Evidence that DNA polymerase δ 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 δ (Pol δ). 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 δ proofreading (pol2-4), consistent with the idea that Pol δ is the major leading strand replicase. Ribonucleotides are also incorporated into the pol2-16 genome in patterns consistent with leading strand replication by Pol δ when Pol δ is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.为在体内探究核DNA复制的酶学机制，我们对携带pol2-16突变的酿酒酵母（Saccharomyces cerevisiae）菌株开展了研究，该突变可使DNA聚合酶δ（DNA polymerase δ，Pol δ）的催化活性丧失。尽管pol2-16突变体能够存活，但其孢子菌落体积极小，倍增时间延长，细胞尺寸大于正常细胞，细胞核形态异常，且抑制突变的积累速度显著加快。上述表型显示该菌株存在严重的生长缺陷，该缺陷与缺失Pol δ校正功能的pol2-4菌株所表现出的生长缺陷存在显著差异，这与Pol δ作为主要前导链复制酶的理论相一致。当Pol δ功能缺失时，核糖核苷酸会以符合Pol δ介导前导链复制的模式掺入pol2-16突变体的基因组中。更为关键的是，复制起点处的核糖核苷酸分布特征表明，在携带全部三种复制酶的菌株中，Pol δ参与了前导链复制的起始过程。我们在此提出两种可能的模型。