Nucleic Acids Research, 2008, Vol. 36, No. 12 4027 Figure 5. Dimeric activation of DNA-PK from eﬀectors containing compatible homopolymeric overhang ends. (A) Eﬀectors with either a 6-base poly-T 30 overhang or a 6-base poly-A 30 overhang were prebound with heterotrimeric DNA-PK and assayed for DNA-PK kinase activation. Assays were conducted with either 1 or 0.5 pmol of each eﬀector per reaction, as indicated, or 0.5 pmol of each eﬀector was incubated together in a single reaction. 0.5 pmol of the poly-T eﬀector is indicated by a white plus symbol, and half a picomole of the poly-A eﬀector is indicated by the black plus symbol. 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. (B) Eﬀectors with either a 6-base poly-T 50 overhang or a 6-base poly-A 50 overhang were assayed for DNA-PK kinase activation as described above. 0.5 pmol of the poly-T eﬀector is indicated by a white plus symbol, and half a pmol of the poly-A eﬀector is indicated by the black plus symbol. eﬀect of the extension sequence on DNA-PK activation was similar, with both sets of eﬀectors showing decreased activation with the A extensions. These eﬀectors were designed such that when individ- ually included in a reaction, the extensions are non- compatible and thus each terminus presumably binds a single DNA-PK. However, if combined, the eﬀectors are capable of annealing, mimicking regions of microhomol- ogy that may occur after processing of termini in prepara- tion for ligation. To perform this assay, each DNA eﬀector was pre-incubated with DNA-PK prior to the addition of 32P-ATP to initiate the kinase reaction. In an eﬀort to increase the level of activation resulting from the relatively poor DNA overhang eﬀectors and to control for total DNA in the combined reaction, individual eﬀector reactions were performed at 2 DNA concentrations, one being equal to the DNA concentration in the combined and the other at 1/2 the DNA concentration of the com- bined reaction. In the combined reactions with the 30 extended eﬀectors, a signiﬁcant increase in activation was observed compared to the individual reactions. If kinase activation were simply additive upon combining the eﬀectors, we would have expected kinase activity of 85 pmol of phosphate transferred in the combined reaction. If we consider an average of the individual reactions at equal concentrations of total DNA we would have expected 62 pmol of phosphate transferred. DNA- PK activity resulting from the eﬀectors with areas of microhomology resulted in close to 190 pmol of phos- phate transferred (Figure 5A). This observed synergy is dependent on the presence of compatible DNA termini and suggests that annealing inﬂuences the activation of DNA-PK. To determine if strand orientation of a microhomol- ogous sequence inﬂuences synergistic activation of DNA-PK, we performed analysis of the 50 extended eﬀectors. Interestingly, while the 50 overhang eﬀectors with microhomology did produce a small amount of synergistic DNA-PK activity, the level of synergy was not as great as that resulting from the 30 overhang eﬀectors with complementary regions (Figure 5B). Additive results predicted a value of 137 pmol of phosphate transferred, while averaging the individual reactions provides a value of 130 pmol of phosphate transferred. The combined reactions resulted in synergistic activity, with 171 pmol of phosphate transferred, but the increase of this activity over the additive and averaged values was not as great as that seen with the 30 compatible overhangs. These results suggest that the 30 end of the DSB is dictating the aligning and microhomology directed ligation. The eﬀectors in Figure 5A have complementary over- hangs, but because of the homopolymeric sequence, these areas have the potential for annealing in diﬀerent registers with 2-, 3-, 4-, 5- or 6 bp annealing. To correct and control for this possibility, eﬀectors depicted in Figure 6 were designed with a complementary mixed sequence on the 30 overhangs. These eﬀectors therefore can only anneal in a single position and thus would contain nicks at each junction following annealing. The kinase assay was conducted as described above, and results show that