DNA ends to be ligated in proximity of each other (17). Such a complex may facilitate recruitment of subsequent proteins and ﬁnal ligation of the DNA. The terminus resulting from an IR-induced DNA DSB can vary in structure, size, and chemistry. In this study, the activation of DNA-PK has been assessed using a variety of DNA molecules diﬀering in structure, chemistry and sequence. It has been shown that DNA-PK is activated by full duplex DNA but not by hairpin struc- tures or supercoiled plasmids (18). Additionally, it has been shown that DNA-PK is preferentially activated by DNA with 30 pyrimidine-rich termini (19), but activity is severely inhibited by cisplatin-DNA adducts (20). Inter- estingly, chemical structures attached to DNA such as biotin do not inhibit kinase activity (21). Analysis of Ku-independent DNA-PK activation has shown that DNA-PKcs is highly activated by DNA with single- strand overhangs (21). Despite this collection of data, it remains unclear what aspects of DNA are important for activation of the kinase. It has been suggested that melting of the DNA terminus may be necessary for DNA-PK to bind in a stable complex with the DNA (22). Melting results in two single- strand ends of DNA, though the role that these ends may play in DNA-PK activation has yet to be elucidated. A model for DNA threading through the kinase and separating of the DNA ends has been proposed, with single strand termini entering an active site on the kinase following synapsis of two DNA bound DNA-PKcs proteins (23). The authors demonstrated formation of a synaptic complex in the absence of sequence homology using blunt-end substrates and cooperative activation of the kinase suggesting that the synaptic complex displays greater catalytic activity. The role of DNA structure and sequence on synaptic complex formation and whether activation requires cis or trans interactions is not known. Our previous results show that DNA sequence bias as a function of strand orientation signiﬁcantly inﬂuences kinase activation (19), suggesting a unique role for each strand of the DNA terminus in DNA-PKcs activation. To investigate the eﬀect of diﬀerent DNA structures on DNA-PK activation, a collection of DNA eﬀectors with diﬀerent structures and sequences was designed. Our results reveal a unique role of each strand of the termini in synaptic activation, which is independent of complex formation. These results have been used to develop a mechanistic model for DNA-PK activation as a function of the DNA termini sequence homology and structure. METHODS DNA effectors Single-strand oligonucleotides were purchased from Integrated DNA Technology (IDT, Coralville, IA, USA) and are presented in Table 1. Oligonucleotides were synthesized with a 50-biotin molecule by IDT, as indicated. The single-strand oligonucleotides were gel-puriﬁed by preparative denaturing 15% polyacrylamide gel electro- phoresis, quantiﬁed and annealed to complementary oligonucleotides, as follows. The 24-bp full duplex DNA Nucleic Acids Research, 2008, Vol. 36, No. 12 4023 Table 1. Oligonucleotide sequences Name Sequence 3024 biotin-TAGATGGACTATCGCAGCACTGTA 3030 biotin-TAGATGGACTATCGCAGCACTGTACGGCTT 3024+6 biotin-TAGATGGACTATCGCAGCACTGTAGCCGAA 3024+6A biotin-TAGATGGACTATCGCAGCACTGTAAAAAAA 3024+6T biotin-TAGATGGACTATCGCAGCACTGTATTTTTT 3024+6mix biotin-TAGATGGACTATCGCAGCACTGTAAAGCCG 5024 TACAGTGCTGCGATAGTCCATCTA 5030 AAGCCGTACAGTGCTGCGATAGTCCATCTA 5024+6 TTCGGCTACAGTGCTGCGATAGTCCATCTA 5024+6A AAAAAATACAGTGCTGCGATAGTCCATCTA 5024+6T TTTTTTTACAGTGCTGCGATAGTCCATCTA All sequences are 50 to 30 with biotin modiﬁcations as indicatetd. eﬀector was created by annealing 3024 to 5024, and the 30-bp full duplex DNA created by annealing oligonucleo- tides 3030 and 5030. The two Y-shaped eﬀectors were prepared by annealing 3024 + 6 to 5030 and 3030 to 5024 + 6. The 30 overhang eﬀector was prepared by anneal- ing oligonucleotide 3030 to 5024. To prepare eﬀectors with 30 overhangs for microhomology studies oligonucleotide 3024 + 6T, 3024 + 6A or 3024 + 6mix were annealed to 5024 to make the 30 overhang eﬀectors containing homopoly- meric Ts, As or with compatible sequences, respectively. Additionally, the 3024 oligonucleotide was annealed to 5030 to make the 50 overhang eﬀector, and 3024 was annealed 5024 + 6T or 5024 + 6A to make the 50 overhang eﬀectors for microhomology studies. Duplex DNA was gel-puriﬁed by native polyacrylamide gel electrophoresis to ensure homogeneity, eluted from gel, precipitated and quantiﬁed. DNA-PK was puriﬁed from HeLa cells, and a series of titrations of both DNA and DNA-PK were performed to determine the appropriate concentrations to use in kinase assays to accurately assess binding and activation. Protein purification Cell-free extracts were prepared from 12 L of HeLa cells as described previously (24) and DNA-PK was puriﬁed as previously described (20). Brieﬂy, extracts were frac- tionated on a 50-ml cisplatin-damaged DNA-Sepharose column, heparin-Sepharose column and Q-Sepharose column. DNA-PK containing fractions were identiﬁed by Coomassie staining of SDS gels, DNA-PK kinase activity and Ku-binding activity. The fractions were pooled, dialyzed, aliquoted and frozen at 708. DNA-PK kinase assays Kinase assays containing a single eﬀector per reaction were performed at 378C in a ﬁnal volume of 20 ml containing 20 mM HEPES, pH 7.5, 8 mM MgCl2, 1 mM DTT, 5% glycerol, 125 mM ATP, [g-32P] ATP (0.5 mCi), 6.3 nM DNA-PK, 5 nM DNA and 500 mM p53 synthetic peptide. Taking into account the buﬀer in which the DNA-PK preparations were dialyzed, the ﬁnal KCl concentration of the reactions ranged from 20 mM to 70 mM. In addition, to determine the eﬀect of ionic strength, a series of DNA-PK activation experiments were performed at varying KCl concentrations and the results demonstrated