Purification and characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA

Publisher Website
Google Scholar
Abstract
There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529,-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5''-O-(3-thiotriphosphate) (ATP-.gamma.-S), 5''-adenylyl methylenediphosphate (AMP-PCP), and 5''-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oliogomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ .simeq. Mn2+ > Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).cntdot.poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay. In the presence of 0.15 M KCl, almost all of the enzyme applied to the column was coeluted with single-stranded circular fd DNA, while in the presence of 1 mM ATP under the same salt condition the amount of the enzyme coeluted with DNA decreased greatly. The effect dissociation of the enzyme from the single-stranded DNA was not observed with other ribonucleoside triphosphates, CTP, GTP and UTP.