The DNA Binding Characteristics of the Trimeric EcoKI Methyltransferase and its Partially Assembled Dimeric Form Determined by Fluorescence Polarisation and DNA Footprinting

• Published in: Journal of molecular biology Vol. 283; no. 5; pp. 947 - 961
• Main Authors: Powell, Lynn M, Connolly, Bernard A, Dryden, David T.F
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 13.11.1998
• Subjects: Base Sequence; Binding Sites - genetics; Dimerization; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA binding; DNA Footprinting; DNA Methylation; DNA methyltransferase; DNA Restriction Enzymes - chemistry; DNA Restriction Enzymes - metabolism; Escherichia coli; Escherichia coli - enzymology; fluorescence anisotropy; Fluorescence Polarization; Fluorescent Dyes; hexachlorofluorescein; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - metabolism; Protein Conformation; S-Adenosylmethionine - metabolism; Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism; Substrate Specificity; UV; fluorescence anisotropy; TBE; hsd; DNA methyltransferase; hexachlorofluorescein; DNase I; DNA footprinting; DNA binding; bp; K d; AdoMet
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1998.2142

The type I DNA restriction and modification systems of enteric bacteria display several enzymatic activities due to their oligomeric structure. Partially assembled forms of the EcoKI enzyme from E. coli K12 can display specific DNA binding properties and modification methyltransferase activity. The heterodimer of one specificity (S) subunit and one modification (M) subunit can only bind DNA whereas the addition of a second modification subunit to form M 2S 1 also confers methyltransferase activity. We have examined the DNA binding specificity of M 1S 1 and M 2S 1 using the change in fluorescence anisotropy which occurs on binding of a DNA probe labelled with a hexachlorofluorescein fluorophore. The dimer has much weaker affinity for the EcoKI target sequence than the trimer and slightly less ability to discriminate against other DNA sequences. Binding of both proteins is strongly dependent on salt concentration. The fluorescence results compare favourably with those obtained with the gel retardation method. DNA footprinting using exonucleaseIII and DNaseI, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer. This indicates that the dimer is a mixture of the two possible forms, M 1S 1 and S 1M 1. The dimer has a footprint on the DNA substrate of the same length as the trimer implying that the modification subunits are located on either side of the DNA helical axis rather than lying along the helical axis.