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

限制性内切酶是分子生物学的主力，它能将DNA分子精确地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，现代实验生物学将是非常不同的。然而，很少有人知道这些酶被正确地称为II型限制性内切酶。什么是I型限制性内切酶？I型限制性内切酶是第一个被遗传学和生物化学分析的限制性内切酶，工作始于1953年，同年沃森和克里克发表了他们著名的DNA结构模型。I型限制性内切酶的分析为II型限制性内切酶的发现和基因工程的诞生铺平了道路。I型限制性内切酶的相对模糊是因为它们不仅是复杂的多功能酶，分子量为440，000，直径约为10 nm，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切割成无实际用途的随机片段。尽管它们的生物化学复杂性，I型限制系统出现在至少50%的细菌中，所以虽然它们在技术上没有用处，但自然界显然对它们有用处。已经表明，在正确的遗传背景下，少于一亿分之一的λ噬菌体（病毒）可以在体内逃脱原型I型酶EcoKI的破坏，EcoKI是本提案的主题。因此，I型限制酶可以是非常有效的细菌防御系统。I型限制性内切酶是智能分子机器，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大长度DNA，并产生高达5 pN的力。由这些酶产生的DNA片段很可能是重组的（即它们可以改变细菌的遗传学），并且I型限制酶的各种催化结构域与经典重组酶RecBCD之间的相似性是显而易见的，RecBCD的结构最近已经解决。要在一种酶中执行如此多的功能，需要复杂的寡聚体结构。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底物组合的任何亚基或结构域，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寻找大型机器（如EcoKI）的结构。虽然电子显微镜的分辨率有限，但它可以与所有现有知识相结合，以产生高分辨率的结构，并帮助我们了解第一台纳米机器是如何工作的。与BB/D522589/1接头。

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.