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

Mark A. Griep, Charles S. McHenry

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5210-5215
Number of pages6
JournalBiochemistry
Volume27
Issue number14
DOIs
Publication statusPublished - Jul 1 1988

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this