Nuclease single-strand specificity

Vogt, V.M. (1973) Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur. /. Biochem., 33, 192. 

For hybridization studies, actinomycin D is nonnaUy included in the reaction mixture in order to prevent the synthesis of a second strand of DNA. However, an Isolated cDNA can readily be converted into a double stranded product by Escherichia coli DNA polymerase. It appears that many cDNAs are trans scribed with short double stranded hairpins at the 3 -OH termini and these are capable of acting as primers for the synthesis of the second strand. The products of the reaction still possess a loop at one end, but since this is single stranded it can be cleaved with the single strand-specific nuclease SI to yield a double stranded DNA. Such double stranded cDNAs, with hmno-oligonucleo-tide 3 - termini, are suitable for cloning by in vitro recomtnnation with a plasmid vector (see p. 195 Figure 3). 

The denaturation/renaturation process can be quantitatively monitored by a number of methods (12,13). The most commonly used ones include (i) binding to hydroxyapatite (only double strands bind in 0.12—0.14 M Na—P buffer), (ii) resistance (or sensitivity) to single strand-specific nucleases, and (iii) optical hyper-chromicity (the breakage of H-bonds and base unstacking result in a 30% increase in UV absorbance at 260 nm). 

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... 

The ability to discriminate between double-stranded and single-stranded regions of a polynucleotide makes the single-strand-specific endonucleases invaluable in fine structural analysis of nucleic acids. This property also makes the single-strand-specific nucleases an important tool in various manipulations of DNA and RNA in recombinant DNA work. 

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... 

Mung bean nuclease, abbreviated here as nuclease MB, is a single-strand-specific endonuclease similar to nuclease SI. Nuclease MB is a glycoprotein with a similar molecular size (39 IcDa) and the functional properties of the two enzymes are also similar. For example, they produce 5 -mononucleotide and 5 -oligonucleotide products. For most practical purposes, nuclease MB can be used interchangeably with nuclease SI. 

The optimal pH for nuclease MB lies between pH 4.7 and 5.3, and is dependent on the NaAc concentration (1). Although the enzyme is less active at higher pH values, reaction conditions at neutral pH (7.0-7.5) may be more useful and even necessary in some applications. At neutral pH, the single-strand specificity of the enzyme, in terms of the relative cleavage rate of supercoiled versus relaxed phage PM2 DNA, increases substantially over that at low pH conditions. 

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). 

Fang, N. Method for detecting and/or quantifying poly(A) RNA and mRNA by hybridization with poly(dX) and digestion with single strand-specific nuclease. Ger. Offen. DE 102009056729, 2011. 

Chen, Y.-T., C.-L. Hsu, and S.-Y. Hou, Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases. Analytical Biochemistry, 2008 375(2) 299-305. 

Several zinc enzymes that catalyse the hydrolysis of phosphoesters have catalytic sites, which contain three metal ions in close proximity (3-7 A from each other). These include (Figure 12.11) alkaline phosphatase, phospholipase C and nuclease PI. In phospholipase C and nuclease PI, which hydrolyse phosphatidylcholine and single-stranded RNA (or DNA), respectively, all three metal ions are Zn2+. However, the third Zn2+ ion is not directly associated with the dizinc unit. In phospholipase C, the Zn-Zn distance in the dizinc centre is 3.3 A, whereas the third Zn is 4.7 and 6.0 A from the other two Zn2+ ions. All three Zn2+ ions are penta-coordinate. Alkaline phosphatase, which is a non-specific phos-phomonoesterase, shows structural similarity to phospholipase C and PI nuclease however,... 

Attempts are being made to design semisynthetic restriction endonucleases specific for single-stranded DNA or RNA. For example, an oligonucleotide with a sequence complementary to a sequence adjacent the linkage that is to be cut can be covalently linked to a relatively nonspecific nuclease. Such an enzyme derived from micrococal nuclease cuts a single-stranded chain of either DNA or RNA adjacent to the double-stranded region of the ES complex.839... 

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