The Dimer of the 0 Subunit of Escherichia coli DNA Polymerase III Holoenzyme Is Dissociated into Monomers upon Binding Magnesium(II)

The β subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg²⁺. Reacting β with fluoresceinmaleimide (FM) resulted in one label per β monomer with full retention of activity. Titration of FM-β with Mg²⁺resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per 13 monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca²⁺or spermidine(3+) but not with the monovalent cations Na⁺and K⁺. the Mg²⁺-induced fluorescence enhancement was specific for FM-β and was not observed with FM-glutathione, dimethoxystilbenemaleimide-β, or pyrenylmaleimide-β. Gel filtration studies indicated that the β dimer-monomer dissociation occurred at physiologically significant β concentrations and that the presence of 10 mM Mg²⁺shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies β dissociation is due to a relief from homoquenching of FM when the β dimer dissociates into monomers.