R-loop-mediated genomic instability is caused by impairment of replication fork progression

Abstract

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress

“…RNase H1 specifically degrades RNA in RNA-DNA hybrids, thus removing R-loops. 22,24 To test the contribution of R-loops to Cyclin E-induced fork slowing, we transiently transfected RNase H1 into Cyclin E-overexpressing and control cells (Figure 3f). RNase H1 transfection had no effect on replication initiation (Figure 3g).…”

“…It has been previously reported that collisions between transcription and replication machineries, as well as RNA-DNA hybrids (R-loops) left behind by the transcription machinery, slow replication fork progression and activate homologous recombination. [22][23][24] Collisions of the replication and transcription machineries have also been implicated in genomic instability at common fragile sites. 25 We speculate that transcription-associated replication stress in Cyclin E-overexpressing cells could be a consequence of increased replication initiation.…”

“…Changes in origin density could also interfere with the spatial coordination of replication initiation and transcription, which may increase collisions between replication and transcription complexes, thereby causing DNA damage and genomic instability. 11,[22][23][24][25] As it is downstream of major oncogenic signaling pathways, Cyclin E seems likely to be an important mediator of oncogene effects on replication. Cyclin E is tumor promoting itself if overexpressed in mice and is found overexpressed in a variety of cancers, which correlates with low disease-free survival.…”

Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

“…In these studies, such transcriptional activity correlated with closer nuclear proximity between translocation partners, which may contribute to the increase in translocation frequency (28 -30). In addition, transcription leads to DNA/RNA hybrids (R-loops), which have been shown to cause persistent DSBs and chromosomal instability (71,72). We suggest that chromosomal DBSs in transcribed loci may also be prone to EJ events that fail to maintain correct end use, which could contribute to such chromosomal rearrangements.…”

LncRNAs: Bridging environmental sensing and gene expression

“…Aberrant formation and/or stabilization of R-loops results in genomic instability as a result of prolonged exposure of a single-stranded DNA to the environment and/or by interfering with DNA replication machinery. 61,62 In contrast, R-loops play beneficial roles by mediating CRISPR interference, 63,64 promoting IgG class switching, 65,66 and by regulating transcription. 67,68 These structures can form in cis 60 or trans 69 in vivo, either by "threading back" of nascent RNAs during transcription by RNA Pol II, through the action of the homologous recombination machinery, or associated RNA-binding proteins as in the case of CRISPR.…”