4028 Nucleic Acids Research, 2008, Vol. 36, No. 12 Figure 6. Activation of DNA-PK with DNA eﬀectors containing compatible mixed sequence overhang ends. Eﬀectors with 6-bp 30 overhang complementary sequences were pre-bound with DNA-PK and assayed for DNA-PK activation. Assays were conducted with 1 pmol or 0.5 pmol of a single DNA eﬀector, or 0.5 pmol of each eﬀector was included together in a reaction, as indicated. The white plus symbol indicates half a pmol of one DNA eﬀector, and the black plus symbol indicates the second eﬀector with a complementary overhang. Results are presented as the picomoles of 32P transferred to the synthetic p53 peptide in a 30-min reaction. Reactions were performed in triplicate and the mean and standard deviation are presented. eﬀectors with 30 overhangs containing a speciﬁc annealing sequence also induce high levels of synergistic activity, 256 pmol of phosphate transferred (Figure 6). This level of activity is much higher than the projected additive and average values, 136 and 108 pmol of phosphate trans- ferred. These results support the conclusion that the 30 end is crucial for microhomology-associated annealing and solidiﬁes the hypothesis that microhomology is important, as the complementary sequence in these eﬀectors is signif- icantly more speciﬁc than the DNA in Figure 5A. This indicates that the dimeric activation observed results from two eﬀectors having the ability to successfully anneal, which requires greater than one complementary base. Synaptic complex formation with DNA effectors containing single-strand overhangs To determine if the greater than additive kinase activity observed with the DNA eﬀectors containing 30 overhangs with regions of microhomology was due to an increase in formation of a synaptic complex between the two DNA eﬀectors and two DNA-PK molecules, we designed a pull- down assay utilizing radiolabeled DNA. The results from this assay (Figure 7) revealed that a synaptic complex is formed in the presence of DNA overhang eﬀectors, Figure 7. DNA-PK binding with DNA eﬀectors containing compatible overhang ends. Five hundred femtomoles of each unlabeled DNA eﬀector was bound to streptavidin magnetic beads. 32P -labeled DNA (indicated by asterisk) and the SA bound unlabeled DNA was incubated with DNA-PK or buﬀer as indicated, and labeled DNA was incubated in a separate tube with DNA-PK or buﬀer for 5 min. The labeled DNA eﬀector prebound with DNA-PK or buﬀer was added to the tubes containing SA-unlabeled DNA and DNA-PK or buﬀer, incubated for 5 min, washed, and counts were read in a scintillation counter. Results are presented as percent of total DNA- DNA-PK complex bound to SA–DNA–DNA–PK complex. as shown by the increase in activity in the reactions containing samples with prebound DNA-DNA-PK com- plexes compared to the reactions that were absent of DNA-PK. Interestingly, DNA overhang eﬀectors formed synaptic complexes regardless of the presence of a region of microhomology within the overhang. This result diﬀers from the kinase activity, where activity was increased with DNA overhang eﬀectors containing regions of micro- homology. These results demonstrate that homology driven dimeric activation seen in our kinase assays is a catalytic eﬀect, and does not result from increased binding and formation of a synaptic complexes. DISCUSSION DNA-PK is a unique protein kinase with a novel mecha- nism of activation. Precisely how DNA serves to activate DNA-PK is not known. Modeling studies suggest that DNA may thread through the catalytic subunit and enzymatic analyses suggest that single-strand ends may be important for kinase activation (16,23,26). The results presented here in Ku-dependent DNA-PK kinase assay show that Y-shaped eﬀectors with preformed single- strand ends, result in activity similar to that observed