Direct visualization of replication and R-loop collision using single-molecule imaging

Abstract

R-loops are three-stranded nucleic acid structures that can cause replication stress by blocking replication fork progression. However, the detailed mechanism underlying the collision of DNA replication forks and R-loops remains elusive. To investigate how R-loops induce replication stress, we use single-molecule fluorescence imaging to directly visualize the collision of replicating Phi29 DNA polymerase (Phi29 DNAp), the simplest replication system, and R-loops. We demonstrate that a single R-loop can block replication, and the blockage is more pronounced when an RNA–DNA hybrid is on the non-template strand. We show that this asymmetry results from secondary structure formation on the non-template strand, which impedes the progression of Phi29 DNAp. We also show that G-quadruplex formation on the displaced single-stranded DNA in an R-loop enhances the replication stalling. Moreover, we observe the collision between Phi29 DNAp and RNA transcripts synthesized by T7 RNA polymerase (T7 RNAp). RNA transcripts cause more stalling because of the presence of T7 RNAp. Our work provides insights into how R-loops impede DNA replication at single-molecule resolution.

Pathological R-loops in bacteria from engineered expression of endogenous antisense RNAs whose synthesis is ordinarily terminated by Rho

“…Co-transcriptional R-loop formation has previously been implicated as an obstacle to DNA replication and as a threat to genome integrity in both bacteria and eukaryotic cells (44, 48, 49, 51, 97, 128–130). It would appear that both co-directional and head-on transcription-replication conflict associated with R-loop formation can affect genome integrity (40, 48, 49, 104, 105, 131), which is supported in the present study as well. Of the two recombineered antisense loci, one is expected to generate head-on (AS_b2459) and the other co-directional (AS_b0397) transcription-replication conflict.…”