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首页> 外文期刊>Nucleic acids research >Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling
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Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling

机译：大肠杆菌β滑动夹具的单个疏水性裂缝对夹具负载，DNA复制和夹具回收的贡献

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摘要

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (βC) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β+), and one βC subunit (β+/βC). The β+/βC heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β+. Moreover, β+/βC interacted normally with Pol III, and stimulated replication to the same extent as did β+. Finally, β+/βC was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.
机译：同型二聚体大肠杆菌β滑动夹包含两个疏水性裂缝，与DNA复制，修复和破坏耐受性相关的蛋白质与之相互作用。 β（β C ）的C端5个残基的缺失破坏了两个裂口，严重损害了夹具与DnaX夹具装载器以及复制性DNA聚合酶Pol III的相互作用。为了确定是否需要两个裂口将钳夹装载到DNA上，刺激Pol III复制并在复制完成后从DNA上取下钳夹，我们开发了一种纯化由一个野生型亚基（β + ）和一个β C 亚基（β + /β C ）。 β + /β C 异二聚体与DnaX钳夹加载器正常相互作用，并且比β + 加载效率更高。此外，β + /β C 与Pol III正常相互作用，并以与β + 相同的程度刺激复制。最后，使用DnaX或DnaX的δ亚基从DNA卸载时，β + /β C 受到严重损害。综上所述，这些发现表明，β钳夹中的单个裂口足以装载和刺激Pol III复制，但是复制完成后从DNA上卸下钳夹需要两个裂口。

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