ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.
ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.
复制标题
大肠杆菌 Rep 解旋酶的 ATP 酶活性很大程度上取决于 DNA 连接和蛋白质寡聚状态。
DOI：
10.1021/bi952959i
复制
发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
Lohman,TM
中科院分区：
文献类型：
--
作者：
Wong,I;Moore,KJ;Bjornson,KP;Hsieh,J;Lohman,TM
E.coliRep解旋酶利用ATP结合和水解产生的能量催化双链DNA的解离。Rep具有二聚体的功能，但只有在结合DNA时才能组装成活性的二聚体。二聚体的每个原型都包含一个dna结合位点，可以与单链(S)或双链(D)dna结合。该二聚体可以在五种连接状态下与两个寡核苷酸结合： ，两个半连接状态，P2S和P2D，以及三个完全连接状态，P2S2，P2D2，和P2SD。我们以前已经证明了这些连接状态的相对稳定性受三磷酸腺苷的结合和水解的变构调节，并提出了一个DNA解离的“主动滚动”模型，其中酶在与三磷酸腺苷水解的催化循环相耦合的过程中通过一系列这些连接状态进行循环[Wong，I.，&Lohman，T.M.，(1992)Science 256,350−355]。Rep蛋白的基础ATPase活性受ss DNA结合和蛋白质二聚体的刺激。我们测量了在每种不同的ss DNA连接状态(PS、P2S和P2S2)下与DT(PT)15结合的Rep的稳态ATPase活性，以与我们以前用未连接的Rep单体(P)[Moore，K.J.M.，&Lohman，T.M.(1994)BioChemical 33,14550]的测量结果进行比较。结果表明，二聚化和SSDNA连接状态对酶活性均有显著影响，酶活性增加了4个数量级以上：P为2.1×10-3s-1，PS为2.17±0.0 4 S-1，P2S为16.5±0.2 S-1，P2S为71±2.5 S-1(20 mM Tris-HCl，pH 7.5，6 mMNaC l，5 mM氯化镁，10%甘油，4°C)。三磷酸腺苷的表观KM：PS为2.0 5±0.1μM，P2S为2.7±0.2μM。这些差异很大的ATPase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位点之间存在协同通信。这些结果进一步强调，当试图仅基于宏观稳态ATPase测量来推断有关解旋酶转位等基本过程的信息时，需要明确考虑解旋酶的寡聚化和DNA连接状态的种群分布。
TheEscherichia coliRep 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 protomer 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)Science256, 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)15in 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 followingkcatvalues for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 × 10-3s-1for P, 2.17 ± 0.04 s-1for PS, 16.5 ± 0.2 s-1for P2S, and 71 ± 2.5 s-1for P2S2(20 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10% glycerol, 4 °C). The apparentKM's for ATP hydrolysis are 2.05 ± 0.1 μM for PS and 2.7 ± 0.2 μM 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 sites 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.