The structure and conformational dynamics of a complex molecular machine: the type 1 DNA restriction enzyme EcoKI

复杂分子机器的结构和构象动力学：1 型 DNA 限制酶 EcoKI

• 批准号: BB/D001870/1
• 负责人: John Trinick
• 金额: $ 50.04万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD001870%2F1
• 关键词: structure; conformational; dynamics; complex; molecular

Restriction enzymes are the workhorses of molecular biology, cutting DNA molecules accurately into the precise fragments required for virtually all molecular biology experiments. They were first purified in 1972 and without them modern experimental biology would be very different. However, few people are aware that these enzymes are properly referred to as type II restriction enzymes. What are the type I restriction enzymes? The type I restriction enzymes were the first restriction enzymes to be analysed genetically and biochemically with work starting in 1953, the same year as Watson and Crick published their famous model for DNA structure. Analysis of type I restriction enzymes paved the way for the discovery of the type II restriction enzymes and the birth of genetic engineering. The relative obscurity of the type I enzymes arises because they are not only complex multifunctional enzymes of huge size (molecular weight 440,000, approximately 10nm in diameter) but also, in stark contrast to the highly commercial and important type II restriction enzymes, the type I enzymes cut DNA into random fragments of no practical use. Despite their biochemical complexity, type I restriction systems appear in at least 50 percent of bacteria so whilst they are not technologically useful, Nature clearly has a use for them. It has been shown that in the correct genetic background that fewer than one in 100 million lambda phage (viruses) could escape destruction in vivo by the archetypal type I enzyme, EcoKI, the subject of this proposal. Therefore, type I restriction enzymes can be very effective bacterial defense systems. It transpires that type I restriction enzymes are smart molecular machines capable of selecting and then performing multiple functions including the maintenance of chromosomal DNA methylation and the manipulation of great lengths of DNA at speeds reaching 1000 base pairs per second and generating forces of up to 5pN. The fragments of DNA produced by these enzymes are likely to be recombinogenic (i.e. they can change the genetics of the bacterium) and similarities between various catalytic domains of type I restriction enzymes and the classic recombination enzyme, RecBCD, whose structure has recently been solved, are apparent. To perform so many functions in one enzyme requires a complex oligomeric structure. However, despite much effort by all of the researchers studying type I restriction enzymes for the past 30 years, no detailed atomic structure has ever been achieved for the entire enzyme or for any subunit or domain with any combination of substrates. It is therefore timely to approach the structure problem by modern electron microscopy by establishing a collaboration between two leading groups in these areas. Electron microscopy is perfectly suited to finding the structure of a large machine such as EcoKI. Although the resolution of electron microscopy is limited, it can be combined with all of the current knowledge to produce a high resolution structure and to help us understand how one of the first nanomachines operates. Joint with BB/D522589/1.

限制性内切酶是分子生物学的中坚力量，它能精确地将DNA分子切割成几乎所有分子生物学实验所需的精确片段。它们在1972年首次被纯化，如果没有它们，现代实验生物学将会大不相同。然而，很少有人知道这些酶被恰当地称为II型限制性内切酶。什么是I型限制性内切酶？1953年，沃森和克里克发表了他们著名的DNA结构模型，第一类限制性内切酶是第一个被用于遗传学和生物化学分析的限制性内切酶。I型内切酶的分析为II型内切酶的发现和基因工程的诞生铺平了道路。I型酶之所以相对默默无闻，不仅是因为它们是复杂的多功能酶，体积巨大（分子量44万，直径约10纳米），而且与高度商业化和重要的II型限制性内切酶形成鲜明对比，I型酶将DNA切割成没有实际用途的随机片段。尽管它们的生化复杂，但至少50%的细菌中存在I型限制系统，因此尽管它们在技术上没有用处，但自然显然对它们有用处。已经证明，在正确的遗传背景下，不到1亿分之一的λ噬菌体（病毒）可以在体内逃脱原型I型酶EcoKI的破坏，这是本提案的主题。因此，I型限制性内切酶是非常有效的细菌防御系统。结果表明，I型限制性内切酶是一种智能分子机器，能够选择并执行多种功能，包括维持染色体DNA甲基化和以每秒1000个碱基对的速度操纵长长度的DNA，并产生高达5pN的力。这些酶产生的DNA片段很可能是重组的（即它们可以改变细菌的遗传），I型限制性内切酶的各种催化结构域与经典的重组酶（RecBCD）之间的相似性是显而易见的，后者的结构最近已被解决。在一种酶中执行如此多的功能需要复杂的低聚结构。然而，尽管在过去的30年里，所有研究I型限制性内切酶的研究人员都付出了很大的努力，但对于整个酶或任何亚基或结构域与底物的任何组合，都没有获得详细的原子结构。因此，通过在这些领域的两个领导小组之间建立合作，利用现代电子显微镜来解决结构问题是及时的。电子显微镜非常适合发现像EcoKI这样的大型机器的结构。虽然电子显微镜的分辨率是有限的，但它可以与当前所有的知识相结合，产生一个高分辨率的结构，并帮助我们了解第一个纳米机器是如何运作的。与BB/D522589/1接头。

项目成果

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance.

• DOI: 10.1093/nar/gkp478
• 发表时间: 2009-08
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: McMahon SA;Roberts GA;Johnson KA;Cooper LP;Liu H;White JH;Carter LG;Sanghvi B;Oke M;Walkinshaw MD;Blakely GW;Naismith JH;Dryden DT
• 通讯作者: Dryden DT

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.

• DOI: 10.1093/nar/gkn988
• 发表时间: 2009-02
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Kennaway CK;Obarska-Kosinska A;White JH;Tuszynska I;Cooper LP;Bujnicki JM;Trinick J;Dryden DT
• 通讯作者: Dryden DT

Single-molecule imaging of Bacteroides fragilis AddAB reveals the highly processive translocation of a single motor helicase.

• DOI: 10.1093/nar/gkq100
• 发表时间: 2010-06
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Reuter M;Parry F;Dryden DT;Blakely GW
• 通讯作者: Blakely GW

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes.

• DOI: 10.1016/j.bbrc.2010.06.069
• 发表时间: 2010-07-23
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Chen, Kai;Roberts, Gareth A.;Stephanou, Augoustinos S.;Cooper, Laurie P.;White, John H.;Dryden, David T. F.
• 通讯作者: Dryden, David T. F.

ArdA proteins from different mobile genetic elements can bind to the EcoKI Type I DNA methyltransferase of E. coli K12.

• DOI: 10.1016/j.bbapap.2013.12.008
• 发表时间: 2014-03
• 期刊: Biochimica et biophysica acta
• 作者: Chen K;Reuter M;Sanghvi B;Roberts GA;Cooper LP;Tilling M;Blakely GW;Dryden DT
• 通讯作者: Dryden DT