Escherichia coli DnaX product, the τ subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity

S. H. Lee, J. R. Walker

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the γ and τ subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the γ and τ subunits of holoenzyme. To characterize biochemically the τ subunit, for which there has been no activity assay, the dnaZX gene was fused to the β-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (τ) were replaced by β-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained β-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-β-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the β-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the γ subunit.

Original languageEnglish (US)
Pages (from-to)2713-2717
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number9
DOIs
StatePublished - Jan 1 1987

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