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.
Highlights
Helicases are conserved proteins found in all kingdoms of life
In the B. subtilis system, purified PcrA and RNA polymerase (RNAP) do clearly interact in the absence of nucleic acids, we found that a DNA/RNA scaffold which mimics an elongation complex was required to form a sufficiently stable complex to resolve using size-exclusion chromatography (SEC) (Figure 1—figure supplement 1A–B)
The UvrD-like family of helicases play a critical role at the interface of such pathways, orchestrating DNA repair as damage arises and helping to minimise conflicts that would otherwise occur between DNA replication and transcription (Dillingham, 2011)
Summary
Helicases are conserved proteins found in all kingdoms of life. They are involved in a wide variety of DNA transactions including replication, repair, recombination, and transcription. The PcrA/UvrD helicase has roles in rolling circle replication, nucleotide excision repair (NER), mismatch repair (MMR), and homologous recombination (HR) (Bruand and Ehrlich, 2000; Husain et al, 1985; Lahue et al, 1989; Merrikh et al, 2015; Petit et al, 1998; Zieg et al, 1978) This adaptability is provided by the many different interaction partners, for example RepC/D, UvrB, MutL and RecA, which recruit the helicase to their respective pathways via physical interactions (Hall et al, 1998; Machon et al, 2010; Manelyte et al, 2009; Veaute et al, 2005).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
