Bacterial restriction-modification (RM) systems act as a form of primitive immune system that aims to prevent foreign DNA from establishing itself within a bacterium. RM systems often function with two complementary activities; DNA methylation to label “self” DNA and DNA cleavage of unlabelled, “non-self” DNA. These two enzymatic activities require strict temporal control to prevent over-methylation or auto-restriction; the endonuclease induced degradation of genomic DNA. In some RM systems a third, controller (C) protein is employed to act as a transcription factor to control the expression of the two enzymes. Previous study, using the Esp1396I RM system as a model, has revealed how the C-protein binds to its DNA operator sequences. Unlike most C-proteins, C.Esp1396I has three binding sites; OM controls the expression of the methyltransferase and OL & OR control the expression of the C-protein and endonuclease genes as a double binding site. The binding affinities for the three sites vary by more than two orders of magnitude despite the small difference in DNA sequence. Crystallographic analysis of C.Esp1396I both as a free protein and bound to its various DNA operator sites has revealed the roles of several highly conserved residues, many of which interact with the DNA.
In this study, several of these DNA binding residues were mutated in an attempt to elucidate their individual roles in the context of protein-DNA complex formation. Mutations made to those residues that interact with the phosphate backbone had a lesser effect on the binding affinity compared to those made to residues that bind to the DNA bases. Crystallographic analysis of the mutant C.Esp1396I proteins showed that the mutations had not affected the overall structure of the protein. However, what was revealed were further details of C.Esp1396I in its free state, especially the high degree of flexibility in a loop region that is key to DNA binding.
During the course of this study, wild type C.Esp1396I was co-crystallised with two different DNA duplexes containing part or all of the OR site. Since there were no biologically relevant interactions in the co-crystal structures with the OR operator sequence, this gave the opportunity to observe the structure of the DNA in the un-bound state. The structure of the complex with a longer DNA fragment showed that the major groove at the OR site is opened up and prepared for C-protein binding when OL is occupied, reducing the binding penalty for this intrinsically weak operator site.
|Date of Award||Feb 2014|
|Supervisor||John McGeehan (Supervisor) & Geoff Kneale (Supervisor)|