RNA transcripts stimulate homologous recombination by forming DR-loops

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Homologous recombination (HR) is a key DNA double-strand break (DSB) repair pathway active during S and G2 cell cycle phases.
• HR proteins are often found at DSBs in transcriptionally active regions, but the underlying mechanism remains unclear.

Purpose of the Study:

• To investigate how local transcription influences homologous recombination (HR).
• To elucidate the role of RNA transcripts and RAD51AP1 in transcription-mediated HR enhancement.

Main Methods:

• Development of a novel assay to evaluate the impact of local transcription on HR.
• Experimental manipulation of RNA transcript tethering near DSBs.
• In vitro assays to assess protein functions and intermediate formation.

Main Results:

• Transcription substantially stimulates HR, with tethered RNA transcripts mimicking this effect.
• The stimulation is sequence- and orientation-dependent, indicating DNA-RNA hybrid involvement.
• RAD51-associated protein 1 (RAD51AP1) is crucial for transcription-enhanced HR and DR-loop formation.

Conclusions:

• Transcription enhances HR primarily through RNA transcripts forming DNA-RNA hybrids (R-loops).
• RAD51AP1 facilitates R-loop formation and is essential for transcription-stimulated HR.
• The study identifies 'DR-loops' (DNA-DNA and DNA-RNA hybrids) as key intermediates that promote RAD51 function in transcribed regions.