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Nucleic Acids Res., Nov 22 2023, gkad1101 | https://doi.org/10.1093/nar/gkad1101 Direct visualization of replication and R-loop collision using single-molecule imaging

Subin Kim ¹, Woo Hee Shin¹, Yujin Kang¹, Hongtae Kim ¹, Ja Yil Lee^1,2

¹Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.

²Institute of Basic Science Center for Genomic Integrity, Ulsan 44919, Republic of Korea.

To whom correspondence should be addressed. : Ja Yil Lee

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.

논문정보

형식Research article
게재일2023년 11월 (BRIC 등록일 2023-11-27)
연구진국내 연구진
분야 생명과학 > 생화학

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