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Papers In Press, published online ahead of print August 22, 2001
J. Biol. Chem, 10.1074/jbc.M106373200
Submitted on July 9, 2001
Revised on August 16, 2001
Accepted on August 21, 2001
Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262
Corresponding Author: charles.mchenry{at}uchsc.edu
The delta and delta' subunits are essential components of the DNA polymerase-III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2 gamma delta’ delta chi psi). The X-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain-III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both N- and C-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. Delta and delta' form a 1:1 heterodimer with a dissociation constant (KD) of 5 x 10-7 M determined by equilibrium sedimentation. The KD determined by surface plasmon resonance was comparable. Domain-III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain-III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain-III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.
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