Wong I, Moore K J, Bjornson K P, Hsieh J, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1996 May 7;35(18):5726-34. doi: 10.1021/bi952959i.
The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.
大肠杆菌Rep解旋酶利用ATP结合和水解所产生的能量催化双链DNA解旋。Rep以二聚体形式发挥作用，但只有在结合DNA时才组装成其活性二聚体形式。二聚体的每个亚基都含有一个可结合单链（S）或双链（D）DNA的DNA结合位点。该二聚体在五种DNA连接状态下最多可结合两个寡脱氧核苷酸：两种半连接状态，P2S和P2D，以及三种完全连接状态，P2S2、P2D2和P2SD。我们之前已经表明，这些连接状态的相对稳定性受到ATP结合和水解的变构调节，并提出了一种DNA解旋的“主动滚动”模型，即酶在与ATP水解催化循环偶联的过程中通过一系列这些连接状态循环[Wong, I., & Lohman, T.M., (1992), Science 256, 350 - 355]。Rep蛋白的基础ATP酶活性受到单链DNA结合和蛋白质二聚化的刺激。我们测量了处于每种不同单链DNA连接状态（PS、P2S和P2S2）下与dT(pT)15结合的Rep的稳态ATP酶活性，以便与我们之前对未连接的Rep单体（P）的测量结果进行比较[Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]。我们发现Rep的ATP酶活性受到二聚化和单链DNA连接状态的显著影响，ATP水解的以下催化常数（kcat）值增加了超过4个数量级：P为2.1×10⁻³ s⁻¹，PS为2.17±0.04 s⁻¹，P2S为16.5±0.2 s⁻¹，P2S2为71±2.5 s⁻¹（20 mM Tris - HCl，pH 7.5，6 mM NaCl，5 mM MgCl₂，10%甘油，4℃）。ATP水解的表观米氏常数（KM）对于PS为2.05±0.1 μM，对于P2S为2.7±0.2 μM。这些差异极大的ATP酶活性反映了DNA连接的变构效应，并表明Rep二聚体两个亚基的ATP和DNA位点之间发生了协同通讯。这些结果进一步强调，在仅基于宏观稳态ATP酶测量试图推断诸如解旋酶易位等基本过程的信息时，需要明确考虑解旋酶寡聚化和DNA连接状态的群体分布。
