Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.
The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition 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)2is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)2complex is able to bind to both the EcoR124I DNA recognition sequence GAAN6RTCG and a symmetrical DNA sequence GAAN7TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)2-DNA complex 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 bound by the (S3/HsdM)2complex.
Smith
M A
MA
Biomolecular Structure Group, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St Michael's Building, White Swan Road, Portsmouth, PO1 2DT, UK.
Read
C M
CM
Kneale
G G
GG
D001483:Base Sequence; D001667:Binding, Competitive; D002942:Circular Dichroism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
D010446:Peptide Fragments; D021122:Protein Subunits; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific