Biopolym. Cell. 2014; 30(1):42-46.
Structure and Function of Biopolymers
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends
1, 2Kosova A. A., 1, 2Khodyreva S. N., 1, 2Lavrik O. I.
  1. Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
    8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
  2. Novosibirsk State University
    2, Pirogova Str., Novosibirsk, Russian Federation, 630090

Abstract

Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyrimidinic (AP) site in the partial DNA duplex containing 5'and 3'-dangling ends (DDE-AP DNA) and mimicking clustered DNA damage. Methods. The Schiff base-dependent cross-linking of a protein to AP DNA (borohydride trapping), MALDI-TOF-MS, chromatography, and gel electrophoresis. Results. A human cell extract protein which forms a major covalent adduct with the AP DNA duplex with dangling ends was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF-MS data. The Ku antigen purified from the HeLa cell extract was shown to form the covalent adducts with the same mobility as observed in cell extracts. Conclusions. The Ku80 subunit of Ku antigen can specifically interact with AP DNA forming the Schiff base-mediated adducts which electrophoretic mobility depends on the structure of DNA ends. The difference in electrophoretic mobility can be caused by the cross-linking of AP DNA to distinct target amino acids that appears to reflect unequal positioning of AP DNAs in the complex with Ku antigen.
Keywords: Ku antigen, apurinic/apyrimidinic site, protein-DNA cross-linking, clustered DNA damages

References

[1] Atamna H, Cheung I, Ames BN. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc Natl Acad Sci USA. 2000; 97(2):686–91.
[2] McCullough AK, Dodson ML, Lloyd RS. Initiation of base excision repair: glycosylase mechanisms and structures. Annu Rev Biochem. 1999; 68:255–85.
[3] Georgakilas AG, O'Neill P, Stewart RD. Induction and repair of clustered DNA lesions: what do we know so far? Radiat Res. 2013; 180(1):100–9.
[4] Ilina ES, Lavrik OI, Khodyreva SN. Ku antigen interacts with abasic sites. Biochim Biophys Acta. 2008; 1784(11):1777–85.
[5] Khodyreva SN, Prasad R, Ilina ES, Sukhanova MV, Kutuzov MM, Liu Y, Hou EW, Wilson SH, Lavrik OI. Apurinic/apyrimidinic (AP) site recognition by the 5'-dRP/AP lyase in poly(ADPribose) polymerase-1 (PARP-1). Proc Natl Acad Sci USA. 2010; 107(51):22090–5.
[6] Prasad R, Liu Y, Deterding LJ, Poltoratsky VP, Kedar PS, Horton JK, Kanno S, Asagoshi K, Hou EW, Khodyreva SN, Lavrik OI, Tomer KB, Yasui A, Wilson SH. HMGB1 is a cofactor in mammalian base excision repair. Mol Cell. 2007; 27(5):829–41.
[7] Gullo C, Au M, Feng G, Teoh G. The biology of Ku and its potential oncogenic role in cancer. Biochim Biophys Acta. 2006; 1765 (2):223–34.
[8] Biade S, Sobol RW, Wilson SH, Matsumoto Y. Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA. J Biol Chem. 1998; 273(2):898–902.
[9] Koike M, Yutoku Y, Koike A. KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair. Exp Cell Res. 2011; 317(16): 2267–5.
[10] Fransson J, Borrebaeck CA. The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis. Int J Cancer. 2006; 119(10):2492–6.