Ku antigen displays the AP lyase activity on a certain type of duplex DNA

摘要

In the search for proteins reactive to apurinic/apyrimidinic (AP) sites, it has been earlier found that proteins of human cell extracts formed the Schiff-base-dependent covalent adduct with an apparent molecular mass of 100 kDa with a partial DNA duplex containing an AP site and 5'- and 3'-protruding ends (DDE-AP DNA). The adduct of such electrophoretic mobility was characteristic of only DDE-AP DNA (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777-1785). The protein in this unusual adduct was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF MS data (Kosova et al., Biopolym. Cell 30 (2014) 42-46). Here we studied the interaction of Ku with DDE-AP DNA in details. Purified Ku (the Ku80 subunit) was shown to form the 100-kDa adduct highly specific for AP DNA with a certain length of protruding ends, base opposite the AP site and AP site location. Ku is capable of AP site cleavage in DDE-AP DNA unlike in analogous AP DNA with blunt ends. Ku cleaves AP sites via beta-elimination and prefers apurinic sites over apyrimidinic ones. The AP site in DDE-DNA can be repaired in an apurinic/apyrimidinic endonuclease-independent manner via the successive action of Ku (cleavage of the AP site), tyrosyl-DNA phosphodiesterase 1 (removal of the 3'-deoxyribose residue), polynucleotide kinase 3'-phosphatase (removal of the 3'-phosphate), DNA polymerase beta (incorporation of dNMP), and DNA ligase (sealing the nick). These results provide a new insight into the role of Ku in the repair of AP sites. (C) 2016 Elsevier B.V. All rights reserved.