Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly.
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.
Song
M S
MS
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
McHenry
C S
CS
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D046911:Macromolecular Substances; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D004258:DNA Polymerase III