Evidence that DNA polymerase ? contributes to initiation of leading strand DNA replication in Saccharomyces cerevisiae

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ? (Pol ?). Although pol2-16 mutants survive, their spore colonies are very tiny, with increased doubling time, larger than normal cells, aberrant nuclei, and rapid suppressor mutation accumulation. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack Pol ? proofreading (pol2-4), consistent with the idea that Pol ? is the major leading strand replicase. Ribonucleotides are also incorporated into the pol2-16 genome in patterns consistent with leading strand replication by Pol ? when Pol ? is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol ? contributes to initiation of leading-strand replication. We describe two possible models.