Dj. Mozzherin et al., Architecture of the active DNA polymerase delta center dot proliferating cell nuclear antigen center dot template-primer complex, J BIOL CHEM, 274(28), 1999, pp. 19862-19867
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.