Boxer L M, Korn D
Biochemistry. 1980 Jun 10;19(12):2623-33. doi: 10.1021/bi00553a014.
We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that 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 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 X 10(5) nmol of ATP hydrolyzed per 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 and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as 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 approximately 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 we have not succeeded directly in demonstrating DNA unwinding by this protein, 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.
我们已将在KB细胞核提取物中鉴定出的单一依赖DNA的ATP酶活性纯化至接近同质状态。该酶的蛋白质结构通过十二烷基硫酸钠凝胶电泳确定，结果显示有一条75000道尔顿的单一蛋白带，它与通过琼脂糖 - ATP色谱法的最后一步或等电聚焦解析的ATP酶活性图谱一致。该酶的等电点为8.5，通过凝胶过滤测得的斯托克斯半径为3.8纳米，在高盐条件下的沉降系数为5.3 S。在低离子强度下，酶活性的沉降系数为7.0 S，这表明它在这些条件下可能会二聚化。纯化后的酶比活性为每小时每毫克蛋白质水解5.9×10⁵纳摩尔ATP，在ATP酶活性的10⁻⁶ - 10⁻⁸排除极限下，它没有内切核酸酶、外切核酸酶、RNA或DNA聚合酶、切口封闭酶和促旋酶活性。该酶只能水解ATP或dATP，生成ADP或dADP加磷酸，但其他NTP和dNTP是该酶相对于ATP的竞争性抑制剂。酶活性需要二价阳离子（Mg²⁺＞Mn²⁺＞Ca²⁺）以及核酸辅因子。单链DNA或脱氧同聚物最有效，但平端线性和带切口的环状双链DNA分子也可被利用，其最大反应速度值约为单链DNA的20%。完整的双链DNA和多聚核糖核苷酸不能支持ATP水解。速度梯度沉降研究证实了动力学分析的结果，并表明该酶与单链DNA和带切口的双链DNA结合，但不与完整的双链DNA结合。尽管我们尚未直接成功证明该蛋白能解开DNA，但初步结果表明，在ATP存在的情况下，该ATP酶可刺激人源α和β DNA聚合酶对带切口的双链DNA底物的反应活性。
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