic acids and protein, the latter then being broken down into amino acids. The nucleic acids are attacked by ribonuclease and deoxyribonuclease enzymes to form nucleotides, which are further hydrolysed by nucleotidases to form nucleosides and phosphates. In the intestines these nucleosides are split by nucleosidases into ribose, deoxy-ribose, purine and pyrimidine bases, which later undergo oxidation and decomposition to ammonia, carbon dioxide and water, to be finally expelled as urea. Nucleotide hydrolysis products are conveniently identified and isolated by chromatographic methods (Chapter 14.2). 

The hydrolysis of polynucleotides by specific nucleases, or by very mild chemical conditions, will produce a mixture of the constituent nucleotides. Furthermore, the conditions of hydrolysis may determine whether a 3 or a 5 nucleotide is obtained. Hydrolysis of RNA with snake venom phosphodiesterase, for example, breaks the linkages at (a) in (11.122) giving a series of mononucleoside 5 phosphates. Alkaline hydrolysis with N NaOH at room tanperature, on the other hand, breaks the linkages at (b), giving initially nucleoside cyclic 2 ,3 phosphates which are further hydrolysed to a mixture of 2 and 3 nucleoside phosphates containing the various bases. 

While some nucleases will depolymerise all polynucleotides, some are specific to RNA (Ribonucleases), and others act only on DNA (Deoxyribonucleases). 

Exonucleases cleave nucleotides from the ends of polynucleotide chains, while Endonucleases produce cleavages at points along the chains. 

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Most reactions of nucleic acid hydrolysis break bonds in the polynucleotide backbone. Such reactions are important because they can be used to manipulate these polymeric molecules. For example, hydrolysis of polynucleotides generates smaller fragments whose nucleotide sequence can be more easily determined. 

Staphylococcal nuclease was discovered by Cunningham and his colleagues in cultures of pathogenic strains of Staphylococcus aureus (/, 2). It was the first nuclease to be found that yielded 3 -nucleotides upon hydrolysis of polynucleotide chains 3-16). Historically, this feature of... 

FIGURE 11.29 The vicinal—OH groups of RNA are susceptible to nucleophilic attack leading to hydrolysis of the phosphodiester bond and fracture of the polynucleotide chain DNA lacks a 2 -OH vicinal to its 3 -0-phosphodiester backbone. Alkaline hydrolysis of RNA results in the formation of a mixture of 2 - and 3 -nucleoside monophosphates. 

RNA is relatively resistant to the effects of dilute acid, but gentle treatment of DNA with 1 mM HCl leads to hydrolysis of purine glycosidic bonds and the loss of purine bases from the DNA. The glycosidic bonds between pyrimidine bases and 2 -deoxyribose are not affected, and, in this case, the polynucleotide s sugar-phosphate backbone remains intact. The purine-free polynucleotide product is called apurinic acid. 

By hydrolysis under very mild alkaline conditions (with a boiling suspension of barium carbonate), ribonucleic acids have been shown to yield small quantities of cyclic phosphates as well as the normal nucleotides.96 These materials were identical electrophoretically with synthetic cyclic phosphates and were readily hydrolyzed to mixtures of 2- and 3-phosphates. Their formation in this way constitutes strong support for Brown and Todd s theory. The precise way in which the alkaline hydrolysis of the polynucleotide occurs has been studied using isotopically labeled water, and the results are in agreement202 with the scheme outlined above. 

It is evident that most future work with irradiated polynucleotides will have to employ techniques such as these. Many pertinent observations were made about the effect of irradiation of the poly U upon enzyme specificity and rate. Irradiation of the polynucleotide drastically reduced the rate of hydrolysis of poly U by RNase. It was observed that RNase could not split the phosphodiester bond on the 3 -OH end of uridylic acid dimer. It was also shown that dehydration of irradiated poly U was accompanied by marked phosphodiester bond breakage and degradation of the polynucleotide. 

Figure 10.3 Enzymatic synthesis of poly(adenylic acid) in self-reproducing oleate liposomes (redrawn from Walde et al., 1994a). (a) The ADP penetrates (sluggishly) the liposome bilayer, (b) in the presence of polynucleotide phosphorylase, ADP is converted in poly(A), which remains entrapped in the liposome, (c) Polycondensation of ADP goes on simultaneously with the self-reproduction of liposomes (A is the membrane precursor, oleic anhydride, which, once added, induces the self-reproduction of liposomes S, surfactant, in this case oleate, which is the hydrolysis product of A on the bilayer E is polynucleotide phosphorylase).

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

The discovery of a small proportion of a nucleoside containing thymine42 in the ribonucleic acid of two strains of Escherichia coli, in Aerobacter aero-genes, and in commercial, yeast-ribonucleic acid emphasizes the point made previously,26-28 namely, that the nucleic acids may contain constituents other than those heretofore identified. Alkaline hydrolysis of the ribonucleic acid from E. coli gave nucleotides42 (probably the 2- and 3-phosphate esters) which were converted to the nucleoside with prostatic phospho-monoesterase.62 Enzymic hydrolysis of the nucleic acid preparation also led to the nucleoside, which was degraded further to thymine by hydrolysis with perchloric acid.42 There can be little doubt that this carbohydrate derivative of thymine is intimately bound as part of the polynucleotide chain of this particular ribonucleic acid. 

The possibilities of N-(dialkylphosphoryl)amino acids for the prebiotic syntheses of peptides and polynucleotides have been studied in a series of papers [24,116-122], However, it must be emphasized that the phosphoryl group does not behave as an amino-activating group, the hydrolysis of which would be coupled to peptide bond formation. Actually, further peptide elongation requires the subsequent hydrolysis of the N-terminal phosphoryl group of the ligated product. In the presence of an amino acid ester, dipeptide esters 16 with an unreacted N-phosphoryl protection are formed, support-... 

Growing membrane systems have been used to obtain artificial infrabiological systems. Walde et al. [47] have carried out the synthesis of polyadenylic acid in self-reproducing vesicles [48], in which the enzyme polynucleotide phosphorylase carried out the synthesis of poly-A, and membrane vesicle multiplication was due to the hydrolysis of externally provided oleic anhydride to oleic acid. The snag is that the enzyme component is not auto-catalytic. Enzymatic RNA replication in vesicles [49] suffers from the same problem. It is also not known whether redistribution of the entrapped enzymes into newly formed vesicles occurs or not. An affirmative answer would be evidence for vesicle reproduction by fission. 

In a given phosphodiester bond, hydrolytic enzymatic cleavage can occur at two locations, indicated by p and d in Figure 10.16. The former is proximal with respect to the 3 -OH group the latter is distal with respect to the 3 -OH. Enzymes that catalyze the hydrolysis of nucleic acids are nucleases (see Table 10.2). Exonucleases remove nucleotides (or nucleosides) from either the 5 or the 3 end of the polynucleotide. These are specific for either the p or the d bond. Thus, an exo-... 

The general scheme for the degradation of nucleic acids has much in common with that of proteins. Nucleotides are produced by hydrolysis of both dietary and endogenous nucleic acids. The endogenous (cellular) polynucleotides are broken down in lysosomes. DNA is not normally turned over rapidly, except after cell death and during DNA repair. RNA is turned over in much the same way as protein. The enzymes involved are the nucleases deoxyribonucleases and ribonucleases hydrolyze DNA and RNA, respectively, to oligonucleotides which can be further hydrolyzed (Fig. 15-18), so eventually purines and pyrimidines are formed. 

In the previous chapters the reactivity of metal ions with the monomer units of nucleic acids has been discussed. This section will deal with the binding of transition metals to the polynucleotides. There are also three types of complexes to be expected the metal-ring, the intermediate and the metal chain complex. The effect of the ribose or deoxyribose residue on the stability constants can be neglected since the reactivity of these sugars with cations is extremely low. However, as it will be seen later, the hydrolysis of polyribonucleotides is markedly facilitated by interaction of metal ions with the 2 —OH groups of the ribose. 

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