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

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Abstract

The β subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg 2+ . Reacting β with fluoresceinmaleimide (FM) resulted in one label per β monomer with full retention of activity. Titration of FM-β with Mg 2+ 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 Ca 2+ or spermidine(3+) but not with the monovalent cations Na + and K + . the Mg 2+ -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 Mg 2+ 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
StatePublished - Jul 1 1988
Externally publishedYes

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DNA Polymerase III
Holoenzymes
Dimers
Magnesium
Escherichia coli
Monomers
Fluorescence
Gels
Monovalent Cations
Spermidine
Titration
Gel Chromatography
Glutathione
Binding Sites
Labels
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

The Dimer of the 0 Subunit of Escherichia coli DNA Polymerase III Holoenzyme Is Dissociated into Monomers upon Binding Magnesium(II). / Griep, Mark A.; McHenry, Charles S.

In: Biochemistry, Vol. 27, No. 14, 01.07.1988, p. 5210-5215.

Research output: Contribution to journalArticle

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AB - The β subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg 2+ . Reacting β with fluoresceinmaleimide (FM) resulted in one label per β monomer with full retention of activity. Titration of FM-β with Mg 2+ 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 Ca 2+ or spermidine(3+) but not with the monovalent cations Na + and K + . the Mg 2+ -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 Mg 2+ 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.

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