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

• Here Ku antigen interaction with AP DNA with protruding ends is studied in details.
• The efficiency of Ku cross-linking with an AP site depends on DNA structure.
• Ku cleaves AP sites in duplex DNA with protruding ends via β-elimination.
• Ku prefers apurinic sites over apyrimidinic ones.
• Ku can initiate the repair of AP sites.

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 β-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 3ʹ-deoxyribose residue), polynucleotide kinase 3ʹ-phosphatase (removal of the 3ʹ-phosphate), DNA polymerase β (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.

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