DNA-PKcs suppress chromosome translocation in human cells
DNA-PKcs is a crucial component of the non-homologous end joining (NHEJ) repair machinery. To investigate its function in human cell lines, we conducted a study using K562 and HEK293T cell lines. We introduced twinned DNA double-strand breaks (DSBs) or genome-wide DSBs into these cell lines via nucleofection and transfection, respectively. Subsequently, we employed high-throughput genome translocation sequencing (HTGTS) to capture the translocation events (i.e., ligation between "prey(s)" and "bait") and the rejoining events (i.e., direct repair within the "bait" locus) under different conditions, including with or without DNA-PKcs inhibition or deletion. We quantified the number of translocation events by normalizing them to the number of rejoining events, denoted as TL. Interestingly, DNA-PKcs inhibition led to an increase in TL, indicating a higher frequency of translocations. However, it is important to note that chromosomal translocations still predominantly relied on NHEJ despite DNA-PKcs inhibition. Furthermore, we observed that DNA-PKcs deletion resulted in an elevated utilization of microhomology in translocation formation. Nevertheless, NHEJ remained the primary mechanism driving translocation events.These findings provide valuable insights into the role of DNA-PKcs in the repair pathways involved in translocation events in human cell lines. The utilization of HTGTS allowed us to comprehensively analyze the effects of DNA-PKcs inhibition and deletion, shedding light on the interplay between NHEJ and alternative repair mechanisms in translocation formation. Overall design: We induced DNA double-strand breaks (DSBs) using CRISPR-Cas9 technology in various cell lines, including K562-derived cell lines (K562-Bcl2, K562-Bcl2-PKcs-/-, and K562-iCas9) and the HEK293T-derived cell line HEK293T-iCas9. These cell lines were subjected to different treatment conditions, including DMSO, DPKi #1 (Nu7441), DPKi #2 (Nu7026), and ATMi. To capture translocation events, we utilized one of the DSB sites, either RAG1L or HBB, as a bait, which allowed us to detect its ligation with the remaining DSB site(s) using high-throughput genome translocation sequencing (HTGTS).