Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.
Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States.
Methods Enzymol. 2021;661:53-75. doi: 10.1016/bs.mie.2021.08.015. Epub 2021 Sep 21.
Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.
在哺乳动物细胞的 S 期，复制叉在通过常染色质和异染色质时会遇到许多挑战。这些挑战包括各种阻碍复制酶解开 DNA 的因素，如替代 DNA 结构、转录复合物和 R 环、DNA-蛋白质复合物以及 DNA 化学加合物。我们对这些事件的了解主要基于对复制压力和 DNA 损伤反应标记物的分析，这些标记物是在复制体停滞之后出现的。为了更直接地研究复制体的后果，我们开发了一种方法来观察复制叉与链间交联（ICL）这种强阻断物的碰撞。该策略基于在 DNA 纤维上可视化复制片段与标记抗原的 ICL 的相遇。我们的研究揭示了在完整 ICL 后出乎意料地重新开始 DNA 合成。此外，同样出乎意料的是，我们发现了两种不同的复制体，一种偏向常染色质，另一种在异染色质中更为突出。在这里，我们介绍了导致这些观察结果的实验细节。
