ИСТИНА

Heterodimer protein Ku consists of two subunits Ku70 and Ku80. It is a DNA-binding protein which has high affinity to DNA ends. Together with proteinkinase subunit DNA-PKcs it plays a key role in the DNA double-strand break repair by the non-homologous end-joining mechanism (NHEJ). In addition, its role was shown in other cellular processes, i.e. transcription, telomere maintenance, V(D)J-recombination, and some others. Protein Ku was identified as a participant in HIV-1 replication at the stages of integration and transcription although the exact mechanism of Ku­-dependent transcriptional regulation is unclear. A new expression vector coding for the heterodimeric Ku, the recombinant protein was purified from E. coli, and its RNA-binding properties were studied using gel-shift assay with synthetic RNAs. For each tested RNA structure, a binding curve was built and Kd was estimated. Ku was not found to interact with a perfect linear double-stranded RNA and interacted weakly with a duplex RNA containing a bulge. The highest Ku affinity was detected toward a hairpin RNA structure containing an extensive single-stranded region or a bulk bulge just near the loop. In vitro Ku interacts with HIV TAR RNA at a bulk bulge region near the loop. Using Ku-depleted cells and dual luciferase reporter system containing firefly luciferase gene under the control of tested promoters and renilla luciferase under the control of human PGK promoter positive regulation of transcription in TAR-independent manner by Ku protein from HIV but not CMV promoter has been observed. Using RNA-immunoprecipitation assay with Ku70/Ku80-Flag proteins a two fold-enrichment of 7SK RNA but not U6 RNA was observed. The binding of Ku to 7SK RNA was verified by EMSA experiment with recombinant protein and 32p-labeled T7 transcripted RNA. Interactions of overexpressed and endogenous Ku with HEXIM1 and Cdk9 proteins of 7SK snRNP complex were detected in co-immunoprecipitation assays in HEK293T cells.