Abstract
R-loops are abundant three-stranded nucleic-acid structures that form in cis during 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.
Highlights
R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription
Our model suggests that these effects are due to the ability of R-loops to absorb negative superhelicity, thereby returning the DNA fiber to a more energetically favorable partially or fully relaxed state
Equilibrium statistical mechanics assesses the frequencies with which alternate molecular conformations occur in a population at equilibrium, based on their energetics [40]
Summary
We used a homogenized random sequence, for which the base-pair energy was set at the constant value of B = +0.23 kcal/mol, corresponding to the average energy of all 16 nearest-neighbor pairs In this sequence and level of superhelicity, short R-loops of lengths up to ∼100 bp were energetically favored over the B form. R-loops of lengths up to 500 bp were energetically favored for a homogenized favorable sequence (B = −0.15 kcal/mol, corresponding to the average energy of all seven favorable nearest-neighbor pairs). R-loops were predicted to occur above σ = +0.1, a physiologically attainable value, in the favorable sequence (B = −0.15 kcal/mol) This behavior is a consequence of the high flexibility of single-stranded DNA.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
