Calf spleen phosphodiesterase

Two diastereoisomers have been identified as radiation induced decomposition products of dC. The synthesis of one of these isomers, (5 5,55,6S)-5, 6-cyclo-5-hydroxy-5,6-dihydro-2 -deoxyuridine (81), and its incorporation into DNA, has been reported The presence of the lesion causes a marked destabilisation of DNA duplexes. The nucleases PI, calf spleen phosphodiesterase and bovine intestinal mucosa phosphodiesterase failed to cleave the lesion, nor was it a substrate for a number of repair enzymes, and it acted as a block to Klenow fragment and Taq polymerases. 

C. M. Bentzley, M. V. Johnston, and B. S. Larsen, Base specificity of oligonucleotide digestion by calf spleen phosphodiesterase with matrix-assisted laser desorption ionization analysis, AwaZ. Biochem. 258, 31-37 (1998). 

Figure 11.18 shows the mass spectrum of the 5 -exonuclease degradation of a DNA 12-mer 5 -d(GCTTXCTCGAGT)-3 digested with calf spleen phosphodiesterase (CSP). Five peaks are observed, which (if the sequence was unknown)... 

Monomers [180]-15a-d were successfully used for the synthesis of several stereodefined oligo(nucleoside [180]phosphorothioate)s. Their stereochemistry and isotopic enrichment were confirmed by a combined enzymatic-mass spectrometry method employing snake venom phosphodiesterase or calf spleen nuclease, and MALDI TOF mass spectrometry. 

The incorporation of the 5R and 5S diastereoisomers of the 5-hydroxyhydan-toin (derived from oxidative damage of dC) derivative (89) has been achieved using phosphoramidite chemistry and mild deprotection. Whilst the ODNs containing either diastereoisomer were digested with nuclease Pi, the hydantoin derivatives were resistant to digestion by calf spleen and bovine intestinal mucosa phosphodiesterases. 

The boranophosphate internucleotide linkage in dimer 2 is quite stable toward cleavage by exonucleases. Enzymatic hydrolysis studies were performed on TpT in vitro (normal dinucleotide), and a mixture of R and S boronated diastereomers of TpBT 2 to ascertain the in vitro exonuclease resistance of the intemucleotide linkage (12). Under conditions where normal dithymidylyl phosphate is >97% cleaved, dimer 2 is >92% stable to both calf spleen and snake venom phosphodiesterase. The boronation confers considerable resistance to these two particular exonucleases. The extent of protection shows that both the R and S chiral forms exhibit nuclease resistance. These experiments are now being repeated for the R and S stereoisomers of the dimer, which we have separated by HPLC. In summary, the P—BH3 group possesses sufficient hydrolytic stability to survive use in biological systems. 

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