Translocating RNA polymerase generates R-loops at DNA double-strand breaks without any additional factors.

The R-loops forming around DNA double-strand breaks (DSBs) within actively transcribed genes play a critical role in the DSB repair process. However, the mechanisms underlying R-loop formation at DSBs remain poorly understood, with diverse proposed models involving protein factors associated with RNA polymerase (RNAP) loading, pausing/backtracking or preexisting transcript RNA invasion. In this single-molecule study using Escherichia coli RNAP, we discovered that transcribing RNAP alone acts as a highly effective DSB sensor, responsible for generation of R-loops upon encountering downstream DSBs, without requiring any additional factors. The R-loop formation efficiency is greatly influenced by DNA end structures, ranging here from 2.8% to 73%, and notably higher on sticky ends with 3' or 5' single-stranded overhangs compared to blunt ends without any overhangs. The R-loops extend unidirectionally upstream from the DSB sites and can reach the transcription start site, interfering with ongoing-round transcription. Furthermore, the extended R-loops can persist and maintain their structures, effectively preventing the efficient initiation of subsequent transcription rounds. Our results are consistent with the bubble extension model rather than the 5'-end invasion model or the middle insertion model. These discoveries provide valuable insights into the initiation of DSB repair on transcription templates across bacteria, archaea and eukaryotes.

Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures.

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop structures during transcription. Using this technique, we found that R-loop formation precedes GQ formation and that there exists a positive feedback loop between G-quadruplex and R-loop formation.

Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation.

G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.