Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.
Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438.
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia. Electronic address: a_anisenko@mail.ru.
Andrey N
AN
Anisenko
Chemistry Department, Lomonosov Moscow State University, Moscow, Russia. Electronic address: e.knyazhanskaya@gmail.com.
Ekaterina S
ES
Knyazhanskaya
Chemistry Department, Lomonosov Moscow State University, Moscow, Russia; Skolkovo Institute of Science and Technology, Skolkovo, Russia. Electronic address: tsz@yandex.ru.
Timofey S
TS
Zatsepin
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia. Electronic address: marina.gottikh@gmail.com.
Marina B
MB
Gottikh
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004270:DNA, Circular; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D000072200:Ku Autoantigen
Chromatography; DNA binding protein; Hairpin RNA; Ku70; Protein-nucleic acid interaction; RNA binding protein