R-loops processing by human apurinic/apyrimidinic endonuclease APE1

R-loops are three-stranded nucleic acid structures that arise during transcription and play important roles in genome regulation, but their unscheduled accumulation can lead to genomic instability. Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme mainly involved in base excision DNA repair. However, APE1, being both nuclear and cytoplasmic enzyme, could be also considered as important participant of RNA metabolism through their endo- and exoribonuclease as well as RNase H activities. This study investigates the ability of APE1 to process R-loop structures through its various enzymatic activities in vitro. We demonstrate that APE1 exhibits RNase H activity on DNA:RNA hybrids within R-loops, with efficiency decreasing as hybrid length increases beyond 16 nucleotides. Additionally, APE1 cleaves abasic sites in both RNA and DNA strands of R-loops via its AP-endonuclease activity. Notably, an abasic site in the RNA strand is efficiently processed followed by robust 3'-5' exoribonuclease degradation, while an abasic site in the DNA strand is cleaved less efficiently and strongly inhibits RNase H activity. This functional versatility reinforces the importance of APE1 in nucleic acid metabolism, offering new insights into its biological significance beyond traditional DNA repair pathways.