Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends

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.

Introduction.The abasic (AP) sites are among the most frequent DNA damages.Up to 50 000 AP sites arise in a mammalian cell per day [1] as a result of spontaneous hydrolysis of N-glycosidic bond or under the action of DNA glycosylases during the base excision repair [2].If unrepaired, the AP sites are mutagenic and cytotoxic.The repair of AP sites in clustered DNA damages presented by combinations of the AP sites, oxidized bases and single strand breaks within 1-2 turns of DNA helix is of particular interest [3].Such lesions arise in DNA under the action of ionizing radiation or radiomimetic drugs.
The cross-linking of proteins to a baseless deoxyribose in DNA via reduction of a Schiff-base intermediate is often called «borohydride trapping» (BHT).BHT in combination with mass spectrometry has been used in a proteomic approach to identify the human pro-teins reactive to AP sites [4][5][6].The set of DNA duplexes containing the AP site in the middle of 32-mer oligonucleotide and different structural features has been used.With all AP DNA, except DNA with both 5'-and 3'-dangling ends of 8 nt (DDE-AP DNA), the predominant products of cross-linking were of the same electrophoretic mobility (an apparent molecular mass of 90 kDa).The protein cross-linked to the AP DNA duplex with blunt ends (BE-AP DNA) was identified as the Ku80 subunit of Ku antigen [4].Ku antigen consisting of two subunits with molecular masses about 70 kDa (Ku70) and 83 kDa (Ku80) is a eukaryotic DNA-binding component of DNA-dependent protein kinase.The main function of Ku antigen is participation in the double-strand break repair by non-homologous end joining [7].In the present work we identified the protein which specifically interacts with DDE-AP DNA forming the product with an apparent molecular mass of 100 kDa.

Materials and methods.
Chromatographic fractionation of cell extract proteins.HEK 293 cell extract prepared as described elsewhere [8] was diluted 4 times with the buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.025 % NP-40 and was applied to a heparin-sepharose column.After washing with the same buffer, the proteins were eluted with linear gradient of NaCl (0.05-1 M) in the same buffer.The presence of a target protein in the fractions was determined by BHT.The fractions were concentrated and supplemented with glycerol (final concentration of 10 %).
Borohydride trapping and MALDI-TOF-MS analysis.All procedures were carried out as described previously [4] with a few exceptions.The concentration of AP DNA in the reaction mixtures was 0.2 mM.The preparative cross-linking for MALDI-TOF-MS analysis was performed using a fractionated cell extract enriched in the target protein by chromatography on heparin-sepharose.The peptides derived from the cross-linked protein were additionally purified on Zip-tip C-18 prior to the MS analysis.
Results and discussion.The BHT experiments using the DDE-and BE-AP DNAs and several human cell extracts, including the HeLa and HEK 293 cells, reveal nearly identical patterns of the cross-linked proteins.Data for HeLa cell extract are shown in Fig. 1.
For identification of the target protein we used the fraction of HEK 293 cell extract proteins eluted from heparin-sepharose at 450-500 mM NaCl and enriched in the target protein.Use of the enriched extract ensures the yield of target protein cross-linking with AP DNA and reduces the likelihood of sample contamination with the proteins nonspecifically bound to DNA.After the electrophoretic separation of the protein covalent adducts with biotinylated DNA bound to streptavidin-coated beads, the gel was stained by Coomassie Brilliant Blue R-250 (Fig. 2, A) and autoradiography was carried out (Fig. 2,  B).Five well-defined protein bands with apparent molecular masses from 100 to 70 kDa (Fig. 2, C) were excised from the gel and subjected to in-gel trypsin digestion.
The bands N 1-4 have the matching radioactive bands, thus corresponding to the protein-DNA covalent adducts.Furthermore, the 100 kDa-band has the highest intensity with the both methods of visualization.Peptides were analyzed by MALDI-TOF-MS and data were searched against a database.The first rank candidates for all the samples with their scores, the number of the matched peptide mass values and the protein sequence coverage are shown in Table 1.
Obviously, Ku80 forms several covalent adducts with DDE-AP DNA.Ku70 is retained on the beads due to the strong interaction with Ku80 [4,9].
The data for the matching peptides in the sample N 1 are shown in Table 2. Interestingly, the set of Ku80 peptides identified in this work is almost overlapped with those obtained in the other works [4,10].This is probably due to an easy ionization of these peptides under the analysis conditions.The appearance of the Ku80 adduct with a lowered electrophoretic mobility characteristic for DDE-AP DNA can reflect the existence of two different modes of Ku80 binding with DNA (and, consequently, different points of interaction with the AP site) or the highly efficient cross-linking of DNA with another Ku80 isoform.Indeed, the lists of candidates for the samples N 1 and 2 include a Ku80 isoform higher molecular mass (93464 Da).The biochemical properties of this isoform resemble those of the conventional Ku80, but it cannot fully replace Ku80 [9].To discriminate these alternatives, we purified the Ku antigen from HeLa cells (see «Materials and methods») to near homogeneity (Fig. 3) and then tested the ability of the purified protein to interact with two aforementioned types of AP DNA (Fig. 4).The patterns of cross-linking of two AP DNAs to the purified Ku antigen and cell extract proteins have the analogous set of products: more abundant 100 kDa and less intensive 90 kDa adducts for DDE-AP DNA (lanes 1, 3) and 90 kDa adduct for BE-AP DNA (lanes 2, 4).
Conclusions.Thus, the Ku80 subunit of Ku antigen specifically interacts with the AP DNA structure with 5'-and 3'-dangling ends and forms a covalent adduct with an apparent molecular mass of 100 kDa after the treatment with NaBH 4 .Further investigations are required to determine the role of these modes of the Ku antigen interaction with the AP sites.This type of damaged DNA structure can be used to monitor the binding activity of Ku antigen in the extracts of various human cells.