PCNA AND DNA-POLYMERASE-DELTA CATALYTIC SUBUNIT FROM SCHIZOSACCHAROMYCES-POMBE DO NOT INTERACT DIRECTLY

DNA polymerase delta (pol delta) is constituted of at least two subuni ts: the catalytic subunit of about 125 kDa (p125), and a subunit of ap proximately 50 kDa (p50) of unknown function. Processivity of pol delt a is dependent on its auxiliary protein PCNA (proliferating cell nucle ar antigen). Contradictory data were reported regarding a direct inter action between p125 and PCNA. We investigated this matter further usin g the baculovirus system to overexpress p125 and PCNA from S. pombe. W e show that the recombinant p125 is active for basal DNA polymerase ac tivity and for 3'-->5' exonuclease activity but is not stimulated by P CNA. Interaction between p125 and PCNA was tested by: (i) co-immunopre cipitation assay using antibodies specific for one or other polypeptid es after co-expression in insect cells, and (ii) a two-hybrid assay. I n both cases, no direct interaction between the two proteins was detec ted. Taken together, our data show that p125 and PCNA do not interact directly. (C) 1997 Academic Press.