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

Abstract

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

“…Analysis of the R-loop-associated RNA by denaturing formaldehyde agarose gel-electrophoresis reveals that most RNA molecules are significantly shorter than 1.4 kb, ranging in size between ∼200-600 nt ( Figure 1E ). Thus, only sections of the Airn sequence form R-loops in vitro , which is consistent with previous single-molecule R-loop sequencing and footprinting data 43 . To characterize the R-loop-containing templates we employed electron-microscopy (EM) after rotary shadow casting ( Figure 1F ).…”

“…To generate DNA templates harboring R-loops of characteristic length and nucleotide composition we inserted a 1.4 kbp fragment derived from the R-loop-forming Airn locus under control of a T7 promoter into yeast replication origin-containing plasmids ( Figure 1A ) 20,43 . Studies in human cells have demonstrated that R-loops formed at this sequence impede normal fork progression 14 .…”

G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders

“…Atomic force microscopy was used to analyze G-loops generated by transcription of a murine immunoglobulin Sc3 switch region cloned into a plasmid,showing that the structure was dependent on the presence of the hybrid duplexes as they disappear upon RNaseH1 treatment ( 77 ). Another study showed that the structural organization of the non-template strand is a fundamental feature of R-loops even though the structural characteristics of the non-template strand were not clearly defined ( 78 ). Interestingly, depletion of telomeric RNA transcripts (TERRA) in human cells resulted in a decrease in telomeric G4 structures suggesting that R-loops may affect G4 formation at telomeres ( 79 ).…”

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

“…To generate DNA templates harboring R-loops of characteristic length and nucleotide composition, we inserted a 1.4 kbp fragment derived from the R-loop-forming Airn locus under control of a T7 promoter into yeast replication origin-containing plasmids ( Figure 1A ; Carrasco-Salas et al, 2019 ; Ginno et al, 2012 ). Studies in human cells have demonstrated that R-loops formed at this sequence impede normal fork progression ( Hamperl et al, 2017 ).…”