Micrococcal nuclease substrates

Among endonucleases which hydrolyze DNA one seldom finds an enzyme that attacks double-stranded and single-stranded substrates with equal ease. If the enzyme shows preference for double-stranded substrates (as DNase I does) autoretardation is observed. This decrease in the reaction rate is caused by the gradual disappearance of the preferred, double-stranded substrate and an increase in the concentration of less susceptible, single-stranded substrate. Differences in rates between the early and terminal phases of the reaction of the order of 1000-fold have been described (< ). The opposite case, autoacceleration, is seen with those enzymes that show preference for the single-stranded structure, e.g., micrococcal nuclease (7). 

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. 

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.

Additional information <1, 8> (<1> protection against heat inactivation by substrates, e.g. calf thymus DNA and ribosomal RNA, both micrococcal nuclease-treated, and 3 -AMP [3] <8> protection against heat inactivation by ATP or 5 -OH-DNA, not 5 -phospho-DNA, at 0.1-0.15 M NaCl [4]) [3, 4]... 

That being so, some of the common nucleases which degrade ordinary polynucleotides could well have evolved to interact primarily with polynucleotides whose nucleoside components are anti. It is of interest, in this regard, that the diesterases from spleen and snake venom, and micrococcal nuclease, do not digest poly F, which is syn also that the specificity of pancreatic RNase for the natural substrates - uridine and cytidine - is based on their normal anti conformation. 

Double-stranded DNAs with 3 -P termini (e.g., the digestion products of micrococcal nuclease or calf spleen nuclease) are substrates for Exo III (1). The phosphatase activity releases inorganic Pj. In contrast, DNAs with 5 -P termini (e.g., the digestion products of E. coli endonuclease) are not substrates. Mononucleotides with 3 -P or 5 -P and small (or acid-soluble) 3 -P oligonucleotides such as d(TTT)p are not substrates. Exo III can also hydrolyze 3 -P glycolate (-O-PO2-OCH2-COOH) to 3 -OH in gamma-irradiated DNAs (23). 

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