Properties of an Endonuclease Activity in Micrococcus Luteus Acting on γ-irradiated DNA and on Apurinic DNA

Summary

A protein fraction from Micrococcus luteus with endonuclease activity against γ-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25,000 was calculated for both endonuclease activities.

The endonuclease activity against γ-irradiated DNA was stimulated five-fold with 5 mM Mg⁺⁺, whereas that against apurinic sites was less dependent on the Mg⁺⁺ concentration. 100 mM KCl inhibited the γ-ray endonuclease, but not the apurinic endonuclease activity. In γ-irradiated DNA the protein recognized 1·65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0·45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a K_m-value of 73 nM.