Cleaved ATP5A1 mediates a poly(ADP-ribose)-dependent regulatory axis for R-Loop homeostasis under oxidative stress

Oxidative stress-induced DNA damage potentiates accumulation of R-loops, DNA:RNA hybrid structures with a displaced DNA strand that are resolved in a poly (ADP-ribose) (PAR)-dependent manner via PARP family enzymes. PARylation orchestrates recruitment of DNA repair factors during the oxidative stress response, in addition to multiple mitochondrial proteins also playing a role to protect the nuclear genome. We previously identified cleaved ATP5A1, a mitochondrial stress-responsive proteoform, as an interactor of R-loop resolving proteins including DHX9 and hnRNPU using immunoprecipitation/mass spectrometry (IP/MS) approach. We further investigated its PAR-dependent role in R-loop regulation using in vitro assays, cell culture, molecular dynamics (MD) simulation, and RNA-seq approaches in Calu-1, a non-small cell lung cancer cell model. To identify the bioactive R-loop resolving isoform of ATP5A1, mass spectrometry peptide fragments from Peptide Atlas were analyzed to identify potential calpain cleavage sites (cleavage windows that span multiple amino acids). Bioinformatic analyses predicted residue 135 as the primary cleavage site, validated by western blot and MD stability profiles. AlphaFold3 structure prediction and MD simulations demonstrated stable interactions of PAR chains with cleaved ATP5A1 (residues 135-553), with RMSD 0.2-0.3 nm and RMSF of stable residues ranging 0.1-0.2 nm, including ADPR monomers and polymers. IP/MS confirmed PARylation of transiently expressed ATP5A1(135-553) following ionizing radiation-induced oxidative stress. Slot blot was used to quantify R-loops in wild-type, ATP5A1 knockout, and cleaved ATP5A1 rescue conditions. RNA-seq of control and ATP5A1 knock-out (KO) cells identified differentially expressed genes enriching for cellular metabolism, DNA repair, and proliferation/cell cycle pathways in a cleaved ATP5A1-linked R-loop response signature. These findings identify cleaved ATP5A1 as a PAR-interacting protein that modulates R-loop homeostasis under oxidative stress, linking mitochondrial retrograde signaling to nuclear stress responses and revealing a potential R-loop therapeutic target in cancers with dysregulated oxidative stress signaling.