Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity
Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity
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DOI：
10.1016/j.jmb.2013.07.021
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发表时间：
2013-11-15
影响因子：
5.6
通讯作者：
Kuzminov, Andrei
中科院分区：
文献类型：
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作者：
Amado, Luciana;Kuzminov, Andrei
染色体DNA复制中间体，显示在体内连接酶缺乏的条件下，是低分子量（LMW）独立的生物体，这表明不连续复制的领先和落后的DNA链。然而，用纯化的酶复制σ结构底物的体外实验显示在完全不存在连接酶的情况下连续合成前导DNA链，支持教科书中的半不连续DNA复制模型。体内和体外结果之间的差异是合理的，提出了各种切除修复事件切口连续合成的前导链合成后，产生观察到的低分子量中间体。在这里，我们表明，在大肠杆菌连接酶缺陷株与所有已知的切除修复途径失活，新的DNA仍然是不连续合成。此外，与链特异性靶标的杂交表明LMW复制中间体来自滞后链和前导链。这些结果支持了大肠杆菌不连续前导链合成的模型。杆菌(C)2013爱思唯尔有限公司保留所有权利。
Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli. (C) 2013 Elsevier Ltd. All rights reserved.