Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia.
Illawarra Health and Medical Research Institute, Wollongong, NSW, 2522, Australia.
Nat Commun. 2020 Mar 20;11(1):1477. doi: 10.1038/s41467-020-15179-y.
In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.
在模式生物大肠杆菌中，UvrAB 损伤监测复合物在全局基因组核苷酸切除修复途径（GGR）中识别螺旋扭曲损伤。或者，在转录偶联修复（TCR）期间，UvrA 被募集到 Mfd 在 RNA 聚合酶因损伤而停滞的位点。最终，UvrA 介导损伤识别，然后由 UvrB 进行验证。在这里，我们通过在缺乏 TCR 的活细胞或野生型细胞中跟踪功能性、荧光标记的 UvrA 分子，来描述 UvrA 与 Mfd 和 UvrB 相互作用的动力学差异。在没有外源性 DNA 损伤的情况下，活细胞中 UvrA 与 Mfd 依赖性或 Mfd 非依赖性相互作用的寿命相当，并且受 UvrB 控制。在 UV 照射后，UvrA 的寿命强烈依赖于 Mfd，并且与 Mfd 的寿命相匹配。总的来说，我们展示了一种非侵入性的、基于成像的方法，用于定量参与细胞中多个途径的损伤识别酶的动力学特征。
