Abstract
Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (∼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124INT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN7TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124INT in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.
Highlights
Restriction–modification (RM) enzymes act as a bacterial defence mechanism against foreign DNA
The two modified SNT subunits, corresponding to just over half an intact S subunit, are able to dimerise effectively via the coiled-coil interface to form a structure that is homologous to the native S subunit containing two target recognition domains (TRDs)’s, but with dyad symmetry
Covalent interactions between the two halves of the S subunit are not required for subunit assembly, for ATP-driven DNA translocation or for DNA cleavage
Summary
Restriction–modification (RM) enzymes act as a bacterial defence mechanism against foreign DNA. Host DNA is fully methylated at specific sequences by a methyltransferase (MTase), protecting its DNA from restriction by the accompanying endonuclease (ENase). Type I RM systems play a role in modulating horizontal gene transfer and are important in the spread of antibiotic resistance genes in bacterial populations [3]. They comprise large multi-subunit enzyme complexes encoded by three hsd (host specificity of DNA) genes, corresponding to three polypeptides: HsdS, responsible for DNA recognition, HsdM for DNA modification and HsdR for cleavage [3]. The ENase requires all three subunits (referred to hereafter as M, S and R) while the MTase requires just the M and S subunits, the stoichiometry being R2M2S and M2S, respectively
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