Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while “riding” the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance “vehicle” for detecting the most frequently occurring replication errors.

核糖核酸酶 HII (RNaseHII) 是从基因组 DNA 中去除错误掺入的单磷酸核糖核苷 (rNMP) 的主要酶。在这里，我们提出了结构、生化和遗传证据，证明核糖核苷酸切除修复（RER）与转录直接偶联。细胞内蛋白质间交联的亲和力下拉和质谱辅助图谱揭示了大多数 RNaseHII 分子与大肠杆菌中的 RNA 聚合酶 (RNAP) 相互作用。RNaseHII 在延伸过程中与 RNAP 结合的冷冻电镜结构，无论有或没有目标 rNMP 底物，都显示出特定的蛋白质-蛋白质相互作用，这些相互作用定义了处于接合和未接合状态的转录偶联 RER (TC-RER) 复合物。RNAP-RNaseHII 相互作用的减弱会损害体内RER 。结构功能数据支持一种模型，其中 RNaseHII 在“骑乘”RNAP 的同时在一维扫描 DNA 以搜索 rNMP。我们进一步证明 TC-RER 占修复事件的很大一部分，从而将 RNAP 建立为检测最常发生的复制错误的监视“工具”。