Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops

  1. Inigo Urrutia-Irazabal
  2. James R Ault
  3. Frank Sobott
  4. Nigel J Savery
  5. Mark S Dillingham

  • Curated by eLife

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    Evaluation Summary:
    The resolution of R-loops that form during collisions between replication and transcription machineries is crucial for cell survival. This is exemplified by the lethality of deletion of PcrA, a helicase that appears to be involved in the resolution of such collisions. Here, the authors aim to characterize the critical regions of PcrA/RNAP interactions and determine the function of such interactions. The manuscript's structural work is refined, elegant and leaves little room for doubt concerning the importance of the CTD PcrA-RNAP molecular interactions. This work moves the field forward in a meaningful way and unravels key aspects of PcrA/UvrD function with regards to interaction and function on RNAP. It will be of interest across the wide field of protein-DNA interactions, both prokaryotic and eukaryotic.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work, we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the β subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimize conflicts between transcription and other processes on DNA including replication.

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  1. Version published to 10.7554/elife.68829 on eLife
  2. Version published to 10.1101/2021.03.15.435481v2 on bioRxiv
  3. eLife

    Reviewer #3 (Public Review):

    In this study the authors investigate the interactions between the Helicase II orthologue PcrA and RNA polymerase. This follows on nicely from previous work that showed that the C-terminal domain (CTD) of PcrA/UvrD mediates a direct interaction with RNAP. So the authors flip the experiment and use hydrogen-deuterium exchange (HDX) mass spec to reveal the location of the interaction between the CTD and RNAP, or more specifically the beta subunit rpoB. Interestingly they discover that the binding motif is conserved across a number of PcrA partner proteins (and others), including UvrB. Further investigation into HDX of full-length revealed protection near the DNA/RNA exit channel. This leads the authors to investigate the role of this interaction. They find using the S9.6 anitbody, specific to RNA/DNA hybrids and helicase assays that PcrA can unwind RNA/DNA hybrids, perhaps those formed as R-loops during transcription. These data are compiled into a model that reiterates the UvrD-like backtracking of RNAP or - based on the HDX data - an alternative that has PcrA cleaning up after RNAP as it processes.

    Overall this is a compelling study that offers good evidence to back up many of the conclusions. The complementary approaches used help to provide wider support of their hypothesis that PcrA is involved in resolving R-loops. This view of PcrA's activity has recently received support by the work from another group (doi: 10.3390/cells10040935). The methods were clear, and appropriate. Using HDX to formulate hypotheses was a strength of this work, and the identification of a Tudor-domain binding motif in PcrA interacting proteins is of significance, because additional proteins were identified from this approach. The main weakness was that no direct evidence for how R-loops are resolved by PcrA was shown. The strongest evidence came from Figure 6A, showing a strong helicase activity on DNA/RNA hybrids, comparable to DNA/DNA, and also from Figure6D/E, showing an increase the DNA/RNA hybrids formed with the inactive PcrA-E224Q. However, this last point does not entirely clarify the mechanism, instead it simply shows that inactive PcrA enables more R-loops to form, this is not supportive of any underlying mechanism. Nonetheless, it is clear that PcrA is involved, by some mechanism, in removing R-loops.

  4. eLife

    Evaluation Summary:
    The resolution of R-loops that form during collisions between replication and transcription machineries is crucial for cell survival. This is exemplified by the lethality of deletion of PcrA, a helicase that appears to be involved in the resolution of such collisions. Here, the authors aim to characterize the critical regions of PcrA/RNAP interactions and determine the function of such interactions. The manuscript's structural work is refined, elegant and leaves little room for doubt concerning the importance of the CTD PcrA-RNAP molecular interactions. This work moves the field forward in a meaningful way and unravels key aspects of PcrA/UvrD function with regards to interaction and function on RNAP. It will be of interest across the wide field of protein-DNA interactions, both prokaryotic and eukaryotic.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  5. eLife

    Reviewer #2 (Public Review):

    Here, the authors aim to characterize the critical regions of PcrA/RNAP interactions and determine the function of such interactions. The manuscript's structural work is refined, elegant and leaves little room for doubt concerning the importance of the CTD PcrA-RNAP molecular interactions. This work moves the field forward in a meaningful way and unravels key aspects of PcrA/UvrD function with regards to interaction and function on RNAP.

    Though the in vitro work and the structural studies are very convincing, the biological connotations of this newly characterized interaction are a bit premature, with the proposed models relying heavily on implications derived from their structural data.

    The authors achieve their goal in a generally successful manner regarding the interaction domains between RpoB and PcrA. However, they focus strongly on the CTD domain. The previously suggested interactions of NTD is not explored and if pursued, could significantly improve our understanding of the structure and function of PcrA/RNAP interactions in a full manner. An additional set of experiments examining the role of NTD here would expand the scope of the study significantly.

    The suggested processing of R-Loops by PcrA through its interaction with RNAP is informative and may be very much relevant to prior findings regarding a role for PcrA in the resolution of replication-transcription conflicts.

  6. eLife

    Reviewer #1 (Public Review):

    UvrD, Rep, and PcrA are bacterial superfamily 1 (SF1) helicase that function as motor proteins during several DNA metabolic processes. Their primary roles are during DNA repair and recombination where they: 1. bind to 3' ssDNA overhangs and translocate on the DNA in a 3'-5' manner, 2. unwind dsDNA, which is coupled to their motor activity, 3. strip or remodel other proteins bound on the DNA. In this paper, the role for the PcrA helicase is proposed based on its interaction with the RNA polymerase complex. This activity occurs during transcription and thus likely serves to resolved stalled transcription events in the cell.

    The authors build on earlier discoveries that show UvrD interacting with the RNAP and controlling its movement. Similarly, the authors have previously reported on the interactions between PcrA and RNAP. While the interaction was shown before, the mechanistic details of how the two protein interacted were not complete and the functional relevance was elusive. In this study, using elegant HDX-MS analysis, they map the binding site on RNAP to a helicase-binding motif. Perturbations in this region interfere with PcrA-RNAP binding. This, in my opinion, is a super important finding as the corollary RNAP binding region on the helicase is conserved in other PcrA interactors. The authors have identified several new PcrA binding proteins and should be an exciting line of investigations going forward.

    The manuscript was a pleasure to read with sound experiments, perfect interpretations of the data (and no overinterpretations). The supporting data are well reasoned and this authors does not find any fault with the experiments, results, and the discussion of the work.

  7. Version published to 10.1101/2021.03.15.435481v1 on bioRxiv