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Molecular Evolution of the Tre Recombinase
Published on: May 29, 2008
聚合酶进化:努力扩展遗传密码的努力
Aaron M Leconte1, Liangjing Chen, Floyd E Romesberg
1Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of the American Chemical Society
|September 8, 2005
概括
研究人员开发了一种DNA聚合酶,以高效地复制具有非自然基对的DNA,特别是PICS自我配对. 这种进步通过改善含有人工核基的DNA的合成和扩展来扩展遗传密码.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 生物化学 生物化学
背景情况:
- 遗传密码,通常是四个字母的字母表,可以使用非自然的核基类比来扩展.
- PICS自我配对是一种稳定,疏水的核基模拟候选人,用于扩展遗传密码.
- 优化DNA聚合酶对于有效地复制非自然基对至关重要.
研究的目的:
- 开发一种DNA聚合酶,能够有效地复制含有PICS自我配对的DNA.
- 增强DNA中非自然核基的结合和扩展.
主要方法:
- 基于活动的菌体显示选择系统被用来进化DNA聚合酶.
- 该系统使用了共显示的聚合酶库和DNA基质与PICS自我配对.
- 通过选择将生物素-dUTP与原料相结合的聚合酶来实现分离,从而使得链状维丁能够被捕获.
主要成果:
- 一个突变的Sf聚合酶,命名为P2,进化了显著增强的PICS自我配对复制能力.
- P2成功地将dPICSTP插入dPICS的对面,并将非自然的原始终端扩展到自然的核酸.
- P2是一种三重突变 (F598I,I614F,Q489H),对含有PICS的DNA原始模板具有较强的亲和力.
结论:
- 进化的P2聚合酶有效地复制含有PICS自我配对的DNA,这是扩展遗传密码的关键步骤.
- 鉴定到的突变表明,一种机制增加了对非自然基对的亲和力.
- 这项工作表明了定向进化的力量,用于创建适合合成生物学应用的聚合酶.
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