MUTANT-DNA POLYMERASE-DELTA FROM THERMOSENSITIVE SCHIZOSACCHAROMYCES-POMBE STRAINS DISPLAY REDUCED STIMULATION BY PROLIFERATING CELL NUCLEAR ANTIGEN

We have isolated and characterized DNA polymerase delta (pol delta) fr om two thermosensitive Schizosaccharomyces pombe strains, pol delta ts 1 and pol delta ts3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 3 7 degrees C pol delta ts1 and pol delta ts3 mutant strains arrest grow th in the S phase of the cell cycle. We show that at low levels of pri mer ends, in vitro stimulation by proliferating cell nuclear antigen ( PCNA) of mutant enzymes is lower than stimulation of wild-type pol del ta. Affinity for primer (3'-OH) ends and processivity of mutant enzyme s do not appear different from wild-type pol delta. In contrast, V-max values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulti ng in reduced velocity of DNA synthesis. In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degrees C, although lo w concentrations stimulate activity at 25 degrees C: suggesting that t his thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.