Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme.
Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.
Griep
M A
MA
Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center 80262.
McHenry
C S
CS
D000838:Anions; D002712:Chlorides / Q000008:administration & dosage / Q000494:pharmacology; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004347:Drug Interactions; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000008:administration & dosage / Q000494:pharmacology*; D018698:Glutamic Acid; D006358:Hot Temperature; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D012492:Salts / Q000008:administration & dosage / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D004277:DNA, Single-Stranded; D005971:Glutamates; D019384:Nucleic Acid Synthesis Inhibitors; D012492:Salts; D018698:Glutamic Acid; D012965:Sodium Chloride; D004258:DNA Polymerase III