The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase

Abstract

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation

“…The PcrA and Mfd translocases physically interact with stalled RNAPs at lesions on the DNA template. PcrA is a pro-backtracking factor by promoting forward RNAP translocation, and Mfd might be an anti-backtracking that dislodges a stalled RNAP, as previously postulated for the isolated protein in vitro (Selby and Sancar, 1993;Ayora et al, 1996;Park et al, 2002;Deaconescu et al, 2006;Epshtein et al, 2014;Sanders et al, 2017;Ho et al, 2018;Le et al, 2018). It is likely that when damaged template bases interfere with RNAP progression, it backtracks and becomes transiently arrested.…”

“…Rep and PcrA play essential roles in the replication of extrachromosomal elements, whereas UvrD and PcrA participate in the resolution of RTCs by poorly understood mechanisms (Boubakri et al, 2010 ; Bruning et al, 2014 ; Epshtein et al, 2014 ; Merrikh et al, 2015 ). In vitro studies reveal that PcrA and UvrD interact with the RNA polymerase (RNAP), Rep interacts with the replicative DNA helicase (DnaB in Proteobacteria) and Srs2 physically interacts with Rad51 and with the PCNA sliding clamp (ortholog of bacterial DnaN) among other proteins (Antony et al, 2009 ; Guy et al, 2009 ; Kaniecki et al, 2017 ; Sanders et al, 2017 ). Absence of Escherichia coli Rep and UvrD renders cells inviable when grown in rich medium (Taucher-Scholtz et al, 1983 ), but lack of Bacillus subtilis PcrA renders cells inviable even when grown in minimal medium (Petit et al, 1998 ; Merrikh et al, 2015 ).…”

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

“…A number of proteins involved in DNA repair were upregulated in the mutant strain (23 genes Table EV5), which may, at least in part, stem from feedback mechanisms increasing the amount of these proteins to help the cell cope with increased number of transcription-replication collisions with mutagenic/DNA damaging effects. These upregulated genes included genes for mismatch repair mutS, mutL (Liu et al, 2016;LeBlanc et al, 2018), nucleotide excision repair pcrA (Sanders et al, 2017), base excision repair mutM, mutT, ung, processing of abasic sites yqfS, exoA, yshC (Lenhart et al, 2012), and genes for restart after replication-transcription collision addA, addB, recA (Shepanek et al, 1989;Krajewski et al, 2014). Interestingly, genes involved in DNA repair after UV damage (UvrABC; Lenhart et al, 2012) were either unchanged or upregulated.…”

Bacillus subtilis HelD, an RNA Polymerase Interacting Helicase, Forms Amyloid-Like Fibrils

“…HelD (also known as YvgS), is one such RNAP binding protein in Bacillus subtilis which belongs to the superfamily I of DNA and RNA helicases ( Delumeau et al, 2011 ; Wiedermannova et al, 2014 ). Helicases are known to interact with the replication and DNA repair machinery ( Tuteja and Tuteja, 2004 ), but recent reports unravel their interactions with the transcription machinery as well, especially with RNAP ( Gwynn et al, 2013 ; Epshtein et al, 2014 ; Wiedermannova et al, 2014 ; Sanders et al, 2017 ). These studies suggest that helicases are diverse enzymes which play important roles in maintaining the genome stability and help in resolving the conflicts between replication and transcription ( Yang, 2010 ).…”

“…These studies suggest that helicases are diverse enzymes which play important roles in maintaining the genome stability and help in resolving the conflicts between replication and transcription ( Yang, 2010 ). Escherichia coli UvrD ( Epshtein et al, 2014 ) and Geobacillus stearothermophilus PcrA ( Gwynn et al, 2013 ; Sanders et al, 2017 ), homologs of B. subtilis HelD has been reported to interact with RNAP, thereby, coupling replication and DNA repair with transcription. Both UvrD and PcrA bind and facilitate the back-tracking of RNAP, thus, recruiting nucleotide excision repair machinery, hence, playing an important role in transcription-coupled DNA repair ( Gwynn et al, 2013 ; Epshtein et al, 2014 ; Sanders et al, 2017 ).…”