The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units

• Published in: PLoS genetics 2015-06, Vol.11 (6), p.e1005289-e1005289
• Main Authors: Merrikh, Christopher N, Brewer, Bonita J, Merrikh, Houra
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Apoptosis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Chromosomes; Conflict resolution; Deoxyribonucleic acid; DNA; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Replication; E coli; Experiments; Gene Expression Regulation, Bacterial; Genes; Genes, Bacterial; Genomes; Mutation; Operon; Proteins; RNA polymerase; RNA, Ribosomal - genetics; RNA, Transfer - genetics; Transcription, Genetic
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1005289

In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA's ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA's C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results are broadly relevant.