Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis

Abstract

SUMMARY Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis. We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B. subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis.

Yra1-bound RNA–DNA hybrids cause orientation-independent transcription–replication collisions and telomere instability

“…We detected R loops regardless of transcriptionreplication orientation, but when Yra1 is overexpressed, an R-loop-dependent increase in recombination occurred in both systems, whether head-on or codirectional, demonstrating that they are present in both situations. Therefore, our data indicate that R loops are formed cotranscriptionally regardless of replication and may be present before the replication fork arrives; that is, it is not the transcription-replication collisions the cause of the R loops (Bermejo et al 2011;Hamperl et al 2017;Lang et al 2017). Consistently, R loops are also observed in the codirectional orientation in a bacterial chromosome too (Lang et al 2017) and are detected in G1 cells in yeast and human cells (Castellano-Pozo et al 2012;Bhatia et al 2014).…”

“…These results argue that RNA-DNA hybrids are an important source of genome instability in systems undergoing head-on transcription-replication conflicts but not in those undergoing codirectional conflicts. This interpretation would be consistent with the recent observation in human cells using similarly designed plasmid-based constructs in which transcription is driven from a bacterial T7 promoter and replication is driven from an EpsteinBarr virus replication origin (Hamperl et al 2017) as well with R-loop-dependent replication impairment observed in the Bacillus subtillis genome (Lang et al 2017). However, none of these results provide any answer to whether R loops are formed only in head-on transcription-replication or after replication.…”

“…Therefore, our data indicate that R loops are formed cotranscriptionally regardless of replication and may be present before the replication fork arrives; that is, it is not the transcription-replication collisions the cause of the R loops (Bermejo et al 2011;Hamperl et al 2017;Lang et al 2017). Consistently, R loops are also observed in the codirectional orientation in a bacterial chromosome too (Lang et al 2017) and are detected in G1 cells in yeast and human cells (Castellano-Pozo et al 2012;Bhatia et al 2014). Therefore, we conclude that R loops are the cause and not the consequence of transcriptionreplication collisions, consistent with the genome-wide accumulation of RNA-DNA hybrids detected in our study ( Fig.…”

“…Details are as in Figure 6D. lethality in RNase H-deficient bacteria (Hamperl et al 2017;Lang et al 2017). Nevertheless, the S9.6 DRIP analysis reveals that RNA-DNA hybrids are formed in LEU2 in not only the head-on but also the codirectional system, although they do not largely compromise the genome integrity.…”

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

“…A single RNAP moving head-on can dislodge the replisome in vitro (26, 50). In vivo, in a head-on collision, the replisome disassembles, the DNA breaks, and local mutation rates more than double (7, 14, 24, 28, 38, 39, 41, 48, 55, 65). Therefore, despite the common assumption in the field, the consequences of head-on conflicts are not necessarily linked to transcription levels.…”