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Endonuclease II of Escherichia coli

II. ENZYME PROPERTIES AND STUDIES ON THE DEGRADATION OF ALKYLATED AND NATIVE DEOXYRIBONUCLEIC ACID

From the ¹ From the Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106

Studies on the degradation of DNA, alkylated with methylmethanesulfonate, by endonuclease II of Escherichia coli are presented. With a DNA gel assay, some of the kinetics of degradation have been examined. With extensively alkylated DNA, only a limited number of double-strand breaks were made. Activity was maximum over a pH range from 8.0 to 9.0. No absolute requirement for added divalent metals was observed. The rate of the reaction was stimulated by added Mg²⁺ or Mn²⁺, but was not inhibited by 8-hydroxyquinoline or several other chelating agents. An exception was ethylenediaminetetraacetic acid. Sulfhydryl reagents and high ionic strength inhibited the enzyme while transfer RNA and caffeine did not. An approximate sedimentation coefficient was 3.6. With lightly alkylated DNA, the enzyme made predominantly single-stranded breaks, and the ratio of single-to double-strand breaks was approximately 3.7:1. It is concluded that the enzyme can hydrolyze a phosphodiester bond near an alkylated base in a native DNA molecule which has no single-strand breaks in this region. With more extensively alkylated DNA, viscosity experiments, which measure double-strand breaks, give an n value of approximately 1.

The activity of endonuclease II of E. coli on several species of native nonalkylated DNA has also been studied. The enzyme makes a limited number of single-strand breaks, with T4 DNA, approximately 3 to 4 per single-strand. Several factors which affect the reaction of the purified enzyme with alkylated DNA also affect the reaction with nonalkylated DNA. These are no absolute metal requirement, normal enzyme activity with 8-hydroxyquinoline but enzyme inhibition by EDTA, and no inhibition by tRNA. There was no induction of an enzyme with properties of endonuclease II after infection with T4.

Enzyme activity was present in a series of mutants deficient in the ultraviolet repair process, in recombination, in DNA synthesis at elevated temperatures, and in host cell restriction as well as in a strain sensitive to methylmethanesulfonate.

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