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首页> 外文学位 >Atomic force microscopy studies of eukaryotic clamp/clamp loader complex and mismatch repair initiation complex.
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Atomic force microscopy studies of eukaryotic clamp/clamp loader complex and mismatch repair initiation complex.

机译：真核钳/钳装载器复合物和错配修复起始复合物的原子力显微镜研究。

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摘要

As an advanced single molecule technique, atomic force microscopy (AFM) is a powerful and versatile tool for high resolution surface imaging and probing physical properties of soft, nonconductive bio-materials in vitro . Imaging of protein-protein and protein-DNA complexes provides structural and conformational information about the interactions of these biomolecular assemblies. In this study, we have used AFM to examine two different protein complexes: the eukaryotic RFC complex function in loading PCNA clamp onto different DNA substrate and eukaryotic MutS homologs function in the initiation of DNA mismatch repair (MMR).;In the study of clamp loader RFC complex, we investigated the effect of nucleotide cofactors on the oligomerization states of RFC interacting with PCNA and DNA substrate. We observed that ATP binding induces a conformational change of RFC and that ATP hydrolysis causes RFC dissociation into small subcomplexes. However, PCNA inhibits the ATP-induced disassembly of RFC. Intriguingly, we found in the presence of ATP, some of the RFC subunits are ejected from DNA substrate, leaving RFC subcomplex bound to the DNA, and it appears that these subcomplexes form stable interaction with PCNA on the DNA. We proposed that this DNA-bound RFC subcomplex tethers PCNA ring at the single strand/double strand junction of primer-template DNA or nick DNA. We further suggest that dissociation of RFC subcomplex from PCNA and DNA substrate is promoted by downstream PCNA-interacting proteins, such as DNA polymerase. In addition to these insights into the complicated potential loading mechanism of PCNA, we observed other RFC-DNA complexes such as RFC-DNA filaments with nicked DNA without nucleotide cofactor and RFC-DNA spider-like complexes containing multiple RFCs and DNAs in the presence of ATP. Although we do not know the physiological role, if any, of such RFC-DNA complexes, these complexes suggest RFC can possess other functions besides as clamp loader, such as helicase.;In the study of MMR initiation complexes, eukaryotic MutS homologs (MutSalpha and MutSbeta), we found, unlike their prokaryotic homologs, eukaryiotic MutS homologs bind different DNA substrates with similar conformation. MutSalpha and MutSbeta both exhibits weak binding specificity to their specific DNA substrates, which makes it more complicated to analyze their specific complexes. However, it appears that eukaryotic MutS homologs do not recognize mismatched bases simply depending on the formation of unbent complexes as seen in the prokaryotic MutS. It is possible they employ other high class mechanism in which the event of recognition of different mismatched DNA substrates happens downstream of mismatch binding.
机译：作为一种先进的单分子技术，原子力显微镜（AFM）是一种功能强大且用途广泛的工具，可用于高分辨率表面成像以及在体外探测柔软的非导电生物材料的物理特性。蛋白质-蛋白质和蛋白质-DNA复合物的成像提供了有关这些生物分子组装体相互作用的结构和构象信息。在这项研究中，我们使用原子力显微镜（AFM）来研究两种不同的蛋白质复合物：将PCNA钳加载到不同DNA底物上的真核RFC复杂功能以及在DNA错配修复（MMR）启动中的真核MutS同源物功能。 RFC复合物，我们研究了核苷酸辅因子对RFC与PCNA和DNA底物相互作用的低聚状态的影响。我们观察到ATP结合诱导RFC的构象变化，并且ATP水解导致RFC分解成小的亚复合物。但是，PCNA抑制ATP诱导的RFC分解。有趣的是，我们发现在ATP的存在下，一些RFC亚基从DNA底物中弹出，而RFC亚复合物与DNA结合，并且看起来这些亚复合物与DNA上的PCNA形成稳定的相互作用。我们提出，这种DNA结合的RFC亚复合物系链PCNA在引物模板DNA或切口DNA的单链/双链连接处环化。我们进一步建议，RFC下游复合物从PCNA和DNA底物中的解离是由下游PCNA相互作用蛋白（例如DNA聚合酶）促进的。除了对PCNA复杂的潜在加载机制的这些见解之外，我们还观察到其他RFC-DNA复合物，例如带有带切口DNA且没有核苷酸辅因子的RFC-DNA细丝，以及在存在DNA的情况下包含多个RFC和DNA的RFC-DNA蜘蛛状复合物。 ATP。尽管我们不知道此类RFC-DNA复合物的生理作用（如果有的话），但这些复合物表明RFC除了钳夹加载程序（例如解旋酶）外还可以具有其他功能。;在MMR起始复合物的研究中，真核MutS同源物（MutSalpha和MutSbeta），我们发现，与其原核同源物不同，真核MutS同源物以相似的构象结合不同的DNA底物。 MutSalpha和MutSbeta都表现出对特定DNA底物的弱结合特异性，这使得分析其特定复合物变得更加复杂。然而，看来真核MutS同源物不能仅仅根据原核MutS中未弯曲复合物的形成就识别不匹配的碱基。他们可能采用其他高级机制，其中识别不同错配DNA底物的事件发生在错配结合的下游。

著录项

  • 作者

    Zhang, Na.;

  • 作者单位

    The University of North Carolina at Chapel Hill.;

  • 授予单位 The University of North Carolina at Chapel Hill.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 183 p.
  • 总页数 183
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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