Characterization of the unique C terminus of the Escherichia coli tau DnaX protein. Monomeric C-tau binds alpha AND DnaB and can partially replace tau in reconstituted replication forks.
A contact between the dimeric tau 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, 643-650). In this report, we exploit the OmpT protease to generate C-tau, a protein containing only the unique C-terminal sequences of tau, free of the sequences shared with the alternative gamma frameshifting product of dnaX. We have established that C-tau is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation. Monomeric C-tau binds the alpha catalytic subunit of DNA polymerase III with a 1:1 stoichiometry. C-tau also binds DnaB, revealed by a coupled immunoblotting method. C-tau restores the rapid replication rate of inefficient forks reconstituted with only the gamma 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-tau, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form.
Dallmann
H G
HG
Department of Biochemistry and Molecular Genetics and Molecular Biology Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Kim
S
S
Pritchard
A E
AE
Marians
K J
KJ
McHenry
C S
CS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D018741:Replication Origin
D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D046911:Macromolecular Substances; D004258:DNA Polymerase III; D004265:DNA Helicases; D053488:DnaB Helicases