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相关概念视频

Gene Evolution - Fast or Slow?02:05

Gene Evolution - Fast or Slow?

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The genomes of eukaryotes are punctuated by long stretches of sequence which do not code for proteins or RNAs. Although some of these regions do contain crucial regulatory sequences, the vast majority of this DNA serves no known function. Typically, these regions of the genome are the ones in which the fastest change, in evolutionary terms, is observed, because there is typically little to no selection pressure acting on these regions to preserve their sequences.
In contrast, regions which code...
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Mutations01:35

Mutations

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Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
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Genome Copying Errors02:46

Genome Copying Errors

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DNA replication is a well-evolved process that copies millions of base pairs with high fidelity during each cell division. Occasionally a wrong base or a long stretch of wrong bases may get added to the daughter strands. If the errors are left unchecked, cells might accumulate several mutations that might endanger their  survival. Therefore, the copying errors are checked and repaired at three levels.
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Mismatch Repair01:20

Mismatch Repair

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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
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In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...
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Proofreading01:31

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase...
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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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脱氨酶驱动的随机突变使得蛋白质进化的有效DNA突变发生成为可能.

Ying Hao1, Tong-Tong Ji1, Shu-Yi Gu2

  • 1College of Chemistry and Molecular Sciences, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan University Wuhan 430071 China bfyuan@whu.edu.cn yqfeng@whu.edu.cn.

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蛋白质进化通过脱氨酶驱动的随机突变 (DRM) 增强,这是一种新的DNA突变发生策略. 由于DRM提供了比易出错的PCR更高的突变频率和多样性,加速了新型蛋白质突变的发现.

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科学领域:

  • 生物化学 生物化学
  • 分子生物学分子生物学
  • 蛋白质工程是指蛋白质工程.

背景情况:

  • 蛋白质进化对于创造具有新功能的蛋白质至关重要.
  • 基因突变是蛋白质进化的关键一步.
  • 目前的方法,如易出错的PCR (epPCR),在突变效率和多样性方面存在局限性.

研究的目的:

  • 开发一种新的DNA突变发生策略,其效率和多样性高于epPCR.
  • 在单一轮的突变发生过程中引入广泛的突变谱.
  • 为了促进新型蛋白质突变物的发现.

主要方法:

  • 开发了使用工程化赛蒂丁脱氨酶A3A-RL和腺脱氨酶ABE8e的脱氨酶驱动随机突变 (DRM).
  • 在两个DNA链上引入了C-to-T,G-to-A,A-to-G和T-to-C突变.
  • 在突变频率和多样性方面,将DRM与epPCR进行比较.

主要成果:

  • 与epPCR相比,DRM显示DNA突变频率高出14.6倍.
  • 而DRM的突变类型多样性是epPCR的27.7倍.
  • 通过DRM,可以更全面地探索蛋白质工程的遗传景观.

结论:

  • DRM是蛋白质进化的强大工具,提供卓越的突变性能力.
  • 这一策略显著增强了新型和改进的蛋白质突变物的发现.
  • DRM为蛋白质工程应用提供高质量的DNA产品.