Interplay between DNA sequence and negative superhelicity drives R-loop structures

Abstract

R-loops are abundant three-stranded nucleic-acid structures that formin cisduring transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms—junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

“…As observed for RNA polymerase II-driven genes, R-loops defined a range of discrete overlapping molecular clusters over the region (Figure 4A). Thus, the formation of such clusters is observed for both RNA Pol I and RNA Pol II-driven R-loops, as well as R-loops generated in vitro by the bacteriophage T3 RNA polymerase [31]. 18S RNA R-loop lengths were within the range measured for protein-coding genes with evidence for kilobase-length structures (Figure 4B).…”

ADAR1 can drive Multiple Myeloma progression by acting both as an RNA editor of specific transcripts and as a DNA mutator of their cognate genes

“…More importantly, our analyses of these matched datasets revealed a concurrence between editing and the acquisition of new mutations at genes whose cognate transcripts are edited, and at specific predetermined locations surrounding the site-to-be-edited. Such locations are defined by the edited adenosine within a transient RNA:DNA hybrid (an R-loop) with an average size range of ~80bp-300bp - (Chen et al, 2019;Malig et al, 2019;Stolz et al, 2019) direct DNA A-to-I deamination would then be centered around the deoxyadenosine whose riboadenosine counterpart is edited in mRNA (e.g. an adenosine in the 3'UTR), converting it to deoxyinosine (hypoxanthine).…”

The sequence of the extruded non-template strand determines the architecture of R-loops

“…Non-denaturing bisulfite probing can be used to map R-loops owing to the presence of bisulfite-reactive single-stranded DNA on the looped out non-template DNA strand (12). Bisulfitemediated deamination of susceptible cytosines to uracils provides a convenient readout for singlestranded DNA that is amenable to high-throughput single molecule DNA sequencing (14). Single Molecule R-loop Footprinting (SMRF-seq) can easily be used to map R-loops generated upon in vitro transcription, without prior S9.6 enrichment.…”

“…Upon library validation, sequencing was performed on pooled barcoded libraries on a Pacific Biosciences RSII instrument. Following sequencing, the Gargamel computational pipeline was used to identify SMRF footprints by tracking significant patches of C to T conversion across high quality circular consensus single reads as described in (14).…”