The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision
The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision
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DOI：
10.1074/jbc.m602320200
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发表时间：
2006-08-11
影响因子：
4.8
通讯作者：
Kuzminov, Andrei
中科院分区：
文献类型：
--
作者：
Amado, Luciana;Kuzminov, Andrei
目前在大肠杆菌中复制DNA的模型假设连续合成前导链，这是基于纯化酶的体外实验。相比之下，在大肠杆菌及其噬菌体中的体内实验中，复制中间产物的成熟被阻止，报告了滞后链和领先链的DNA合成不连续。为了解决这种差异，我们分析了来自Thya(+)大肠杆菌细胞的新生DNA物种，这些DNA物种在连接酶缺乏的条件下复制它们的DNA，以阻止复制中间产物的成熟。我们在这里报道了从连接酶缺陷细胞中分离出来的大部分新合成的DNA的长度在0.3到3kb之间，有一小部分长于11kb，但比染色体短。通过用RecBCD突变阻断线性DNA处理或用ung突变阻断尿嘧啶切除，复制中间产物的低分子量不变。这些结果与之前提出的在大肠杆菌中不连续复制前导链的说法是一致的。
The current model of DNA replication in Escherichia coli postulates continuous synthesis of the leading strand, based on in vitro experiments with purified enzymes. In contrast, in vivo experiments in E. coli and its bacteriophages, in which maturation of replication intermediates was blocked, report discontinuous DNA synthesis of both the lagging and the leading strands. To address this discrepancy, we analyzed nascent DNA species from ThyA(+) E. coli cells replicating their DNA in ligase-deficient conditions to block maturation of replication intermediates. We report here that the bulk of the newly synthesized DNA isolated from ligase-deficient cells have a length between 0.3 and 3 kb, with a minor fraction being longer that 11 kb but shorter than the chromosome. The low molecular weight of the replication intermediates is unchanged by blocking linear DNA processing with a recBCD mutation or by blocking uracil excision with an ung mutation. These results are consistent with the previously proposed discontinuous replication of the leading strand in E. coli.