THE DNA-BINDING CHARACTERISTICS OF THE TRIMERIC ECOKI METHYLTRANSFERASE AND ITS PARTIALLY ASSEMBLED DIMERIC FORM DETERMINED BY FLUORESCENCEPOLARIZATION AND DNA FOOTPRINTING

The type I DNA restriction and modification systems of enteric bacteri a display several enzymatic activities due to their oligomeric structu re. Partially assembled forms of the EcoKI enzyme from E. coli K12 can display specific DNA binding properties and modification methyltransf erase 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,S, also confers methyltransferas e activity. We have examined the DNA binding specificity of M,S, and M ,S, using the change in fluorescence anisotropy which occurs on bindin g of a DNA probe labelled with a hexachlorofluorescein fluorophore. Th e dimer has much weaker affinity for the EcoKI target sequence than th e trimer and slightly less ability to discriminate against other DNA s equences. Binding of both proteins is strongly dependent on salt conce ntration. The fluorescence results compare favourably with those obtai ned with the gel retardation method. DNA footprinting using exonucleas eIII and DNaseI, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer. This ind icates that the dimer is a mixture of the two possible forms, M,S, and S,M,. The dimer has a footprint on the DNA substrate of the same leng th as the trimer implying that the modification subunits are located o n either side of the DNA helical axis rather than lying along the heli cal axis. (C) 1998 Academic Press.