Nuclease cleavage preferences

Ribonuclease II [EC 3.1.13.1], also called exoribo-nuclease II, catalyzes the exonucleolytic cleavage of the polynucleic acid, preferring single-stranded RNA, in the 3 - to 5 -direction to yield 5 -phosphomononucleotides. The enzyme processes 3 -terminal extra-nucleotides of monomeric tRNA precursors, following the action of ribonuclease P. Similar enzymes include RNase Q, RNase BN, RNase PHI, and RNase Y. Ribonuclease T2 [EC 3.1.27.1] is also known as ribonuclease II. 

In vivo, cleavage of P-0 bonds are performed by enzymes such as phosphatases, phosphodiesterases, phosphohydrolases, nucleases, DNases and RNases (see Section 13.1.1). In vitro, cleavage of a P- O bond is often a trivial synthetic step. Even for an easy step, enzymes attract increasing attention. The enzymatic reactions are preferred when regio- or stereoselectivity is required, and when the substrates are temperature or pH sensitive. Many phosphate analogs have been tested as substrates of enzymes that hydrolyze phosphoryl groups. These analogs are often accepted as substrates for the enzymes, and such reactions could be synthetically valuable. Typical examples are presented in the tables. 

Endo- and exonucleases have been used successfully with nucleic acids and their analogs for organic synthetic purposes. For example, ATP was synthesized from AMP for use in cofactor recycling (Table 13-9, entry 1). The AMP was obtained from yeast RNA by cleavage with the nuclease Pi yielding a mixture of nucleoside monophosphates11011. In another report1731, nucleoside diphosphates were obtained by hydrolysis of RNA with nuclease Pi and a polynucleotide phosphorylase (the diphosphates are preferred because the diphosphates were more easily transformed to the nucleoside triphosphates than the monophosphates). 

RNA is also a substrate for the nuclease activity of Cu(phen)2. The copper complex shows a preference for single-stranded loops relative to double-stranded A-structures present in RNA, but the detailed mechanism of cleavage is still unknown (33). 

Since the residues that carry out an enzyme s catalyic function reside in the active site, and since Serratia nuclease and l-Ppol nuclease share much the same active site makeup, it would be reasonable to conclude that they would share many functional properties in common. As noted, they do share the same sissicile bond, the 3 0-P bond, and the same preference for cleaving A form DNA as indicated by a preference for cleavage in GC rich regions by Serratia nuclease. On the other hand they have many differences as well. One nuclease cleaves both DNA and RNA in double and single stiranded forms the other cleaves only a very large (15 bp) and specific DNA site. The Serratia enzyme is extremely fast, 15-foId faster than DNase I, while VPpol has a modest kcat value. 

This system has also been shown to be dependent on the secondary structure of DNA, the A, B, and Z forms reacting at different rates [150]. The likely explanation is that the faster reacting B DNA forms a more stable complex with the catalyst. This artificial DNase activity has also been compared with cleavage by micrococcal nuclease, and shown to recognize the same sites but not all those cleaved by DNase 1, again implying some local conformational preferences [151]. Chromatin structure has also been probed [152]. 

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