Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY, 10016, USA.
Howard Hughes Medical Institute, NYU Grossman School of Medicine, New York, New York, 10016, USA.
Nat Commun. 2022 Mar 30;13(1):1702. doi: 10.1038/s41467-022-28871-y.
Global Genomic Repair (GGR) and Transcription-Coupled Repair (TCR) have been viewed, respectively, as major and minor sub-pathways of the nucleotide excision repair (NER) process that removes bulky lesions from the genome. Here we applied a next generation sequencing assay, CPD-seq, in E. coli to measure the levels of cyclobutane pyrimidine dimer (CPD) lesions before, during, and after UV-induced genotoxic stress, and, therefore, to determine the rate of genomic recovery by NER at a single nucleotide resolution. We find that active transcription is necessary for the repair of not only the template strand (TS), but also the non-template strand (NTS), and that the bulk of TCR is independent of Mfd - a DNA translocase that is thought to be necessary and sufficient for TCR in bacteria. We further show that repair of both TS and NTS is enhanced by increased readthrough past Rho-dependent terminators. We demonstrate that UV-induced genotoxic stress promotes global antitermination so that TCR is more accessible to the antisense, intergenic, and other low transcribed regions. Overall, our data suggest that GGR and TCR are essentially the same process required for complete repair of the bacterial genome.
全球基因组修复 (GGR) 和转录偶联修复 (TCR) 分别被视为核苷酸切除修复 (NER) 过程中的主要和次要亚途径，该过程可从基因组中去除大体积损伤。在这里，我们在大肠杆菌中应用下一代测序测定法 CPD-seq 来测量在 UV 诱导的遗传毒性应激之前、期间和之后的环丁烷嘧啶二聚体 (CPD) 损伤水平，从而以单核苷酸分辨率确定 NER 引起的基因组恢复率。我们发现，活跃的转录不仅对于模板链 (TS) 的修复是必需的，而且对于非模板链 (NTS) 的修复也是必需的，并且大部分 TCR 不依赖于 Mfd——一种被认为在细菌中对于 TCR 是必需和充分的 DNA 转位酶。我们进一步表明，通过增加 Rho 依赖性终止子的通读，TS 和 NTS 的修复都得到了增强。我们证明，UV 诱导的遗传毒性应激促进了全局抗终止，从而使 TCR 更容易接近反义、基因间和其他低转录区域。总的来说，我们的数据表明，GGR 和 TCR 本质上是完成细菌基因组完整修复所需的相同过程。
