Quinet Annabel, Carvajal-Maldonado Denisse, Lemacon Delphine, Vindigni Alessandro
Saint Louis University School of Medicine, St. Louis, MO, United States.
Saint Louis University School of Medicine, St. Louis, MO, United States.
Methods Enzymol. 2017;591:55-82. doi: 10.1016/bs.mie.2017.03.019. Epub 2017 May 3.
Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach. We also review how DNA fiber analysis can be used to shed light upon several replication parameters including fork progression, restart, termination, and new origin firing. Next, we discuss a modified DNA fiber protocol to monitor the presence of single-stranded DNA (ssDNA) gaps on ongoing replication forks. ssDNA gaps are very common intermediates of several replication stress response mechanisms, but they cannot be detected by standard DNA fiber approaches due to the resolution limits of this technique. We discuss a novel strategy that relies on the use of an ssDNA-specific endonuclease to nick the ssDNA gaps and generate shorter DNA fibers that can be used as readout for the presence of ssDNA gaps. Finally, we describe a follow-up DNA fiber approach that can be used to study how ssDNA gaps are repaired postreplicatively.
了解基因毒性应激诱导后复制应激反应的机制正迅速成为细胞存活和人类疾病研究的核心主题。DNA纤维分析是在单分子分辨率下全基因组研究复制叉动力学变化的最强大工具之一。这种方法依赖于许多生物体将胸腺嘧啶类似物掺入复制DNA的能力，并且被广泛用于研究基因毒性剂如何干扰DNA复制。在这里，我们回顾了制备DNA纤维的不同方法，并讨论了每种方法的重要局限性。我们还回顾了DNA纤维分析如何用于阐明几个复制参数，包括叉进展、重启、终止和新起点激发。接下来，我们讨论一种改良的DNA纤维方案，以监测正在进行的复制叉上单链DNA（ssDNA）间隙的存在。ssDNA间隙是几种复制应激反应机制中非常常见的中间体，但由于该技术的分辨率限制，它们无法通过标准DNA纤维方法检测到。我们讨论了一种新策略，该策略依赖于使用ssDNA特异性核酸内切酶切割ssDNA间隙并生成更短的DNA纤维，这些纤维可用于作为ssDNA间隙存在的读数。最后，我们描述了一种后续DNA纤维方法，可用于研究ssDNA间隙如何在复制后进行修复。
