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DNA-PKcs suppress chromosome translocation in human cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE232940 | GEO | 2023/09/01

REPOSITORIES: GEO

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Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.

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DNA-PKcs suppress chromosome translocation in human cells

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