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5'-P-forming exonuclease from Actinomyces sp. of coelicolor group, strain No. 5 (exonuclease A 5). Purification and some properties.

A new exonuclease named exonuclease A5 has been isolated from the culture medium of Actinomyces sp. of the coelicolor group, strain No. 5 (classification after Krassilnikov). The enzyme exists in the culture broth, as a complex with some other component(s), from which it can be obtained in a free state by non-ionogenic detergent OP-7 treatment. Its purification procedure consists of the gel-filtration of the salted out protein on Sephadex G-75 and the double chromatography on DEAE-Sephadex A-50 at two different pH values. The specific activity of the purified enzyme reaches as a rule 2000 units per mg of protein. The enzyme is free of nucleosidase and deaminase. The nuclease/phosphatase ratio of the best preparations corresponds to 2000—4000. The exonuclease A5 splits RNA and denatured DNA into 5′-mononucleotides and does not hydrolyze bis-p-nitrophenylphosphate. The cleavage begins from the 3′-end of the polynucleotide chain. The enzyme does not discriminate many modified nucleotides. It hydrolyses T-even phage DNA as well as tRNA, native or chemically modified. Polynucleotide chains containing no modified bases are split more rapidly however. 
 
A peculiarity of the enzyme is its small sensitivity to the 3′-terminal P. An oligonucleotide with 3′-terminal P is split with the enzyme into mononucleosidediphosphate (3′-end), dinucleosidemonophosphate (5′-end) and 5′-mononucleotides. Thus the nucleotide sequence of tri- and tetranucleotides can be readily established. Dinucleosidemonophosphates are split only with large enzyme amounts. Dinucleotides with 3′-terminal P are resistant to its attack. The oligonucleotide sequence bearing 5′-terminal P can be completely split into 5′-mononucleotides, the last internucleotide bond at the 5′-end being easily attacked. 
 
The above mentioned properties of exonuclease A5 will probably make it useful for determining the nucleotide composition of polynucleotides containing unusual bases, as well as for end analysis of nucleic acid fragments.