Investigating Role of Tyrosyl-DNA Phosphodiesterase 1 (TDP1) In Non-Homologous End Joining (NHEJ)

The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. Major DSB repair pathways in mammalian cells include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggest that NHEJ includes mechanisms to minimize error. In yeast, mutations of tyrosyl-DNA phosphodiesterase 1 (TDP1) reduced NHEJ fidelity. We are investigating the role of TDP1 in NHEJ in human cells. Using affinity capture chromatography, we found human TDP1 physically interacted with the required NHEJ protein -XLF. This interaction also stimulated DNA binding for both TDP1 and XLF, and formation of TDP1:XLF:DNA complexes. TDP1 can remove adducts from DNA 3’ ends, and TDP1:XLF interactions stimulated this activity on double-stranded, but not on single-stranded DNA. To investigate role of TDP1 in NHEJ in human cells, we used CRISPR/Cas9-mediated genome editing to generate TDP1-knockout HEK 293 cells, which showed an expected increase sensitivity to Topoisomerase 1 poisoning and ionizing radiation. Using a chromosomally- integrated end-joining reporter substrate, we observed an average 4-fold reduction in repair of I-SceI-induced DSBs in TDP1-KO cells as compared to wild type cells. These data indicate that, in human cells, TDP1 contributes to repair of DSBs that lack 3’ end damage. NextGen sequencing of end-joining junctions generated in this reporter system showed that TDP1 deficiency resulted in increased use of microhomology in joining. Within the N-terminal domain of TDP1, phosphorylation at serine 81 (S81) has been reported to regulate interaction with DNA repair factors, including DNA ligase III, XRCC4 and PARP1. We observed that the TDP1-S81 phosphomimetic, TDP1-S81E, had 10-fold reduced XLF binding, and ectopic expression of TDP1-S81E in TDP1-knockout cells failed to restore NHEJ activity. These data suggest that phosphorylation of TDP1-S81 regulates TDP1 participation in NHEJ, and may also direct TDP1 towards DNA ligase III-related pathways. Our observations support the hypothesis that TDP1 participates in mammalian NHEJ, and contribute important details to our understanding of DNA repair.