Base-pair resolution reveals clustered R-loops and DNA damage-susceptible R-loops.

R-loops are pervasive triplex nucleic acid structures across diverse organisms, yet their biological functions remain incompletely understood. Here, we develop R-loop identification assisted by nucleases and sequencing (RIAN-seq), a nuclease-assisted, antibody-free sequencing technology, to map R-loops at base-pair resolution. By digesting single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), and double-stranded DNA (dsDNA) with nuclease P1, T5 exonuclease, and lambda exonuclease while preserving RNA:DNA hybrids, RIAN-seq achieves unprecedented precision in identifying the position and size of R-loops, detecting an order of magnitude more R-loops than existing methods. Approximately 50% of RNA:DNA hybrids span between 60 and 130 bp, with many forming previously undetectable clusters. Clustered R-loops at promoters recruit zinc-finger proteins VEZF1 and SP5, enhancing transcription in a number-dependent manner and resisting transcriptional perturbation. Conversely, R-loops featuring the Y(C/T)M(A/C)CAG motif at both ends contribute to DNA damage, a phenomenon conserved from yeast to mammalian cells. Our findings reveal a dual role for R-loops: clustered R-loops promote gene expression, while YMCAG-associated R-loops compromise genome stability.