Structural and enzymological characterization of deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

Abstract

The single DNA-dependent ATPase activity that was identified in extracts of [human oral epidermoid carcinoma] KB cell nuclei was purified to near homogeneity. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75,000 daltons; this was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI [isoelectric point] of 8.5, a Stokes'' radius by gel filtration of 3.8 nm and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 .times. 105 nmol of ATP hydrolyzed/h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing and gyrase activities at exclusion limits of 10-6-10-8 of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs [nucleotide triphosphates] and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ > Mn2+ > Ca2+) and a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values .apprx. 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses, and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA, but not to intact duplex DNA. Although direct demonstration of DNA unwinding by this protein was unsuccessful, preliminary results suggest that, in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases .alpha. and .beta. on nicked duplex DNA substrates.