Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo
Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo
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DOI：
10.1093/nar/gky1242
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发表时间：
2019-02-28
影响因子：
14.9
通讯作者：
Smith, Duncan J.
中科院分区：
文献类型：
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作者：
Kahli, Malik;Osmundson, Joseph S.;Smith, Duncan J.
在连接之前，在真核生物的滞后链上合成的每个冈崎片段必须经过核酸水解处理。核酸酶切割发生在由 DNA 聚合酶 delta 进行链置换合成而生成的 5 个瓣结构中。至少三种 DNA 核酸酶：Rad27 (Fen1)、Dna2 和 Exo1 与冈崎片段瓣的加工有关。然而，无论是单个核酸酶对滞后链合成的贡献，还是在它们缺失的情况下形成的 DNA 中间体的结构，都尚未在体内得到完全确定。通过有条件地消耗滞后链核酸酶并直接分析酿酒酵母体内合成的冈崎片段，我们对 Rad27、Dna2 和 Exo1 对滞后链合成的影响进行了系统评估。我们发现 Rad27 处理大部分滞后链瓣，其中 Exo1 做出了显着的额外贡献，但 Dna2 没有。当核酸酶裂解受损时，我们观察到链置换合成减少，而不是像某些模型预测的那样广泛产生长冈崎片段 5 瓣。此外，使用细胞周期限制的构建体，我们证明了冈崎片段的溶核加工和连接都可以与DNA复制脱钩，并延迟到大部分基因组的合成完成后。
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), Dna2and 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 fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces 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 cell cycle-restricted 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.