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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >How a holoenzyme for DNA replication is formed
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How a holoenzyme for DNA replication is formed

机译：用于DNA复制的全酶是如何形成的

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摘要

Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45-gp44/62-DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconver-sion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps. Two populations of the gp45-gp44/62-DNA complex are formed on the end-blocked DNA that are poised to form the holoenzyme with the polymerase. In the absence of a polymerase, the two clamp populations dissociated from the DNA along with gp44/62 with distinct rates. In the presence of polymerase, holoenzyme assembly involved the recruitment of the polymerase to the gp45-gp44/62-DNA complex mediated by the chaperoning activity of gp44/62. This transient multiprotein complex then decomposed through an ATP hydrolysis-dependent exit of gp44/62 leaving the holoenzyme on DNA. The rate of dissociation of the holoenzyme from the DNA is sensitive to whether the DNA ends are blocked, underscoring its mobility on the DNA.
机译：使用基于FRET的单分子和整体动力学研究方法，将gp44 / 62钳装载器将噬菌体T4滑动钳gp45装载到DNA上形成全酶，并研究其解体途径。 gp44 / 62介导的gp45组装到DNA上涉及gp45-gp44 / 62-DNA复合物的限速构象重排。单分子测量揭示了中间体在gp45上的负载及其相互转化，表明该组装不协调，但分解为许多小的动力学步骤。在最终封闭的DNA上形成了两个gp45-gp44 / 62-DNA复合物群体，它们准备与聚合酶形成全酶。在没有聚合酶的情况下，两个钳位群体与gp44 / 62一起以不同的速率与DNA分离。在存在聚合酶的情况下，全酶组装涉及将聚合酶募集到由gp44 / 62的伴侣活性介导的gp45-gp44 / 62-DNA复合物中。然后，这种短暂的多蛋白复合物通过gp44 / 62的ATP水解依赖性出口而分解，从而在DNA上留下了全酶。完整酶与DNA的解离速率对DNA末端是否被封闭敏感，从而突出了其在DNA上的迁移性。

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    Senthil K. Perumal; Wenhui Ren; Tae-Hee Lee; Stephen J. Benkovic;

  • 作者单位

    Department of Chemistry, Pennsylvania State University, University Park, PA 16802;

    Department of Chemistry, Pennsylvania State University, University Park, PA 16802;

    Department of Chemistry, Pennsylvania State University, University Park, PA 16802;

    Department of Chemistry, Pennsylvania State University, University Park, PA 16802;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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  • 正文语种 eng
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