ИСТИНА

The trimeric complex DNA-PK, composed of DNA-binding heterodimer Ku and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), is a participant of DNA double-strand breaks repair pathway through the non-homologous end joining (NHEJ). The Ku heterodimer formed by Ku70 and Ku80 subunits acts as the main sensor of DSBs which triggers a cascade of phosphorylation events required for the subsequent DSB repair. In addition to NHEJ, Ku is involved in various cellular processes such as V(D)J recombination, AP-site repair, telomere maintenance, apoptosis, transcription, and translation. Also, Ku is suggested to participate in human immunodeficiency virus-1 (HIV-1) replication at the stages of integration and transcription although the exact mechanism of Ku-dependent transcriptional regulationis unclear. To clarify the way of Ku-mediated regulation of HIV transcription a set of HEK 293T derived sublines with a stable depletion of either Ku70, Ku80 or DNA-PKcs subunits was established using CRISPR/Cas9 technology. Then using a luciferase reporter system with firefly luciferase under the control of promoters (viral promoters CMV, SV40, TK and HIV promoter LTR and also cellular promoter PGK), we observed a strong reduction in luciferase expression from all tested promoters under depletion of Ku70 and especially Ku80 subunit. Surprisingly, the influence of DNA-PKcs knockout on the transcription efficiency from all promoters and particular HIV promoter was not detected. To elucidate the influence of the DNA-PK subunits on the cellular transcription, we performed a transcriptome analysis of wild type HEK 293T cells and those with depletion of either Ku70, Ku80 or DNA-PKcs. The genes regulated by each subunit were defined, and the genes dependent on the all DNA-PK complex were separated. The work was supported by the RSF grant 17-14-01107.