PCNA and DNA Polymerase δ Catalytic Subunit fromSchizosaccharomyces pombeDo Not Interact Directly

Abstract DNA polymerase δ (pol δ) is constituted of at least two subunits: the catalytic subunit of about 125 kDa (p125), and a subunit of approximately 50 kDa (p50) of unknown function. Processivity of pol δ is dependent on its auxiliary protein PCNA (proliferating cell nuclear antigen). Contradictory data were reported regarding a direct interaction between p125 and PCNA. We investigated this matter further using the baculovirus system to overexpress p125 and PCNA from S. pombe. We show that the recombinant p125 is active for basal DNA polymerase activity and for 3′→5′ exonuclease activity but is not stimulated by PCNA. Interaction between p125 and PCNA was tested by: (i) co-immunoprecipitation assay using antibodies specific for one or other polypeptides after co-expression in insect cells, and (ii) a two-hybrid assay. In both cases, no direct interaction between the two proteins was detected. Taken together, our data show that p125 and PCNA do not interact directly.