Phage nucleases

Disposable gloves should be worn when you are handling the enzyme container. Remove the enzyme from the freezer just before you need it. Store the enzyme in an ice bucket when it is outside the freezer. The enzyme should never be stored at room temperature. Because of high cost, digestion by restriction enzymes is carried out on a microscale level. A typical reaction mixture will contain about 1 fig or less of DNA and 1 unit of enzyme in the appropriate incubation buffer. One unit is the amount of enzyme that will degrade 1 fig of A. phage DNA in 1 hour at the optimal temperature and pH. The total reaction volume is usually between 20 and 50 fiL. Incubation is most often carried out at the recommended temperature for about 1 hour. The reaction is stopped by adding EDTA solution, which complexes divalent metal ions essential for nuclease activity. 

Martin-Bertram 1981,1982 Martin-Bertram et al. 1983 for DNA isolated from y-irradiated yeast cells see Andrews et al. 1984). While such lesions, detected by SI nuclease ( Sl-nuclease-sensitive sites ), are not observed with A phage DNA y-irradiated in aqueous solutions (Martin-Bertram et al. 1984), they become prominent when the solutions is highly scavenged (Junker et al. 1984). Under such conditions, the damage caused by the direct effect plays a role because the indirect effect is largely suppressed (Ward 1981). 

Light J, Lerner RA, Random mutagenesis of staphylococcal nuclease and phage display selection, Bioorg. Med. Chem., 3 955-967, 1995. 

Fig. 5.12. Strategy for the selection of phage-displayed staphylococcal nuclease (SNase) with a DNA substrate derivative featuring a biotin (Bt) on one end and a basic peptide on the other [65], The substrate is attached to the phage via an acidic peptide and the phage is captured with immobilised streptavidin (Sv).

The 3 phosphorothioate residues, introduced by the phosphoramidite method, were shown to significantly protect the oligomer from the action of nucleases. This hybrid, in which the ribonucleotide portion is complementary to the leader sequence of phage fl coat protein mRNA, was used to study the formation of the initiation complex in prokaryotic translation. 

A. Phage Cro protein has been ctmverted into an operator-specific nuclease by replacing the C-terminal alanine of the wild type protein with cysteine. Alkylation of the sulphydryl group with S-(iodoacetamido)-i,10-phenanthroline resulted in a semisynthetic nuclease which... 

We have mentioned how DNA polymerase I from Escherichia coli extends the DNA chain, but how does it initiate the chain A clue to the mechanism was obtained when it was observed that rifampicin, an inhibitor of RNA polymerase, also inhibits the conversion of a phage single stranded into a double stranded DNA circle. This suggested that DNA synthesis is initiated by RNA priming. It is now established that the initiation of DNA synthesis by DNA polymerase I requires RNA and DNA polymerase, ribose and deoxyribose triphosphates, an unwinding protein and other factors whose roles are still unclear. The initiation step seems to involve a transcriptional operation by RNA polymerase. The newly synthesized RNA serves as primer for DNA polymerase I, yielding an RNA-DNA complex and the primary RNA is excised by a nuclease [236, 237]. 

Whereas the structure of mRNA determines its susceptibility to degradative enzymes, the detailed mechanisms are complex. In prokaryotes, the enzymes involved include two endonucleases (RNase E and RNase III) and two exonucleases (polynucleotide phosphorylase and RNase II). Other nucleases may be active in particular cases such as phage infection. In eukaryotes, a major pathway involves removal of the 3 poly(A) tail (deadenylation), followed by removal of the 5 cap, which renders the mRNA susceptible to rapid endonucleolytic degradation in the 5 3 ... 

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. 

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