Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells
Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells
复制标题
直接可视化从冈崎片段中去除 RNA-DNA 引物为真核细胞中的瓣切割和核酸外切途径提供支持
DOI：
10.1074/jbc.m116.758599
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发表时间：
2017-03-24
影响因子：
4.8
通讯作者：
Kong, Daochun
中科院分区：
文献类型：
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作者：
Liu, Bochao;Hu, Jiazhi;Kong, Daochun
在真核细胞的DNA复制过程中，被称为冈崎片段的短单链DNA片段首先在滞后链上合成。冈崎片段来源于类似于35个核苷酸长的RNA-DNA引物。冈崎片段合成后，必须去除这些引物以允许片段连接成连续的滞后链。迄今为止，去除RNADNA引物的酶机制模型几乎完全来自生化重建研究和一些遗传相互作用测定，并且几乎没有直接证据证实这些模型。阐明冈崎片段加工的一个障碍是缺乏可以直接检查体内引物去除的方法。在这项研究中，我们开发了一种电子显微镜分析，可以可视化核甘酸瓣结构的DNA复制叉在裂殖酵母（裂殖酵母）。通过该试验，我们首次证明了在冈崎片段体内加工过程中皮瓣结构的产生。野生型细胞中瓣的平均和中值长度分别类似于51和类似于41个核苷酸。我们还使用酵母突变体来研究删除关键DNA复制核酸酶对这些瓣结构的影响。我们的研究结果提供了直接的体内证据，以前提出的皮瓣切割途径和关键功能的DNA 2和Fen 1在切割这些皮瓣。此外，我们还发现了另一种先前提出的外切核酸溶解途径的证据，该途径涉及外切核酸酶RNase H2和Exo 1对RNA-DNA引物的消化。综上所述，我们的观察表明冈崎片段成熟的双重机制，在滞后链合成和建立
During DNA replication in eukaryotic cells, short single stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from similar to 35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNADNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo. In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo. The mean and median lengths of the flaps in wild-type cells were similar to 51 and similar to 41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish