Architecture of the active DNA polymerase δ·proliferating cell nuclear antigen·template-primer complex

Abstract

The relative positions of components of the DNA-dependent DNA polymerase δ (pol δ)·proliferating cell nuclear antigen (PCNA)·DNA complex were studied. We have shown that pol δ incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol δ was nearly totally (95%) inhibited by a biotin·streptavidin complex located at the 3'-end of a template with a 15- mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin·streptavidin complex. Quantification of results with primers of varying length suggested that pol δ interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol δ, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located 'behind' pol δ in the polymerization complex during DNA synthesis and that only the large subunit of pol δ (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.