Cell
Volume 186, Issue 11, 25 May 2023, Pages 2425-2437.e21
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Article
RNA polymerase drives ribonucleotide excision DNA repair in E. coli

https://doi.org/10.1016/j.cell.2023.04.029Get rights and content
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Highlights

  • Majority of RNaseHII molecules interact with RNA polymerase (RNAP) in E. coli cells

  • RNaseHII-RNAP interaction facilitates ribonucleotide excision repair (RER)

  • RER is the second transcription-coupled (TC) DNA repair pathway in E. coli

  • Cryo-EM structures of the TC-RER complex provide mechanistic insights

Summary

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.

Keywords

RNA polymerase
transcription elongation
DNA repair
RNaseHII
cryo-EM

Data and code availability

Coordinates for the structural models of EC18–RNaseHII and EC18(rGMP)–RNaseHII(E17A) have been deposited to the Protein Data Bank under PDB accession numbers 7UWE and 7UWH, respectively. The cryo-EM density maps for EC18–RNaseHII and EC18(rGMP)–RNaseHII(E17A) have also been deposited to the Electron Microscopy Data Bank under accession number EMD-26830 and EMD-26832, respectively. All deposited data is publicly available as of the date of publication.

This paper does not report the original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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