Mechanism of loading the Escherichia coli DNA polymerase III β sliding clamp on DNA -: Bona fide primer/templates preferentially trigger the γ complex to hydrolyze ATP and load the clamp
Mechanism of loading the Escherichia coli DNA polymerase III β sliding clamp on DNA -: Bona fide primer/templates preferentially trigger the γ complex to hydrolyze ATP and load the clamp
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DOI：
10.1074/jbc.m211741200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Bloom, LB
中科院分区：
文献类型：
--
作者：
Ason, B;Handayani, R;Bloom, LB
大肠杆菌DNA聚合酶III γ复合物夹加载器将环形β滑动夹组装到DNA上。核心聚合酶通过β连接到模板上，使基因组能够进行性复制。在这里，我们调查的DNA底物特异性的钳加载反应，通过测量的前稳态动力学的DNA结合和ATP水解使用的延伸熟练和缺陷的引物/模板DNA。在存在或不存在β的情况下，ATP结合的钳加载剂结合延伸熟练的和缺陷的DNA底物。然而，延伸熟练的DNA优先触发γ复合物释放β到DNA上，伴随着ATP的水解。结合到延伸熟练的DNA将γ复合物从高亲和力ATP结合状态转化为对DNA具有低10倍亲和力的ADP结合状态。稳态结合试验具有误导性，表明γ复合物更积极地与不可延伸的引物/模板DNA结合，因为再循环到高亲和力结合状态是限速的。前稳态旋转各向异性数据揭示了γ复合物与可延伸引物/模板的动态缔合-解离，导致截然相反的结论。对可延伸DNA的强烈有利的动态识别不需要β的存在。因此，γ复合物使用ATP结合和水解作为调节其与DNA相互作用的机制，其中ATP结合形式以高亲和力与DNA结合，但擅长延伸的DNA底物优先触发ATP水解并转化为低亲和力状态。
The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA. The core polymerase is tethered to the template by beta, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the presteady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta. However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10'-fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to nonextendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Presteady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of beta. Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.