Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5’ flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5’ flaps, as predicted by some models. Further, using cyclin-controlled constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.在连接反应完成前，真核生物后随链上合成的每一段冈崎片段（Okazaki fragment）均需经核酸酶水解加工。核酸酶切割发生在DNA聚合酶δ（DNA polymerase delta）通过链置换合成所形成的5'侧翼结构（5’ flap）的背景下。目前已有至少三种DNA核酸酶——Rad27（Fen1）、Dna2及Exo1——被证实参与冈崎片段侧翼结构的加工过程。然而，无论是单个核酸酶在后随链合成中的具体贡献，还是缺失这些核酸酶时所形成的DNA中间产物的结构，均未在活体（in vivo）条件下得到明确界定。本研究通过条件性敲除后随链相关核酸酶，并直接分析酿酒酵母（S. cerevisiae）体内合成的冈崎片段，对Rad27、Dna2和Exo1在后随链合成中的影响开展了系统性评估。研究发现，Rad27负责加工绝大多数后随链侧翼结构，Exo1可额外发挥显著辅助作用，而Dna2则无相关功能。当核酸酶切割功能受损时，我们观察到链置换合成水平下降，而非部分模型所预测的广泛产生长片段冈崎片段5'侧翼结构。此外，通过使用受细胞周期蛋白（cyclin）调控的重组构建体，我们证实冈崎片段的核酸酶水解加工与连接反应均可脱离DNA复制过程，并被延迟至全基因组绝大多数区域合成完成之后。