R-loops are three-stranded nucleic acid structures formed when an RNA transcript hybridizes back to its complementary DNA template strand, leaving the non-template strand as single-stranded DNA. While R-loops occur naturally and serve physiological functions in transcription regulation and antibody gene diversification, their uncontrolled accumulation is harmful. The single-stranded DNA region created by R-loops is chemically unstable and more vulnerable to enzymatic attack, which makes it prone to mutations, breaks, and other forms of genomic damage.

One major source of genome instability arises from conflicts between replication and transcription machinery. When replication forks encounter R-loops, they can stall or collapse, particularly during head-on collisions. This leads to replication stress and double-strand breaks, which are highly deleterious to chromosome integrity. Such replication–transcription conflicts are recognized as a major pathway through which persistent R-loops contribute to chromosomal fragility and rearrangements.

Additionally, R-loops interfere with DNA repair pathways. Proteins that normally safeguard genome integrity, such as BRCA1/2 and Fanconi anemia proteins, are often recruited to resolve R-loop–induced damage. However, excess or unresolved R-loops can sequester repair enzymes or block homologous recombination and non-homologous end joining, resulting in error-prone repair. The consequence is the accumulation of mutations, deletions, or translocations, further driving genome instability.

Finally, R-loops frequently accumulate at vulnerable genomic regions, such as telomeres, repetitive sequences, and common fragile sites. At these loci, persistent R-loops trigger chronic activation of the DNA damage response, chromosomal rearrangements, and in some cases, aneuploidy. To counteract this, cells rely on surveillance factors such as RNase H enzymes and helicases like Senataxin to resolve RNA:DNA hybrids. When these safeguards fail or are overwhelmed, R-loop accumulation becomes a potent driver of genomic instability, linking it to cancer, neurodegeneration, and other human diseases.