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

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.

The type I restriction enzymes were, as their name suggests, the first restriction enzymes to be analysed genetically and biochemically with work starting in 1953. 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 type I enzymes are complex multifunctional enzymes of huge size ( one third the size of the ribosome, molecular weight 440,000, approximately 10nm in diameter) and, in stark contrast to the type II restriction enzymes, the type I enzymes cut DNA into random fragments. Despite their biochemical complexity, the genes for type I restriction systems appear in at least 50 per cent of bacterial genomes 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 could escape restriction in vivo by the archetypal type I enzyme, EcoKI, the subject of this proposal. 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 and similarities between various catalytic domains of type I restriction enzymes and the classic RecBCD enzyme, whose structure has recently been solved, are apparent. To perform so many functions in one enzyme requires a complex oligomeric structure, figure 3, which has been largely derived by Dryden and his long-term collaborator Prof. Noreen Murray FRS (recently retired) from extensive genetic, biochemical and biophysical analyses. However, despite much effort by all of the researchers studying different type I restriction enzymes for the past 30 years, no crystal structure information 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. Excellent preliminary EM images have been obtained showing much structural detail and additional spectroscopic data shows complex behaviour as the enzyme goes through its various reaction cycles (binding, methylation, translocation and cleavage). EM analysis can give structures reaching 7 angstrom resolution at various points during the reaction. Combining this with the extensive biophysical data and the experimentally validated structural models of domains will define the location of domains and subunits within the EM structure and lead to a complete structural model of this complex nanomachine. Joint with BB/D522589/1.