Department of BioSciences, Rice University, Houston, Texas, United States.
Department of BioSciences, Rice University, Houston, Texas, United States.
Methods Enzymol. 2022;672:103-123. doi: 10.1016/bs.mie.2022.03.009. Epub 2022 Apr 5.
Replicative helicase and polymerase form the leading-strand replisome that unwinds parental DNA and performs continuous leading-strand DNA synthesis. Uncoupling of the helicase-polymerase complex results in replication stress, replication errors, and genome instability. Although numerous replisomes from different biological systems have been reconstituted and characterized, structural investigations of the leading-strand replisome complex are hindered by its large size and dynamics. We have determined the first replisome structure on a fork substrate with bacteriophage T7 replisome as a model system. Here, we summarized our protocols to prepare and characterize the coupled T7 replisome complex. Similar methods can potentially be applied for structural investigations of more complicated replisomes.
复制解旋酶和聚合酶形成前导链复制体，解开亲本 DNA 并进行连续的前导链 DNA 合成。解旋酶-聚合酶复合物的解偶联会导致复制应激、复制错误和基因组不稳定。尽管已经重建和表征了来自不同生物系统的许多复制体，但由于其较大的尺寸和动态性，对前导链复制体复合物的结构研究受到了阻碍。我们已经确定了第一个带有噬菌体 T7 复制体作为模型系统的叉底物上的复制体结构。在这里，我们总结了制备和表征偶联 T7 复制体复合物的方案。类似的方法可能适用于更复杂的复制体的结构研究。
