ИСТИНА

Heterodimer protein Ku consists of two subunits Ku70 and Ku80. It is a DNA-binding protein showing a high affinity to DNA ends, which plays a key role in the DNA double-strand break repair by 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. Ku was identified as a participant of HIV-1 replication at the stages of integration and/or transcription. There are only some shreds of evidence of the human Ku protein interaction with RNA: Ku binds to telomerase RNA, HIV-1 TAR RNA, a hairpin at the 5’-end of p53 mRNA. We developed a simple method for Ku heterodimer expression in the prokaryotic system and performed a systematic in vitro analysis of Ku/RNA binding using gel-shift assay with this recombinant human Ku protein and synthetic RNAs. We identified RNA structural determinants important for interactions with the Ku protein. Ku was not found to interact with a perfect linear double-stranded RNA and weakly interacts 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. A double-stranded DNA displaces RNA ligands from the Ku/RNA complexes, and this fact indicates a common binding site for the double-stranded DNA and hairpin RNA binding. Interestingly, Ku tightly binds to the second loop of nuclear non-coding 7SK RNA (7SK-L2), which is an essential cellular transcription regulator participating in particular in HIV-1 transcription. The 7SK-L2 structure resembles the structure of HIV-1 TAR RNA, and Ku interacts with both RNAs with a comparable affinity. These interactions might be important for understanding the mechanism of Ku influence on transcription of cellular and HIV genes.