Single-molecule visualization of the collision between replication and R-loop

Subin Kim¹, Woo-Hee Shin¹, Yujin Kang¹, JaYil Lee^1*

¹Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea

* Presenter:JaYil Lee, email:biojayil@unist.ac.kr

Phi29 DNA polymerase is a bacteriophage polymerase which has high processivity and high fidelity because of its unique structure. Although it is currently being used in various biotechniques, the detailed mechanism underlying DNA replication by Phi29 DNAp remains poorly understood. Here we investigate biophysical properties of phi29 DNA polymerase using a novel single-molecule imaging technique DNA curtain, which enables the visualization of replication events of individual Phi29 DNAp molecules in real time. In addition, we observed the collision between replicating phi29 DNAp and a single R-loop, which is a three-stranded nucleic acid structure consisting of a DNA-RNA hybrid and a displaced single-stranded DNA. R-loop can be occurred by various reasons such as replication stress, DNA damage, fork collision, and RNA polymerase pausing. Due to its special structure, the R-loop can interfere with DNA replication and cause genomic instability. Phi29 DNAp can pass the R-loop having DNA-hybrid on template strand during replication. However, more than 60% of phi29 DNAp is stalled when the phi29 DNAp encounters R-loop with DNA-RNA hybrid on non-template strand, and it is confirmed by observing the remaining RNA after replication. We also tested the collision with D-loop, and the results are similar to R-loop. It suggests that hybridized structure on non-template strand inhibits the progression of Phi29 DNAp replication. Moreover, reducing the length of R-loop RNA and making mismatch of the DNA-RNA hybrid do not affect the stalling ratio. Those results propose that three-stranded structure may affect the replication stalling. In addition, R-loop with G-quadruplex structure showed higher stalling portion, which suggests that G-quadruplex does stabilize R-loop structure. At last, we visualized the transcription-replication conflicts and compared to artificial R-loop. This study can give us the clue about how does the R-loop can act as a DNA replication stress.

Keywords: single-molecule, DNA curtain, replication, r-loop, transcription-replication conflict