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Title: Biochemical and biophysical characterisation of the genetically engineered Type I restriction-modification system, EcoR124I NT
Author: Taylor, James Edward Nathan
Awarding Body: University of Portsmouth
Current Institution: University of Portsmouth
Date of Award: 2005
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The EcoR124INT restriction-modification (R-M) system contains the genes HsdS3, HsdM and HsdR. S3 encodes the N-terminal domain of the wild-type S subunit and has been shown to dimerise in solution (Smith et al., 1998). Following purification of the subunits of the EcoR124INT R-M system, complexes of the methyltransferase S3/M and restriction endonuclease S3/M/R were formed and shown to have activity in vitro, methylating and hydrolysing a symmetrical DNA recognition sequence, respectively. The DNA mimic OCR (overcome classical restriction) protein inhibited the methyltransferase activity in vitro, with maximum inhibition at a 1: 2 molar ratio of (S3/M)2 to an ocr dimer. Dynamic light scattering (DLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments showed S3 to exist as a dimer and S11 (the central conserved domain of S) to exist as a tetramer in solution. M was found to be dimeric in solution, whilst the R protein was monomeric. A complex of S3/M was found to have a stoichiometry (S3/M)2 and a complex of S3/M/R had a stoichiometry of S3/M/R1, even when a 2: 1 molar ratio of R to S3/M, was added. Small angle neutron scattering (SANS) experiments provided values for the radius of gyration (Rg), which for S3 was comparable to that calculated for the recently published crystal structure of the S subunit from Methanococcus jannaschii (Kim et al., 2005). These experiments also showed a decrease in the Dmax in the presence of the 30 bp DNA recognition sequence from 200A to 140A, suggesting a similar conformational change in the positioning of the subunits as has been detected for the wild-type M. EcoR124I and a related type 1 1/2 system AhdI. This change following DNA binding was also observed by SV experiments. Furthermore ab initio modelling from the SANS data has provided a low-resolution structure for the EcoR124INT MTase and its complex with DNA.
Supervisor: Kneale, Geoff Sponsor: Institute of Biomedical and Biomolecular Science, University of Portsmouth
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Restriction-modification, type I DNA methyltransferase; DNA restriction enzymes; DNA translocation; analytical ultracentrifugation; SANS; small-angle neutron scattering; contrast variation; molecular modeling; DNA translocases; protein; DNA; macromolecular complexes