Balanced act of a leading strand DNA polymerase specific domain and its exonuclease domain promotes genome-wide replication fork symmetry

During genome replication, the leading strand DNA polymerase conducts continuous synthesis over long stretches of DNA within each replicon. Processive DNA synthesis supports symmetric progression of sister replication forks and efficient genome duplication. To address the mechanisms underlying leading strand polymerase-mediated synthesis, we examine one of its conserved domains, referred to as POPS, in the budding yeast Pol2 enzyme. We provide evidence that POPS supports replication fork symmetry and efficient genome replication via balancing the function of the Pol2 exonuclease domain. We found that the defective growth, slow S phase progression, and impaired genome synthesis associated with a POPS mutation were rescued by abolishing the Pol2 exonuclease activity. The suppressive effects further extended to the increased DNA re-arrangements in the POPS mutant and its negative genetic interactions with mutants of other genome maintenance factors. Significantly, our single molecule replicon-seq data demonstrate that the POPS mutant exhibited genome-wide replication fork asymmetry, and this defect was improved by eliminating the Pol2 exonuclease activity, thus providing a basis for its rescuing of a range of POPS mutant phenotypes. Collectively, these data suggest a model in which balanced activity between a unique Pol2 catalytic domain, and its exonuclease domain facilitates replication fork symmetry and genome maintenance.在基因组复制（genome replication）过程中，前导链DNA聚合酶（leading strand DNA polymerase）可在每个复制子（replicon）内的长段DNA上开展持续性合成。持续性DNA合成（processive DNA synthesis）能够维持姐妹复制叉（sister replication forks）的对称行进，支持高效的基因组复制。为探究前导链聚合酶介导合成的潜在机制，我们对出芽酵母Pol2酶中一个被命名为POPS的保守结构域（conserved domain）展开了研究。我们的研究证据表明，POPS可通过平衡Pol2核酸外切酶结构域（exonuclease domain）的功能，维持复制叉对称并促进高效基因组复制。我们发现，消除Pol2的核酸外切酶活性，可挽救POPS突变体所伴随的生长缺陷、S期（S phase）进程迟缓以及基因组合成受损等表型。这一挽救效应还延伸至POPS突变体中加剧的DNA重排（DNA re-arrangements），以及其与其他基因组维持因子突变体之间的负遗传相互作用（negative genetic interactions）。尤为关键的是，我们的单分子复制子测序（single molecule replicon-seq）数据显示，POPS突变体呈现全基因组范围内的复制叉不对称性，而消除Pol2的核酸外切酶活性可改善这一缺陷，这为其挽救一系列POPS突变体表型提供了理论依据。综上，上述数据支持了如下模型：独特的Pol2催化结构域与其核酸外切酶结构域之间的活性平衡，可促进复制叉对称与基因组维持。