Abstract
The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template. The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains. The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta'. In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels. The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III. Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex. A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.
Highlights
The ␦Ј subunit is a C-shaped molecule comprised of three structural domains determined by x-ray crystallography [9, 18]
Different concentrations (0.83, 2.3, and 5.0 M) of full-length ␦Ј tagged at its COOH-terminal end, ␦ЈC, were incubated with followed by gel filtration, and binding was monitored by evaluating Coomassie-stained SDS-polyacrylamide gels of Superose 6 gel filtration fractions (Fig. 1)
Domain III of ␦Ј Contains the DnaX-binding Site and the Elements Required for Cooperative DnaX Complex Formation—we examined the interactions of DnaX to the truncated ␦Ј proteins lacking specific domains in the presence of ␦ and
Summary
Materials, and Buffers—Centricon-10 microconcentrators were obtained from Amicon (Millipore, Bedford, MA). Samples containing fusion proteins and DnaX alone and in various combinations with or without ␦ ϩ were incubated in a total volume of 300 –500 l in Buffer A at 15 °C for 30 min, injected onto the column at 4 °C. Protein concentrations were not constant during these binding studies because significant dilution occurs during gel filtration For this reason, KD values were calculated as a range varying from the concentration of components in the applied sample (300 l) to the diluted elution volume (1.2–1.6 ml) [31]. Biotin Blot—After separation by SDS-PAGE, proteins were transferred onto the transfer membranes and developed as described [14]
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