DNA primase acts as a molecular brake in DNA replication
DNA primase acts as a molecular brake in DNA replication
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DOI：
10.1038/nature04317
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发表时间：
2006-02-02
期刊：
影响因子：
64.8
通讯作者：
van Oijen, AM
中科院分区：
文献类型：
--
作者：
Lee, JB;Hite, RK;van Oijen, AM
DNA复制的一个标志性特征是引导链上核苷酸的连续聚合与滞后链上DNA的不连续合成之间的协调（1）。这种同步化需要一系列精确定时的酶促步骤，这些步骤控制RNA引物的合成、滞后链DNA聚合酶的回收以及冈崎片段的产生。引物酶合成RNA引物的速率比分叉处DNA聚合酶的DNA合成速率低2-4个数量级。此外，滞后链DNA聚合酶从完成的冈崎片段到新引物的再循环固有地比核苷酸聚合的速率慢（5）。已经提出了不同的模型来解释这些缓慢的酶促步骤如何在滞后链发生而不失去与连续和快速的前导链合成的协调（6-8）。尽管如此，一个清晰的画面仍然难以捉摸。在这里，我们使用单分子技术来研究从噬菌体T7的多蛋白复制复合物的动力学和特征的影响，引发酶活性叉进展。我们观察到引物在滞后链上的合成引起高度进行性的前导链合成的短暂暂停。在前导链和滞后链合成的存在下，我们观察到滞后链上复制环的形成和释放。在环形成之前，引发酶充当分子制动器并暂时停止复制叉的进展。这一观察结果表明，在滞后链上的缓慢酶促步骤期间，防止前导链合成超过滞后链合成的机制。
A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand(1). This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower(2-4) than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization(5). Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis(6-8). Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading- strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.