[Purification and various properties of exonuclease from bacteriophage T4]

Exonuclease A was isolated from bacteriophage T4-infected cells of E. coli. The molecular mass of the enzyme is approximately 42,000 Da, pH optimum is 7-8.5, pI is 4.05. The enzyme activity depends on Mg2+, the optimal concentration of Mg2+ being 1-5 mM. The enzyme splits one- and two-helical DNA in the direction of 3'----5' and is a deoxyribonuclease splitting 5'-deoxynucleotides. The enzyme shows a practically equal affinity for one and two-helical DNA. The Km value for one- and two-helical DNA is 10 +/- 1 and 11 +/- 1 pmole of chain DNA, respectively. The Vmax value for one- and two-helical DNA is 61 +/- 5 and 45 +/- 5 pmole of nucleotides per min. Exonuclease A may be used for preparing substrates for DNA-polymerase T4 and Klenow fragment, i.e., during labeling of DNA at 3'-ends.