LINE-1 ORF2p endonuclease activity links DNA damage signalling to enhancer associated chromatin remodeling

Aberrant expression of Long Interspersed Element-1 (LINE-1/L1) retrotransposons is increasingly linked to genomic instability in cancer, particularly in the context of compromised p53 function. The endonuclease (EN) domain of the LINE-1 ORF2 protein (ORF2p) generates DNA strand breaks, yet its role in shaping downstream chromatin and transcriptional responses remains poorly defined. Here, we investigate the impact of ORF2p-EN activity on DNA damage response signalling and epigenomic remodeling in isogenic wild-type and p53-deficient A375 melanoma cells using a L1 expression system. ORF2p-EN expression induced activation of ATM dependent DNA damage signalling, as evidenced by increased {gamma}H2AX accumulation. This response was accompanied by altered levels of the DNA repair factor XRCC5, consistent with engagement of non-homologous end joining pathways. Notably, EN-associated DNA damage correlated with increased p300 activity and enhanced H3K27ac enrichment at regulatory regions, suggesting coupling between DNA damage signalling and chromatin acetylation. Pharmacological inhibition of ATM attenuated both {gamma}H2AX accumulation and H3K27ac levels, supporting a model in which chromatin remodeling occurs downstream of DNA damage signalling. Collectively, our findings position ORF2p endonuclease activity as an initiating source of DNA damage that is functionally linked to chromatin remodeling and transcriptional reprogramming, with p53 acting as a critical modulator of this axis. These results provide mechanistic insight into how LINE-1 activation may contribute to oncogenic gene expression programs in cancer.