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首页> 外文学位 >Crystal structure-based mutagenesis and characterization of the RecBCD protein from Escherichia coli.
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Crystal structure-based mutagenesis and characterization of the RecBCD protein from Escherichia coli.

机译：基于晶体结构的诱变和表征来自大肠杆菌的RecBCD蛋白。

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摘要

The recently published crystal structure of RecBCD enzyme complexed with a DNA hairpin revealed several striking structures: a putative "chi-scanning site" in RecC, an "arm" in RecB and a "pin" in RecC. I applied site-directed mutagenesis to identify their functions.;First, I established the important residues responsible for chi recognition in the channel passed through by the 3'-ended ssDNA in RecC. Residues in the channel were mutagenized to alanines individually and three types of mutants were identified based on genetic screening. Here, biochemically, the "Lost-recognition" mutants lose the ability to respond to chi. The "Relaxed-specificity" and "Reduced-specificity" mutants still recognize chi; moreover, they are able to recognize some single base mutants of chi with different efficiencies. These findings provide new information on how RecBCD enzyme recognizes and interacts with the chi sequence.;Second, I determined the role of the "arm" structure in RecB that contacts the incoming duplex DNA. When the "arm" structure was deleted, the resultant recBarm-CD cells are UV resistant while the recBarm-C cells are sensitive to UV irradiation. Consistently, in vitro, the RecBarm- CD holoenzyme is fully functional, displaying wild-type level helicase and nuclease activities, although the affinity for duplex DNA ends is lower than RecBCD. Moreover, RecBarm-CD enzyme could respond to chi as RecBCD does, suggesting the "armless" RecB is active during translocation. However, the affinity of RecBarm-C protein for dsDNA ends is dramatically lower than RecBC protein. These results suggest that the "arm" plays a direct role in dsDNA binding.;Third, the methionine "pin" in RecC at the junction between ssDNA and dsDNA was studied. The methionine dyad was replaced with two alanines. The recBCpin-D cells are UV resistant in vivo. Consistently, purified RecBCpin-D protein possesses wild-type level of dsDNA-dependent ATPase, helicase and nuclease activities, as well as chi response. The implications of these results on the proposed function of the "pin" are discussed.;Fourth, I studied the translocation polarity of RecBCD enzyme. The nuclease deficient mutant RecBD1080ACD enzyme could bypass gaps on either strand of the duplex DNA, suggesting that RecBCD translocates on both strands of DNA.
机译：最近发表的与DNA发夹复合的RecBCD酶的晶体结构揭示了几个惊人的结构：RecC中一个假定的“ chi扫描位点”，RecB中的一个“臂”和RecC中的一个“销”。我应用了定点诱变来鉴定它们的功能。首先，我在RecC中3'端ssDNA所通过的通道中建立了重要的残基，用于识别chi。通道中的残基分别诱变为丙氨酸，并根据遗传筛选鉴定出三种类型的突变体。在生物化学上，“失落识别”突变体失去了对chi的反应能力。 “特异性降低”和“特异性降低”的突变体仍然可以识别chi。而且，他们能够识别一些不同效率的chi单碱基突变体。这些发现为RecBCD酶如何识别chi序列以及如何与chi序列相互作用提供了新信息。第二，我确定了RecB中“臂”结构与进入的双链DNA接触的作用。当删除“臂”结构时，所得的recBarm-CD细胞具有抗紫外线能力，而recBarm-C细胞则对紫外线辐射敏感。一致地，尽管对双链DNA末端的亲和力低于RecBCD，但RecBarm-CD全酶在体外始终发挥功能，显示出野生型水平的解旋酶和核酸酶活性。此外，RecBarm-CD酶可以像RecBCD一样对chi作出反应，这表明“无臂” RecB在转运过程中是活跃的。但是，RecBarm-C蛋白对dsDNA末端的亲和力大大低于RecBC蛋白。这些结果表明“臂”在dsDNA结合中起直接作用。第三，研究了在ResC中ssDNA和dsDNA之间的甲硫氨酸“ pin”。蛋氨酸二聚体被两个丙氨酸取代。 recBCpin-D细胞在体内具有抗紫外线能力。一致地，纯化的RecBCpin-D蛋白具有野生型水平的dsDNA依赖性ATPase，解旋酶和核酸酶活性，以及​​chi应答。讨论了这些结果对拟议中的“ pin”功能的影响。第四，我研究了RecBCD酶的易位极性。核酸酶缺陷型RecBD1080ACD酶可以绕过双链DNA任一条链上的缺口，表明RecBCD在DNA的两条链上都易位。

著录项

  • 作者

    Yang, Liang.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 254 p.
  • 总页数 254
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:25

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