Molecular mechanism and energetics of clamp assembly in Escherichia coli -: The role of ATP hydrolysis when γ complex loads β on DNA
Molecular mechanism and energetics of clamp assembly in Escherichia coli -: The role of ATP hydrolysis when γ complex loads β on DNA
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DOI：
10.1074/jbc.m910441199
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发表时间：
2000-09-15
影响因子：
4.8
通讯作者：
Goodman, MF
中科院分区：
文献类型：
--
作者：
Bertram, JG;Bloom, LB;Goodman, MF
大肠杆菌DNA聚合酶III全酶是一种多亚基复合物，含有β滑动夹和夹加载γ复合物。γ复合物需要ATP将β加载到DNA上。利用双色荧光光谱方法研究该系统，其中组装（红色荧光; X-罗丹明标记的DNA各向异性测定）和ATP水解（绿色荧光;磷酸盐结合蛋白测定）以毫秒计时分辨率同时测量。两个时间相关的停止流动信号揭示了预组装的β. γ复合物复合物在ATP水解独立步骤中快速结合引物/模板DNA。一旦结合，两个ATP分子迅速水解（类似于34 s（-1））。水解后，γ复合物从DNA中解离（类似于22 s（-1））。一旦解离，下一个加载循环就会受到严重影响，导致稳态ATP水解速率最大值仅接近3 s（-1）。两个单一的网站β二聚体接口突变体进行了检查，受损的稳态ATP水解率。这些突变体的前稳态相关动力学揭示了与野生型基本相同的模式。各向异性的数据表明，这些突变体降低ATP水解的稳态速率，引起的“卡住”的二元-三元复合物的引物/模板DNA上的积累。
Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta.gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (similar to 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA (similar to 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only similar to 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.