Architecture of the active DNA polymerase delta center dot proliferating cell nuclear antigen center dot template-primer complex

The relative positions of components of the DNA-dependent DNA polymerase de lta (pol delta) proliferating cell nuclear antigen (PCNA) DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a t emplate biotin-streptavidin complex located 5' (downstream) to the replicat ing complex in the presence or absence of PCNA. PCNA-dependent synthesis ca talyzed by pol delta was nearly totally (95%) inhibited by a biotin strepta vidin complex located at the 3'-end of a template with a 15-mer primer (ups tream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affec ted by the biotin streptavidin complex. Quantification of results with prim ers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer term inus. Using UV photocross-linking, we determined that the 125-kDa subunit o f pol delta, but not the 50-kDa subunit, interacted with a photosensitive r esidue of a substrate oligonucleotide, Interaction apparently takes place t hrough the C terminus of p125, Based on these results, we conclude that PCN A is located "behind" pol delta in the polymerization complex during DNA sy nthesis and that only the large subunit of pol delta (two-subunit form) int eracts directly with DNA. A detailed model of the enzymatically active comp lex is proposed.