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

An endonuclease with DNA single-strand specificity has been 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 molecular weight 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 eightfold greater than that observed with denatured DNA, although the Km for both substrates is 1.74 X 10(-5) M. The relative rates of hydrolysis of homopolymers by the endonuclease are: poly(dG) greater than poly(dT) greater than poly(dA) greater than 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.