Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR1241

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kD a consisting of two modification subunits, HsdM, and a single specificity s ubunit, HsdS. Studies have been largely restricted to the HsdM subunit or t o the intact methyltransferase since the HsdS subunit is insoluble when ove r-expressed independently of HsdM. Two soluble fragments of the HsdS subuni t have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S 3) comprising the N-terminal target recognition domain together with the ce ntral conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 s ubunit exists principally as a dimer of 50 kDa. Gel retardation and competi tion assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is requi red for effective DNA binding. Cooperative binding is observed and at low e nzyme concentration, the multisubunit complex dissociates, leading to a los s of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA se quence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease Ill footprinting of the (S3/HsdM)(2) -DNA comple x indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence b ound by the (S3/HsdM)(2) complex. (C) 2001 Academic Press.