Purification and characterization of a DNA single strand specific endonuclease from human cells

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Abstract
An endonuclease with DNA single-strand specificity was purified from KB cells. The enzyme has a pH optimum at 9.2, requires Mg2+ for activity and is inhibited by mono- or divalent cations. Its sedimentation coefficient of 4.6 S is based on sucrose gradient sedimentation, and it has a MW of 54,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme specifically catalyzes the endonucleolytic cleavage of denatured DNA, yielding acid-soluble oligonucleotides which contain 5''-phosphoryl termini. The rate of hydrolysis of poly(dT) is approximately 8-fold greater than that observed with denatured DNA, although the Km for both substrates is 1.74 .times. 10-5 M. The relative rates of hydrolysis of homopolymers by the endonuclease are: poly(dG) > poly(dT) > poly(dA) > Poly(dC). Purified enzyme preparations also hydrolyze poly(U), releasing acid-soluble products. This activity cosediments in sucrose gradients with the DNA endonuclease activity, suggesting that both activities are contained in the same enzyme molecule.