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The influence of quaternary structure on the function of the Type IC restriction endonuclease EcoR124IDr. Pavel Janscak1 and Keith Firman2Dr Keith Firman, Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth, PO1 2DT, United Kingdom Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, 14240 Prague 4, Czech Republic. Abstract:Type I restriction endonucleases are composed of three different subunits of which HsdS subunit determines DNA specificity, HsdM subunit is responsible for DNA methylation and HsdR subunit is required for restriction. HsdM subunit together with HsdS subunit can produce an independent trimeric DNA methyltransferase with stoichiometry of M2S1. Gel retardation analysis of EcoR124I restriction endonuclease, bound to a 39-mer DNA oligoduplex containing one EcoR124I recognition site, revealed that the purified protein was a mixture of two stoichiometric species forming two different specific DNA-protein complexes. Subunit analysis of protein isolated from both complexes has shown that the larger species has a stoichiometry of R2M2S1. The smaller species has a stoichiometry R1M2S1 and appears to be an intermediate in the endonuclease assembly pathway from the trimeric methylase and HsdR subunit. Enzyme assays have shown that only the R2M2S1 complex is capable of DNA cleavage, however, R1M2S1 complex still retains ATPase and DNA methylation activities. Measuring the equilibrium dissociation constants of the subunit complexes has shown that the M2S1 methylase and R1M2S1 are very strong complexes with a Kd values lower than 10-9 M while the R2M2S1 species is a much weaker complex and it dissociates to R1M2S1 and free HsdR subunit with a Kd of 2.2 x 10-7M. The anti-restriction polypeptide Stp, encoded by bacteriophage T4, appears to operate through a major structural change in the endonuclease. The relevance of the stoichiometry to the above situations will be discussed in terms of the quaternary structure of the endonuclease.
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