The Mammalian DNA Polymerase δ−Proliferating Cell Nuclear Antigen−Template-Primer Complex: Molecular Characterization by Direct Binding

Abstract

Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase δ (pol δ), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol δ, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol δ·PCNA· template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol δ, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol δ·template-primer complex.