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首页> 外文期刊>Journal of Molecular Biology >Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit.
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Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit.

机译：双重功能，夹具打开和底漆模板识别，定义了一个关键的夹具装载器子单元。

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摘要

Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E.coli gamma complex clamp loader and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that the delta subunit in the gamma complex makes close contact with the primer-template junction. Tryptophan 279 in the delta C-terminal domain lies near the 3'-OH primer end and may play a key role inprimer-template recognition. Previous studies have shown that delta also binds and opens the beta clamp (hydrophobic residues in the N-terminal domain of delta contact beta. The clamp-binding and DNA-binding sites on delta appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S.cerevisiae RFC complex suggests that the dual functionality observed for delta in the gamma complex may be true also for clamp loaders from other organisms.
机译：钳加载器蛋白催化DNA上圆形滑动钳的装配，以使DNA进行连续复制。在反应过程中，夹具装载器结合引物模板DNA，并将其放置在夹具的中心，以在两者之间形成拓扑联系。钳装载器是多种蛋白质的复合物，例如五种蛋白质大肠埃希氏菌，酿酒酵母和人钳装载器，以及两种蛋白热球菌和嗜热自养甲烷杆菌钳装载器，到目前为止，是负责结合和选择引物的位点-模板DNA作为夹具组装的目标仍然未知。为了解决此问题，我们使用紫外线诱导的蛋白质-DNA交联和质谱分析了大肠杆菌伽玛复合物钳状装载剂与DNA之间的相互作用。结果表明，γ-复合物中的δ亚基与引物-模板连接紧密接触。 δC-末端结构域中的色氨酸279位于3'-OH引物末端附近，并可能在引物模板识别中起关键作用。先前的研究表明，δ也会结合并打开β钳位（δ的N末端结构域的疏水残基与beta接触。δ上的钳位结合位点和DNA结合位点的位置似乎便于引物模板进入中心对酿酒酵母RFC复合物的类似分析表明，在γ复合物中观察到的δ的双重功能对于其他生物的夹持物装载器也可能是正确的。

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