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Ribozymes02:47

Ribozymes

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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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Restriction Enzymes01:11

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Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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DNAzyme-dependent Analysis of rRNA 2’-O-Methylation
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水溶性エンド核溶性リボ酵素 (HYER) は,配列特異のDNA分裂のためにプログラム可能である.

Zi-Xian Liu1, Shouyue Zhang1, Han-Zhou Zhu1

  • 1Beijing Advanced Innovation Center for Structural Biology, State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Science (New York, N.Y.)
|February 1, 2024
PubMed
まとめ

研究者達はDNAを切断できる 触媒性RNAの一種である HYER を発見しました これらの天然のリボ酵素は精密なDNA操作とゲノム編集の応用の可能性を示しています

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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
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関連する実験動画

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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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科学分野:

  • 分子生物学
  • 生物化学
  • 遺伝学

背景:

  • リボ酵素は,細胞プロセスにおいて重要な役割を果たす触媒RNA分子である.
  • 天然のリボ酵素は,DNA操作を含むRNA触媒を超えた可能性がますます認識されています.

研究 の 目的:

  • 配列特異的なDNAエンドヌクレアゼとして機能する自然リボ酵素を特定し,特徴づけること.
  • 新しいDNA操作ツールとして リボ酵素の再利用を 探求するためです

主な方法:

  • 細菌II-C群のイントロンを中心に,研究者はイントロンにコードされたタンパク質が欠けているシステムをスクリーニングしました.
  • 様々な核酸基板に対する識別されたリボ酵素 (HYERs) の分裂活性をテストするために,in vitroアッセイが使用された.
  • HYER1のDNA結合と触媒機構の構造的基礎を決定するために,冷凍電子顕微鏡を用いた.
  • 合理的な設計戦略を適用し,特異性と機能性の向上したHYERのバリエーションを設計しました.

主要な成果:

  • HYER (Hydrolytic Endonucleolytic Ribozymes) と呼ばれるいくつかのイントロン系が,RNA,単鎖DNA,および双鎖DNA (dsDNA) を分割するものであることが確認された.
  • HYER1は,哺乳類のゲノムでdsDNAの断絶を誘導する能力をインビトロで示した.
  • クリオ電子顕微鏡では,Mg2+依存の水解ポケットとDNA結合能力を持つHYER1のホモジメ構造を明らかにした.
  • エンジニアリングされたHYERの変種は DNA操作の特異性と柔軟性を向上させました

結論:

  • 自然のリボ酵素,特にII-C群のHYERは,DNAエンドヌクレアース活性を持っています.
  • これらのHYERは精密なDNA分裂のために設計され,ゲノム編集とバイオテクノロジーの有望な新しいプラットフォームを提供します.