Breaking bad: R-loops and genome integrity

Abstract

R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded (ss) DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin (Ig) class switching, and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. We discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability, and their potential contribution to the origin of diseases including cancer and neurodegenerative disorders.

Keywords: R-loop; epigenomic stability; gene expression; genomic instability.

Figure 1. Physiological roles of R-loops in cells

R-loops are involved in bacterial and mitochondrial DNA replication, class-switch recombination, gene expression, and DNA repair. G4 quadruplexes are proposed to form on the non-template strand.

Figure 2. Formation of R-loop-mediated single-strand breaks

AID/APOBEC enzymes can deaminate cytosine residues on the non-template DNA, but also on the template DNA once the RNA molecule is displaced by the RNA exosome. The base excision repair enzyme uracil DNA glycosylase then excises the uracil base to create an abasic site, which can be processed by apurinic-apyrimidinic endonucleases to create a single-strand break (SSB).

Figure 3. Model for the processing of R-loops into DSBs

(A) The endonucleases XPF and XPG can excise the RNA-DNA hybrid of an R-loop. The single-stranded gap may be converted into a DSB either by the action of structure-specific endonucleases, or by encounter with the replication fork, which can also involve structure-specific endonucleases. (B) The endonucleases XPF and XPG can recognize both strands of an R-loop and generate a DSB. (C) The TC-NER machinery may be recruited to R-loops during replication, and the concerted action of the endonucleases XPF and XPG with other structure-specific endonucleases may generate a DSB.

Figure 4. R-loops are associated with chromatin marks

(A) R-loops influence both transcriptional initiation and termination. R-loops located at promoters prevent DNA methylation, which leads to the establishment of active chromatin marks and transcriptional initiation. By contrast, R-loops located at termination regions induce the formation of heterochromatin promoting transcriptional termination. In addition, the RNA/DNA helicase Senataxin resolve R-loops at termination regions to promote transcriptional termination. It is still unknown if these two mechanisms are interconnected. (B) R-loops can form at genes with trinucleotide repeats and induce the formation of chromatin marks that silence the gene of interest. R-loops formed at these genes might also prevent further trinucleotide expansions. Gene silencing and expansion can lead to disease.