Carboxyl-terminal domain III of the delta' subunit of the DNA polymerase III holoenzyme binds delta.
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 (tau2gammadeltadelta'chipsi). 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 NH2- and COOH-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 (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) 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.
Song
M S
MS
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Dallmann
H G
HG
McHenry
C S
CS
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D004258:DNA Polymerase III / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004258:DNA Polymerase III