Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa

Abstract

An endonuclease specific for single-stranded DNA and RNA was purified from Neurospora crassa conidia by the method of Linn and Lehman (J. Biol. Chem. 240, 1287 (1965)). The activity of the enzyme was measured by following the rate of release of acid-soluble material at 37 °C from either denatured (single-stranded) or native DNA. After an initial lag period, the length of which was inversely proportional to enzyme concentration, the release of acid-soluble material occurred at a rate which was directly proportional to enzyme concentration. Freshly purified enzyme catalyzed the release of acid-soluble material from denatured DNA 50–70 times faster than from native DNA. This ratio of activities increased to approximately 200 following storage at 0–4 °C. Throughout this "ageing" period 2 × 10−4 M ATP inhibited the activity of the enzyme toward both denatured and native DNA by 50%. The following nucleoside phosphates, at a concentration of 4 × 10−4 M, had no effect on the activity of the endonuclease toward denatured DNA: ADP, AMP, ITP, XTP, CTP, dTTP, and UTP. GTP at 4 × 10−4 M inhibited this activity by 15%. Both ATP and dATP at 1 × 10−4 M inhibited endonuclease activity against denatured DNA by about 25%. The inhibition by ATP was noncompetitive over the range of concentration 0.11–0.75 mg DNA per milliliter. Specific inhibition of nuclease activity by ATP has not been previously reported.Several other properties of the endonuclease were examined. There is evidence that endonuclease action was inhibited by accumulation of the products of digestion. The enzyme lost activity toward denatured DNA in a medium containing 0.3 M NaCl but activity was partially restored in the presence of 4 × 10−3 M mercaptoethanol. The endonuclease did not digest DNA–RNA hybrid to any appreciable extent.