ABSTRACT
Deoxyribonuclease 4 from Aspergillus nidulans was purified to over 70% homogeneity. It contains a polypeptide of Mr about 30000, and behaves as a dimer, but with some evidence of dissociation on gel filtration and ultracentrifugation. The pH optimum is 7-9. Activity is supported by metal ions in the order (Mn2+ + Ca2+) greater than Mn2+ approximately equal to (Mg2+ + Ca2+) much greater than Mg2+. Mn2+ is optimal at 10-20 mM. DNAase 4 strongly prefers native DNA, for which the Km is about 0.5 mM, and on which it acts as an endonuclease. The specific activity is about 2000 mumol of nucleotide made acid-soluble in 30 min at 37 degrees C per mg of protein. Action on denatured DNA, which has a lower optimal Mn2+ concentration and a different time course from its action on native DNA, may be due to partial renaturation of the DNA used. It has no action on RNA. With native DNA the enzyme gave mainly, or entirely, double-strand cleavages by a single-hit mechanism, with either Mn2+ or Mg2+. The enzyme has no strongly preferred sequences. Action stops, or becomes very slow, when 50-60% acid-solubility has been reached. In a near-limit digest, mononucleotides were absent, dinucleotides to at least heptanucleotides occurred in similar weight yields, there was an excess of chains of 10-11 (or rather longer) and a rapid decline at greater lengths. Products have 3'-OH, 5'-P termini. The products and kinetics can be understood in terms of the enzyme's causing non-staggered double-strand cleavages randomly in DNA but subject to a requirement for at least two base pairs at one side of the cleavage site and at least 10 (or perhaps rather more) base pairs on the other side of the site.
Subject(s)
Aspergillus nidulans/enzymology , Deoxyribonucleases/metabolism , Cations, Divalent , Centrifugation, Density Gradient , DNA/metabolism , Deoxyribonucleases/isolation & purification , Hydrogen-Ion Concentration , Kinetics , Macromolecular Substances , Nucleic Acid Denaturation , Oligonucleotides/metabolism , Substrate SpecificitySubject(s)
Deoxyribonucleotides , Iron , Chemical Phenomena , Chemistry , DNA , Hydrogen-Ion Concentration , KineticsABSTRACT
Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methanesulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methanesulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis. In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism. The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.
