Emerging roles for R-loop structures in the management of topological stress

Abstract

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.

Keywords: DNA structure; DNA topoisomerase; DNA topology; DNA transcription; R-loop; chromosomes; gene transcription; genome stability; supercoiling; superhelicity; topological stress.

Figure 1.

Superhelicity introduces torsional stress and modifies DNA shape. A covalently closed circular DNA molecule is shown at the top in a relaxed state. The introduction of four negative supercoils causes undertwist (Tw) stress in the molecule, which can be expressed as a change in twist shown at the bottom as strand opening or as a change in overall shape shown at the right by the formation of a negatively supercoiled writhed (Wr) structure.

Figure 2.

Transcription generates superhelical stresses that can be mitigated by R-loop formation. Transcription-driven supercoiling leads to the formation of dual waves of positive (downstream) and negative (upstream) superhelicity depicted here as interlinked plectonemic structures (A) or toroidal structures (B). C, topological disruptions caused by transcription are shown as undertwist (upstream) and overtwist (downstream). As the RNA polymerase (light blue) translocates forward, an R-loop initiates and extends. R-loop formation relaxes the upstream negative superhelical stress by absorbing undertwist within the strand opening that accompanies the formation of long R-loops. In addition, the displaced looped out ssDNA strand may wrap around the RNA:DNA hybrid in a left-handed helical fashion (bottom), further absorbing negative superhelicity.

Figure 3.

Nucleosomes and R-loops share in topological relief duties. A, positive supercoiling (red) traveling ahead of the translocating RNAP (shown here localized to the linker DNA) destabilizes nucleosomes, favoring their extrusion. Nucleosome release frees one negative supercoil, mitigating the buildup of positive superhelicity. Reassociation of the nucleosome behind the polymerase is favored by the transcription-driven negative supercoiling; sequestration of one negative supercoil upon nucleosome reformation relieves that stress. B, the formation of an R-loop results in two main consequences. First, nucleosome redeposition is inhibited. Second, the accompanying relaxation of the upstream region may weaken surrounding nucleosome-DNA contacts.

Figure 4.

R-loop formation mediates long-range reversible topological relaxation. The consequences of R-loop formation and resolution on the properties of the local chromatin environment are described. When R-loops are resolved, large stores of negative superhelicity are released into the chromatin fiber, driving increased chromatin contacts, increased non-B DNA structure formation (bubble DNA, cruciform, and Z DNA are depicted), and increased protein-DNA interactions. See text for details.