Lee Jong-Bong, Hite Richard K, Hamdan Samir M, Xie X Sunney, Richardson Charles C, van Oijen Antoine M
Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
Nature. 2006 Feb 2;439(7076):621-4. doi: 10.1038/nature04317.
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. 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 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. 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. 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.
DNA复制的一个标志性特征是前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。这种同步需要一系列精确计时的酶促步骤，这些步骤控制RNA引物的合成、滞后链DNA聚合酶的循环利用以及冈崎片段的产生。引发酶合成RNA引物的速度比复制叉处DNA聚合酶合成DNA的速度低几个数量级。此外，滞后链DNA聚合酶从完成的冈崎片段循环到新引物的过程本质上比核苷酸聚合的速度慢。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上发生，而又不会与连续且快速的前导链合成失去协调。尽管如此，清晰的图景仍然难以捉摸。在这里，我们使用单分子技术来研究噬菌体T7的多蛋白复制复合物的动力学，并表征引发酶活性对复制叉进展的影响。我们观察到滞后链上引物的合成会导致高度连续的前导链合成短暂暂停。在同时存在前导链和滞后链合成的情况下，我们观察到滞后链上复制环的形成和释放。在环形成之前，引发酶起到分子刹车的作用，暂时阻止复制叉的前进。这一观察结果提示了一种机制，可防止在滞后链上的缓慢酶促步骤期间前导链合成超过滞后链合成。
