Synthetic peptides to selected sequences in human DNA polymerase delta (pol delta) were used to identify the region involved in the interaction of pol delta to proliferating cell nuclear antigen. Peptides corresponding to sequences in five regions in the amino terminus of human pol delta and three in the carboxyl terminus, which are conserved with the yeast homologs of pol delta, were tested. These studies showed that the peptide corresponding to the N2 region (residues 129-149) selectively and specifically inhibited the PCNA stimulation of pol delta. This inhibition was relieved by titration with excess PCNA. The identification of the N-2 region as being involved in PCNA binding was supported by studies that demonstrated that the N2 peptide could bind PCNA. Deletion mutants of pol delta expressed in Sf9 cells provided evidence that the binding region for PCNA was located in the first 182 residues of the amino terminus. These studies provide reasonable evidence that residues within the region 129-149 of pol delta are involved in the binding site for PCNA.

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

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