Further purification and characterization of T4 endonuclease V

Abstract

T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.