L1 Transposon Intermediates as Drivers of Cancer

In this study, we mapped R-loops by performing immunoprecipitation experiments using the GFP antibody on wild-type and p53 knockout A375 cell lines expressing the RNaseH D210N-GFP (RNH-mutant), which selectively binds to R-loops without resolving them. Our DRIP-seq data, combined with reverse transcriptase inhibitor assays, reveal that the loss of p53 significantly increases LINE1 mRNA-genomic DNA hybrids (cis-LINE1 R-loops) and LINE1 mRNA-complementary DNA hybrids (trans-LINE1 R-loops), both of which are key intermediates in LINE1 retrotransposition. The formation of trans-LINE1 R-loops was further validated using a reverse transcriptase inhibitor assay. Notably, full-length LINE1 elements capable of retrotransposition showed the highest levels of R-loop formation. These findings highlight the critical role of p53 in regulating LINE1 derived DNA-RNA hybrid formation.