Abstract
The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau protein. Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance. Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V).
Highlights
The subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the ␣ subunit
We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged proteins
In systems using the reconstituted DNA polymerase III holoenzyme, subunitϪDnaB interactions stimulate the rate of helicase unwinding more than 10-fold to levels approaching the rate of fork progression in vivo
Summary
Strains—E. coli DH5␣ and HB101 were used for initial molecular cloning procedures and plasmid propagation. For N-⌬413 and N-⌬496, supernatant proteins were precipitated by adding 0.36 g of ammonium sulfate to each milliliter of cell lysates For these two fusion proteins, the imidazole concentrations in the binding and washing steps were 2 and 15 mM, respectively. Protein Sequencing—After digestion, the selected biotinylated fragments were purified from others by using Ni2ϩ-NTA chromatography with the same buffers used for N-⌬1 except that urea was added to the binding and washing buffer at 8 M final concentration. These purified biotinylated fragments were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes in 10 mM CAPS (pH 11.0) and 10% methanol at constant current (0.4 A) for 3 h. B, construction of C-terminal fusion expression plasmid with PA1 promoter
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