Genome-wide mapping of endogenous topoisomerase cleavage complexes reveals TOP1 at the interface of DNA topological regulation and genome instability

Topoisomerase cleavage complexes (TOPcc) are covalent protein-DNA intermediates formed transiently during topoisomerase catalysis and constitute a major class of DNA-protein crosslinks in human cells, yet their endogenous genomic distributions remain difficult to resolve. Here, we developed protein-DNA adduct immunoprecipitation followed by sequencing (pDAIP-seq), a highly sensitive method for detecting endogenous TOPcc that enables their genome-wide profiling under normal physiological conditions without stabilization by topoisomerase poisons. Using pDAIP-seq, we found that camptothecin treatment, required by existing TOP1cc profiling methods, not only increased TOP1cc abundance but also substantially altered its genomic distribution. Endogenous TOP1cc were globally reduced upon transcriptional suppression and increased upon TOP2A depletion, supporting TOP1cc regulation by the maintenance of genome-wide DNA topological homeostasis. Interestingly, both the regional distribution and nucleotide-scale positioning of TOP1cc, including the positions of TOP1 catalytic cleavage sites, were largely specified by local DNA sequence context. By extending pDAIP-seq to capture transient covalent protein-DNA intermediates involved in DNA repair, including TDP1-, PARP1-, and KU70-DNA adducts, we further identified endogenous TOP1cc as a potential major source of spontaneous DNA breaks in human cells. Finally, pDAIP-seq also enabled genome-wide mapping of TOP2cc, TOP3cc, and SPO11-DNA covalent intermediates in mouse testis undergoing programmed meiotic DNA cleavage. Together, these findings establish pDAIP-seq as a broadly applicable method for the mapping of covalent protein-DNA intermediates and reveal endogenous TOP1cc as a key intermediate between DNA topological regulation and human genome instability.