Evidence that DNA polymerase d 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 d (Pol d). 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 d proofreading (pol2-4), consistent with the idea that Pol d is the major leading strand replicase. Ribonucleotides are also incorporated into the pol2-16 genome in patterns consistent with leading strand replication by Pol d when Pol d is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol d contributes to initiation of leading-strand replication. We describe two possible models. Overall design: Examination of location of ribonucleotides in multiple yeast strains bearing different DNA polymerase variants, either proficient or deficient in rnh201.