Structure and Dynamics of the Methyl-transeferase subunit of the E.coli restriction-modification system EcoR124I

Sudhir Kumar Pal1,2, Dhiraj Sinha1,2, David Reha1,2, Rudiger Ettrich1,2

1Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences, Zamek-136, 373 33, Nove Hrady, Czech Republic 1

2Faculty of Sciences, University of South Bohemia, Ceske Budejovice, Czech Republic 2

EcoR124I belongs to the IC family of type I restriction-modification (R–M) enzymes of E. Coli. Type I R–M enzymes are pentameric proteins comprised of 2 motor subunits (HsdR), and a trimeric methyltransferase (MTase) that includes the specificity subunit. On hemimethylated dsDNA, the enzyme complex acts solely as conventional adenine methylases. On unmethylated DNA translocation occurs independently on the motor subunits that translocate dsDNA towards the stationary enzyme. Recombination between Target Recognition Domains (TRDs) generates new sequence specificities and is a powerful driver of Type I R–M system diversification. Due to the lack of structural information, the overall atomistic mechanism of DNA recognition and modification is unknown. Based on early genetic studies a paradigm was established that mutations within the hsdS gene produce an r- m- phenotype, as do mutations within the hsdM gene while mutations in the hsdR caused Mod+ Res- (1). Res- Mod- phenotype was presumed to reflect a loss of DNA-binding ability of the HsdS subunit responsible for DNA recognition which than must prevent both restriction and modification functions. However, two point mutants (K184N) (2) and (K384N) that are mutated in the conserved helical domain of the HsdS subunit were found to have an r- m+ phenotype in vivo (including the double substitution), which could only be a result of subunits assembly defect. We generate and refine three-dimensional homology model(s) of the EcoR124I HsdS subunit and study of dynamics of WT and mutant HsdS to predict the overall affect of the experimental mutations on the dynamical behaviour and the conformational space sampled.

1. Hubacek, J. & Glover, S.W., Journal of Molecular Biology, 1970, 50, 11 1-127.

2. Obarska,A., Blundell,A., et al., Nucleic AcidsResearch, 2006, 34.