Expression and characterisation of the N-terminal fragment of the HsdS subunit of M.EcoR124I.

Abstract

The type IC modification methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two copies of the modification subunit, HsdM, and a single DNA specificity subunit, HsdS. Studies to date have been largely restricted to the HsdM subunit or the intact methyltransferase, since the HsdS subunit is insoluble when expressed independently of HsdM. Using PCR, we have cloned and expressed 13 fragments of the gene for the HsdS subunit, including the sequences encoding each of the variable and conserved domains and various combinations of these. Only two of these fragments were found to be soluble, a 8.6 kDa fragment (S11) comprising the central conserved domain and a 25 kDa N-terminal fragment (S3) containing the N-terminal variable domain and the central conserved domain. Analysis of the larger of these fragments by gel retardation shows that the protein binds DNA in the presence of HsdM at a subunit stoichiometry of 1:1. Gel filtration and CD spectroscopy indicate that the protein is monomeric and predominantly alpha-helical.