Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase. 3′- or 5′-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10^-5 to 10^-7 relative to the ATPase activity. The enzyme is a monomer of M_r 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 Å and a frictional coefficient of 1.32. In the presence of Mg²⁺ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The K_m for dsDNA or ssDNA is 2.2 μM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.