Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites

• Published in: Biochimie Vol. 132; pp. 85 - 93
• Main Authors: Anisenko, Andrey N., Knyazhanskaya, Ekaterina S., Zatsepin, Timofey S., Gottikh, Marina B.
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01-01-2017
• Subjects: Base Sequence; Binding Sites - genetics; Binding, Competitive; Blotting, Western; Chromatography; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA binding protein; DNA, Circular - chemistry; DNA, Circular - genetics; DNA, Circular - metabolism; Hairpin RNA; Humans; Kinetics; Ku Autoantigen - chemistry; Ku Autoantigen - genetics; Ku Autoantigen - metabolism; Ku70; Nucleic Acid Conformation; Nucleotide Motifs - genetics; Protein Binding; Protein Domains; Protein-nucleic acid interaction; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA binding protein; Sequence Homology, Nucleic Acid; Ku70; dsRNA; DNA binding protein; RNA binding protein; Protein-nucleic acid interaction; GST; LTR; DRaCALA; Chromatography; HIV TAR RNA; SEC; hpRNA; IRES; Hairpin RNA; NHEJ; ssDNA; dsDNA
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2016.11.001

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.
•A two-site nucleic acid binding mode for Ku70 is proposed.•Hairpin RNA is bound within the central region of Ku70.•dsDNA is bound in the C-terminal region of Ku70 probably in the SAP domain.