Further characterization of deoxyribonucleases from vaccinia virus

Beatriz G.T. Pogo, Michael T. O'Shea

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9 Scopus citations

Abstract

Further characterization was made of DNases present in vaccinia virus. The nature of the enzymes as they occur within cores and following solubilization was determined. Two activities were identified hydrolyzing single-stranded (ss) DNA but at exclusively a pH optimum of 4.5 or 7.8, respectively. Neither activity has any requirement for ions or cofactors. The pH 7.8 enzyme was activated preferentially by heating cores to 50°. Analysis of the products of hydrolysis by means of DEAE-paper chromatography confirmed that the pH 4.5 activity was an exonuclease and the pH 7.8 enzyme an endonuclease. The exonuclease could act on the 5′-terminus of the DNA. Both nucleases could hydrolyze poly(dT), poly(dA), and poly(dC) to a varying degree but had no effect on poly(dG). Since the oligonucleotides arising as a product of endonuclease action did not serve as a substrate for the exonuclease, it is concluded that the two enzymes probably do not act in concert. Solubilization of both DNases was achieved by treatment of cores with salts or urea. With 0.5 M NaCl, most of the pH 4.5 activity but only 10-20% of the pH 7.8 was released. The presence of 6-8 M urea caused the solubilization of both enzymes. When in their soluble state, the nucleases could be separated by means of isoelectric focusing in either gel or liquid milieu and retain the exo- or endonucleolytic activities. The pH 4.5 DNase had an isoelectric point or pI of approximately 4.5, and the pH 7.8 DNase had a pI of approximately 3.7. Each activity was contained in a single protein. Further analysis of the isolated enzymes, using sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis, revealed each to be a polypeptide of MW 50,000. Taken together, the evidence indicates that vaccinia cores contain two DNases with independent modes of action associated with separate proteins of similar molecular weights but different pIs.

Original languageEnglish
Pages (from-to)56-66
Number of pages11
JournalVirology
Volume77
Issue number1
DOIs
StatePublished - Mar 1977
Externally publishedYes

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