The Major Roles of DNA Polymerases Epsilon and Delta at the Eukaryotic Replication Fork Are Evolutionarily Conserved

Coordinated replication of eukaryotic genomes is intrinsically asymmetric, with continuous leading strand synthesis preceding discontinuous lagging strand synthesis. Here we provide two types of evidence indicating that, in fission yeast, these two biosynthetic tasks are performed by two different replicases. First, in Schizosaccharomyces pombe strains encoding a polδ-L591M mutator allele, base substitutions in reporter genes placed in opposite orientations relative to a well-characterized replication origin are strand-specific and distributed in patterns implying that Polδ is primarily involved in lagging strand replication. Second, in strains encoding a polε-M630F allele and lacking the ability to repair rNMPs in DNA due to a defect in RNase H2, rNMPs are selectively observed in nascent leading strand DNA. The latter observation demonstrates that abundant rNMP incorporation during replication can be tolerated and that they are normally removed in an RNase H2-dependent manner. This provides strong physical evidence that Polε is the primary leading strand replicase. Collectively, these data and earlier results in budding yeast indicate that the major roles of Polδ and Polε at the eukaryotic replication fork are evolutionarily conserved.

真核生物基因组的协同复制本质上具有不对称性，即连续合成的前导链（leading strand）先于不连续合成的后随链（lagging strand）完成。本研究提供两类证据，表明在裂殖酵母中，这两项生物合成任务由两种不同的复制酶完成。其一，在携带DNA聚合酶δ（Polδ）L591M突变体等位基因的粟酒裂殖酵母（Schizosaccharomyces pombe）菌株中，相对于特征明确的复制起点（replication origin）以反向插入的报告基因所发生的碱基替换具有链特异性，且其分布模式暗示Polδ主要参与后随链复制。其二，在携带DNA聚合酶ε（Polε）M630F突变体等位基因且因核糖核酸酶H2（RNase H2）缺陷而无法修复DNA中核糖核苷酸单磷酸（rNMPs）的菌株中，核糖核苷酸单磷酸选择性地出现在新生前导链DNA中。这一发现证明，复制过程中大量掺入的核糖核苷酸单磷酸可被耐受，且通常通过RNase H2依赖的方式被清除，为Polε作为主要前导链复制酶提供了强有力的实验证据。综上，本研究数据结合芽殖酵母的既往研究结果表明，真核复制叉处Polδ与Polε的主要功能在进化上是保守的。