4026 Nucleic Acids Research, 2008, Vol. 36, No. 12 Figure 3. Titration of DNA eﬀectors containing 30 and 50 overhangs. DNA with a 30 or 50 6-base overhang, as described in ‘Methods’ section, were assayed for DNA-PK kinase activation. Reactions were incubated with increasing concentrations of each eﬀector. 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. Figure 4. Autophosphorylation of DNA-PK by overhang and Y-shaped eﬀectors. DNA-PK autophosphorylation was measured with 24- and 30-bp full duplex DNA eﬀectors, 30 and 50 overhang eﬀectors and Y-shaped eﬀectors. Reactions were performed as described in Figure 1, initiated with 1.0 mCi [g-32P] ATP, and stopped with SDS. Reaction products were separated by 8% SDS–PAGE. The gel was dried and exposed to a PhosphorImager. a SDS denaturing gel and 32P-labeled DNA-PK was visualized with a PhosphoImager . DNA-PK auto- phosphorylation resulting from the overhang eﬀectors, both 30 and 50, was signiﬁcantly reduced when com- pared with the full duplex 30-bp eﬀector (Figure 4). Furthermore, the kinetics of autophosphorylation of DNA-PK was determined with the 30-bp eﬀector and the 30 overhang eﬀector (Supplementary Data). The results conﬁrm the decreased autophosphorylation activ- ity obtained with the 30 overhang eﬀector compared to the full duplex DNA (Figure 4). The level of autopho- sphorylation presented in Figure 4 comparing the over- hang eﬀectors and the 30-bp duplex eﬀector mimics the level of peptide phosphorylation observed in peptide- based kinase assays (Figure 2). Interestingly, the level of autophosphorylation resulting from the 24-bp eﬀector is the same as that from the 30-bp eﬀector (Figure 4). If DNA-PK autophosphorylation resulting from the full duplex sets of DNA were mimicking the peptide phos- phorylation observed in the kinase assays, a lower amount of autophosphorylation from the 24-bp eﬀector would be expected. Importantly, there is no diﬀerence in autopho- sphorylation between the 30 overhang and 50 overhang DNA eﬀectors. These results demonstrate that the lower activity resulting from the 30 overhang compared to the 50 overhang eﬀector is not due to a disparity in autophos- phorylation. In contrast, the increase in autophosphory- lation resulting from the 24 bp may account for the decrease in peptide phosphorylation from the 24-bp DNA compared to the 30-bp duplex eﬀector (Figure 2). To ensure these results were consistent with the peptide kinase assays, reactions were performed in the presence and absence of the peptide substrate and similar results were obtained under both conditions (data not shown). Thus, length dependence may play a role in inﬂuencing autophosphorylation. Influence of DNA effectors containing regions of microhomology After determining that a single-strand overhang on a duplex DNA eﬀector does not increase DNA-PK acti- vity over a full duplex eﬀector, we designed a set of DNA eﬀectors with homopolymeric overhang strands to determine if sequence bias on a single-strand overhang diﬀerentially activates DNA-PK. One series of eﬀectors contained 30-6 base extensions of either Ts or As (Figure 5A). The second series were identical except the extensions were on the 50 strand of the accessible terminus (Figure 5B). Assays were performed at two DNA concen- trations as indicated in the ﬁgure. Results show that DNA-PK activity with the 30A extension was slightly decreased compared to the activity observed in reactions containing the 30A extended eﬀector, an eﬀect observed at both DNA concentrations (Figure 5A). These results are similar to our previously published results regarding sequence bias and DNA-PK activation. The diﬀerence in activation between the two is not as dramatic as we observed with full duplex eﬀectors containing 30T or 30A, but the general pattern is consistent with our previous results (19). Analysis of the 50 extended DNA eﬀectors also revealed a decreased activation with the A-containing eﬀector compared to the T-containing eﬀector (Figure 5B). Consistent with our previous results, activation with the 5-extensions is greater compared to the 30-extension, independent of sequence. Interestingly, the