Helicases and Genomic Integrity Section, Translational Gerontology Branch, National Institute on Aging, NIH, Baltimore, MD, United States.
Methods Enzymol. 2022;672:153-171. doi: 10.1016/bs.mie.2022.04.003. Epub 2022 Apr 28.
Helicases, DNA translocases, nucleases and DNA-binding proteins play integral roles in protecting replication forks in human cells. Perturbations to replication fork dynamics can be caused by genetic loss of key factor(s) or exposure to replication stress inducing agents that perturb the nucleotide pool, stabilize unusual DNA secondary structures, or inhibit protein function (typically catalytic activity performed by a DNA polymerase, nuclease or helicase). DNA fiber analysis is a highly resourceful and facile experimental approach to study the molecular dynamics of replication forks in living cells. In this chapter, we provide a detailed list of reagents, equipment and experimental strategies to perform DNA fiber experiments. We have utilized these approaches to characterize the role of the Werner syndrome helicase (WRN) to protect replication forks in cells that are deficient in the tumor suppressor and genome stability factor BRCA2.
解旋酶、DNA 转位酶、核酸酶和 DNA 结合蛋白在保护人类细胞中的复制叉方面发挥着重要作用。复制叉动力学的扰动可能是由关键因子的遗传缺失或暴露于复制应激诱导剂引起的，这些诱导剂会扰乱核苷酸池、稳定异常的 DNA 二级结构或抑制蛋白质功能（通常是由 DNA 聚合酶、核酸酶或解旋酶执行的催化活性）。DNA 纤维分析是一种非常有帮助且简单的实验方法，可用于研究活细胞中复制叉的分子动力学。在本章中，我们提供了详细的试剂、设备和实验策略列表，以进行 DNA 纤维实验。我们利用这些方法来描述 Werner 综合征解旋酶 (WRN) 在 BRCA2 肿瘤抑制因子和基因组稳定性因子缺陷的细胞中保护复制叉的作用。
