Characterization of the unique C terminus of the Escherichia coli τ DnaX protein: Monomeric C-τ binds α and DnaB and can partially replace τ in reconstituted replication forks Journal Article
| Authors: | Dallmann, H. G.; Kim, S.; Pritchard, A. E.; Marians, K. J.; McHenry, C. S. |
| Article Title: | Characterization of the unique C terminus of the Escherichia coli τ DnaX protein: Monomeric C-τ binds α and DnaB and can partially replace τ in reconstituted replication forks |
| Abstract: | A contact between the dimeric τ subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643650). In this report, we exploit the OmpT protease to generate C-τ, a protein containing only the unique C- terminal sequences of τ, free of the sequences shared with the alternative γ frameshifting product of dnaX. We have established that C-τ is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation. Monomeric C-τ binds the α catalytic subunit of DNA polymerase III with a 1:1 stoichiometry. C-τ also binds DnaB, revealed by a coupled immunoblotting method. C-τ restores the rapid replication rate of inefficient forks reconstituted with only the γ dnaX gene product. The acceleration of the DnaB helicase can be observed in the absence of primase, when only leading- strand replication occurs. This indicates that C-τ, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form. |
| Keywords: | dna binding protein; nonhuman; dna replication; carboxy terminal sequence; enzyme activity; bacteria (microorganisms); bacterial proteins; kinetics; escherichia coli; dimerization; protein structure, quaternary; molecular weight; protein determination; dna helicases; protein dna interaction; macromolecular substances; stoichiometry; dna directed dna polymerase gamma; dna b; replication origin; dnab helicases; dna polymerase iii; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 20 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-05-19 |
| Start Page: | 15512 |
| End Page: | 15519 |
| Language: | English |
| DOI: | 10.1074/jbc.M909257199 |
| PUBMED: | 10748120 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
