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Related Experiment Video

Updated: Jul 10, 2025

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

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

Subin Kim1, Woo Hee Shin1, Yujin Kang1

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

Nucleic Acids Research
|November 23, 2023
PubMed
Summary
This summary is machine-generated.

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R-loops, structures of RNA and DNA, can stall DNA replication forks. This study reveals how R-loops impede replication using single-molecule imaging, identifying secondary structures and G-quadruplexes as key blocking factors.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biophysics

Background:

  • R-loops are nucleic acid structures formed by an RNA-DNA hybrid and a displaced DNA strand.
  • R-loops can interfere with DNA replication, leading to replication stress.
  • The precise mechanisms by which R-loops impede replication forks are not fully understood.

Purpose of the Study:

  • To investigate the mechanisms by which R-loops induce replication stress at the single-molecule level.
  • To visualize the direct collision between a DNA polymerase and R-loops.
  • To understand how structural features of R-loops affect replication fork progression.

Main Methods:

  • Single-molecule fluorescence imaging was employed to directly observe interactions.
  • The Phi29 DNA polymerase (Phi29 DNAp) was used as a simplified replication system.

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  • R-loops were constructed and visualized in conjunction with the replication machinery.

    • Main Results:

    • A single R-loop can effectively block replication fork progression.
    • Replication blockage is exacerbated when the RNA-DNA hybrid is on the non-template strand due to secondary structure formation.
    • G-quadruplex formation on the displaced single-stranded DNA further enhances replication stalling.
    • Collisions with RNA transcripts, particularly those associated with T7 RNA polymerase (T7 RNAp), caused significant stalling.

    Conclusions:

    • R-loops impede DNA replication through mechanisms involving secondary structure formation and G-quadruplexes.
    • The orientation of the RNA-DNA hybrid within the R-loop influences the degree of replication blockage.
    • Direct visualization provides critical insights into R-loop-mediated replication stress at high resolution.