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J. Biol. Chem., Vol. 276, Issue 44, 40668-40679, November 2, 2001

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Carboxyl-terminal Domain III of the ' Subunit of the DNA Polymerase III Holoenzyme Binds ^*

Min-Sun Song, H. Garry Dallmann, and Charles S. McHenry

From the Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262

The  and ' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (₂'). The x-ray crystal structure of ' 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 -binding domain of ' to a carboxyl-terminal domain III by quantifying the interaction of  with a series of ' 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 NH₂- and COOH-terminal-tagged full-length ' were soluble and had specific activities comparable with that of native '.  and ' form a 1:1 heterodimer with a dissociation constant (K_D) of 5 × 10⁷ M determined by equilibrium sedimentation. The K_D determined by surface plasmon resonance was comparable. Domain III alone bound  at an affinity comparable to that of wild type ', whereas proteins lacking domain III did not bind . Using a panel of domain-specific anti-' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with -' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.

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