4024 Nucleic Acids Research, 2008, Vol. 36, No. 12 no signiﬁcant eﬀect over the range of concentrations tested (Supplementary Data). Biotinylated duplex DNA was incubated with 1 ng streptavidin (SA)/fmol DNA and incubated on ice for 5 min. DNA-PK and reaction buﬀer containing peptide were added to DNA and incubated on ice for 5 min. Reactions were initiated with addition of ATP, incubated at 378C for 15 or 30 min, as indicated, and terminated by addition of 20 ml of 30% acetic acid. Reaction products were spotted on P81 phosphocellulose ﬁlter paper. The paper was washed ﬁve times, 5 min. each, in 15% acetic acid, once in 100% methanol and allowed to dry. Samples were quantiﬁed by PhosphorImager analy- sis using ImageQuant software (Molecular Dynamics). Each DNA eﬀector used in the assays to analyze dimeric activation was bound with SA then DNA-PK was added to the DNA and allowed to incubate for 5 min on ice. Each eﬀector was then added alone to a tube or mixed with the complementary prebound eﬀector and allowed to incubate on ice for 5 min. Reaction buﬀer and peptide were then added to the DNA-DNA-PK mix, the reaction was initiated with ATP and the assay was completed as described above. DNA-PK autophosphorylation assay Kinase assays were performed as described above with the following modiﬁcations. Reactions contained 20 nM DNA-PK and 1.0 mCi [g-32P] ATP. Where indicated, reactions were performed in a time-dependent manner (Supplementary Data). Reactions were terminated with addition of SDS buﬀer and loaded onto an 8% SDS– PAGE gel. Following electrophoresis, the gel was dried and visualized by PhosphorImager analysis. DNA-PK pull-down assay DNA synaptic complex formation was determined by incubating 500 fmol of each biotinylated DNA eﬀector with 10 ml slurry of SA magnetic beads (Promega) in 500 ml of 10 mM Tris-pH 7.5, 1 mM EDTA, 5% glycerol, and 0.1% BSA for 30 min to allow for binding to occur. SA-DNA magnetic beads were washed three times to remove excess DNA. DNA-PK was bound to the SA- DNA as well as to a nonbiotinylated DNA eﬀector with a [g-32P] ATP 50 label in a 20 ml reaction containing 20 mM HEPES, pH 7.5, 8 mM MgCl2, 1 mM DTT and 5% glycerol. Following a 5-min incubation period, the DNA-PK-50 labeled DNA eﬀector complex was added to the SA-DNA-DNA-PK bead mixture and allowed to incubate at room temperature for an additional 5 min. The beads were collected in a magnetic separator and the supernatant was removed, beads were gently washed two times in reaction buﬀer, and the bound DNA quantiﬁed via scintillation counting. RESULTS DNA effectors used to assess the influence of DNA structure on activation of DNA-PK We have previously demonstrated that DNA-PK is diﬀer- entially activated by 30 pyrimidine and 50 purine-rich DNA termini, suggesting that one strand terminus may be more important for activation than the other. Therefore, we have designed a series of DNA eﬀectors with diﬀerent termini to determine how each strand, 30 or 50, contributes to activation of DNA-PK. DNA eﬀectors were designed with one strand containing a biotinylated 50 terminus so that when incubated with SA, only one termini from each duplex eﬀector is rendered accessible for DNA-PK binding (Figure 1). Eﬀectors were designed as fully duplex, or contained either a 30 or 50 overhang strand, or Y-shaped termini, all located on the accessible end (Figure 1). Using these DNA eﬀectors and puriﬁed DNA-PK, we were able to conduct a series of in vitro reactions to determine how these diﬀerent DNA structures inﬂuence activation of DNA-PKcs in Ku-dependent reactions. Effect of DNA termini on activation of DNA-PK It has previously been shown that when the DNA-PKcs is puriﬁed away from Ku, kinase activity is dramatically stimulated when DNA eﬀectors have single-stranded overhangs compared to full duplex eﬀectors (21). How- ever, the eﬀect of single-strand overhang ends on acti- vation of the heterotrimeric complex, Ku and DNA-PKcs, has not been investigated. DNA eﬀectors described in Figure 1 and Table 1 were utilized, and all experiments included a 30-bp control to ensure that diﬀerences were not a result of experimental variation. Results show that although DNA-PK is activated by a 24-bp duplex eﬀector, a 30-bp duplex eﬀector results in a greater degree of activation (Figure 2). This result was expected, as previous publications have demonstrated that DNA-PK activity is length dependent, with longer DNA eﬀectors resulting in greater kinase activity (5,21). Analysis of a DNA eﬀector comprised of 24 bp of duplex DNA with an additional 6 base overhang DNA on either the 30 or the 50 termini resulted in no greater activity than that from a full duplex DNA eﬀector with 24 bp (Figure 2A), and was in fact Figure 1. DNA eﬀectors used in this study. Duplex DNA eﬀectors were prepared from gel puriﬁed single strand oligonucleotides. The addition of streptavidin, as indicated by the oval, results in blocking of the 50 biotinylated terminus, indicated by the B. The eﬀectors generated have either a 30 overhang, 50 overhang or Y-shaped structure at the DNA-PK accessible terminus.