SRP476030 PRJNA1049124 GSE249453 DNA-PKcs Suppresses Illegitimate Chromosome Rearrangements Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ predominantly involved in human and A-EJ in mice. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in human is not known. We find partial DNA-PKcs inhibition in human cell lines leads to increased genome-wide translocations composed mostly of direct joints, indicating the continued involvement of dampened NHEJ in these processes. In contrast, complete DNA-PKcs inhibition and genetic inhibition DNA-PKcs kinase domain substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse pro-B cell lines, impair the recombination of RAG1/2-mediated DNA double-strand breaks (DSBs). This DNA-PKcs-deficient repair mechanism exhibited reduced V(D)J recombination efficiency, increased end resection, decreased polymerase-mediated insertions, loss of recombination fidelity and generated relatively higher rates of chromosome translocation as a consequence of dysregulated coding and signal end joining. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement in both species. Overall design: We induced DNA double-strand breaks (DSBs) using CRISPR-Cas9 technology in various cell lines, including K562-derived cell lines (K562-Bcl2 and K562-Bcl2-PKcs-/-) and the HCT116-derived cell lines (HCT116 DNA-PKcs+/-, HCT116 DNA-PKcs-/- and HCT116 DNA-PKcs KD/-). These cell lines were subjected to different treatment conditions. To capture translocation events, we utilized a DSB site either RAG1L located on chr11, as a bait, which allowed us to detect its ligation with the remaining DSB site(s) using high-throughput genome translocation sequencing (HTGTS). In parallel, we also captured the V-J recombination and chromosome translocations in murine DNA-PKcs catalytically dead v-Abl Pro-B cells using JK1CE and JK1SE as baits, respectively. GSE249453 pubmed 38412274