Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids.
Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids.
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枯草芽孢杆菌编码裂解RNA-DNA杂种的离散皮瓣核酸内切酶。
DOI：
10.1371/journal.pgen.1010585
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发表时间：
2023-05
期刊：
影响因子：
4.5
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中科院分区：
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目前细菌中Okazaki片段成熟的模型是通过RNaseH切割RNA，然后通过DNA聚合酶I(Pol I)进行链置换合成和5‘RNA翻盖去除。Pol I被认为是通过位于蛋白质N末端的5‘-3’翻盖内切/外切核酸酶(FEN)结构域去除RNA。除Pol I外，许多细菌还编码第二个不依赖Pol I的Fen。POL I和POL I非依赖性FENS对DNA复制和基因组稳定性的贡献尚不清楚。在这项工作中，我们纯化了枯草芽孢杆菌Pol I和Fen，然后在各种RNA-DNA杂化和DNA纯底物上对这些蛋白质进行了检测。我们发现，在镍双瓣、5‘单瓣和镍RNA-DNA杂化底物上，Fen的活性远高于Pol I。我们发现枯草杆菌PolI的5‘核酸酶活性很弱，即使在DNA合成过程中，当模拟Okazaki片段中间体形成5’折叠底物时也是如此。在纯DNA底物上对Pol I和Fen的检测表明，在所测试的大多数底物上，Fen比Pol I更具活性。进一步的实验表明，通过表达C端聚合酶结构域可以完全挽救ΔPolA表型，而N端5‘核酸酶结构域的表达不能补充ΔPola。缺乏FEN的细胞(ΔFENA)表现出与RNaseHIII缺陷相关的表型，为FEN参与冈崎片段加工提供了遗传学证据。根据这些结果，我们提出了一个模型，其中细胞使用Fen去除RNAi，而上游冈崎片段通过POL I合成延伸。我们的模型类似于真核生物中的冈崎片段处理，其中POLδ催化链置换合成，然后使用FEN-1进行5‘翻盖切割。我们的工作共同强调了在从细菌到人类的各种细胞中冈崎片段处理的有序步骤的保守。具有5‘折叠内切/外切核酸酶(FEN)活性的蛋白质在所有细胞生命中对DNA复制和修复起着至关重要的作用。在细菌中，DNA聚合酶I被认为是参与冈崎片段加工的中心酶，利用其DNA聚合酶和5‘核酸酶活性来产生并移除冈崎片段的5’单链RNA片段。许多细菌基因组除了Pol I外，还编码第二个离散的Fen。我们证明Fen是枯草杆菌用来去除引物的主要5‘核酸酶。Fen在大多数底物上的活性超过Pol I，包括几种模仿Okazaki片段中间体的底物。此外，我们还提供了遗传学证据，表明Fen参与了Okazaki片段的处理，并且在体内重要的是Pol I的DNA聚合酶结构域，而不是它的5‘核酸酶结构域。根据我们的结果，我们提出了一个新的模型，用于处理枯草杆菌中的冈崎片段，这可能在比先前所认识的更广泛的细菌群中流行。
The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5′ RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5′-3′ flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5′ single flap, and nicked RNA-DNA hybrid substrates. We show that the 5′ nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5′ flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5′ nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5′ flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human. Proteins with 5′ flap endo/exonuclease (FEN) activity provide an essential contribution to DNA replication and repair in all cellular life. In bacteria, DNA polymerase I is thought to be the central enzyme involved in Okazaki fragment processing, using its DNA polymerase and 5′ nuclease activities to generate and then remove the 5′ ssRNA segment of an Okazaki fragment. Many bacterial genomes encode a second, discrete FEN in addition to Pol I. We show that FEN is the primary 5′ nuclease used by B. subtilis for primer removal. FEN activity exceeds that of Pol I on most substrates, including several that mimic Okazaki fragment intermediates. Additionally, we provide genetic evidence showing that FEN is involved in Okazaki fragment processing and that it is the DNA polymerase domain of Pol I rather than its 5′ nuclease domain that is important in vivo. With our results, we propose a new model for Okazaki fragment processing in B. subtilis, which may be prevalent in a wider group of bacteria than previously appreciated.
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影响因子：
--
作者：
Finger, L David;Atack, John M;Shen, Binghui
通讯作者：
Shen, Binghui
影响因子：
3.2
作者：
Fukushima, Sanae;Itaya, Mitsuhiro;Yoshikawa, Hirofumi
通讯作者：
Yoshikawa, Hirofumi
DOI：
10.1073/pnas.94.5.1619
发表时间：
1997-03-04
影响因子：
11.1
作者：
Joyce, CM
通讯作者：
Joyce, CM
影响因子：
3.6
作者：
Duigou, S;Ehrlich, SD;Noirot-Gros, MF
通讯作者：
Noirot-Gros, MF
影响因子：
14.9
作者：
Hernandez-Tamayo, Rogelio;Oviedo-Bocanegra, Luis M.;Graumann, Peter L.
通讯作者：
Graumann, Peter L.