Nucleic Acids Research, 2008, Vol. 36, No. 12 4025 Figure 2. Eﬀect of DNA overhangs on DNA-PK activation. DNA-PK kinase assays were conducted with annealed DNA eﬀectors, as described in ‘Materials and methods’ section. Brieﬂy, 24- and 30-bp full duplex DNA eﬀectors, 30 and 50 6-bp overhang eﬀectors and Y-shaped eﬀectors were assayed. Reactions contained 6.3 nM DNA-PK and 5 nM SA-DNA, as indicated. 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. consistently reduced compared to the 24-bp duplex. These results suggest that in the presence of Ku, DNA-PKcs does not require DNA with a single-strand overhang for maximal activation. To determine if this inhibition was due to the presence of a single stranded terminus, eﬀectors were designed with 24 bases of complementary DNA, and an addi- tional 6 bases of noncomplementary sequence on each strand at the terminus (Y-eﬀectors). Analysis of kinase activation revealed that maximal DNA-PK activation was nearly completely restored with Y-eﬀectors, suggest- ing that single-stranded ends per se are not inhibitory (Figure 2A). In order to rule out any potential diﬀerences in DNA-PK activation resulting from sequence or orien- tation dependent activation, we created the identical structure but varied the sequence on each strand. These results again demonstrated DNA-PK activation by the Y-DNA eﬀectors is comparable to the fully duplex DNA eﬀectors and any minor diﬀerences can be attributed to strand orientation-dependent sequence bias. To fully establish this point, similar eﬀectors with homopolymeric 6-base extensions were constructed. The analysis of these eﬀectors is presented in Figure 2B and again demonstrates that Y-shaped eﬀectors to not result in greater activation of DNA-PK compared do fully duplex DNA eﬀectors of identical length. Together, these results demonstrate that in the presence of Ku, DNA-PKcs does not require single-strand termini for maximal activation. The results from Figure 2 suggest that DNA-PK is not preferentially activated by single-strand overhang eﬀectors compared to full duplex eﬀectors of the same length and in fact display reduced activation. Statistical analysis of those results revealed statistically signiﬁcant diﬀerence with a P-value <0.05 in comparisons with each set of eﬀectors. We hypothesized that if there were truly relevant diﬀerences in activation with the varying overhang eﬀec- tors these may become accentuated as a function of DNA concentration. In our standard kinase assay reaction with fully duplex DNA eﬀectors, the 5-nM DNA concentra- tion is saturating (data not shown). DNA-PK activity was assayed with increasing concentrations of the 30 over- hang eﬀector or the 50 overhang eﬀector (Figure 3) and a substantial diﬀerence in activity over the entire range of eﬀector concentrations was observed. These results conﬁrm that DNA-PK exhibits lower activity from a 30 overhang DNA eﬀector compared to the 50 overhang eﬀector. The ﬁnding that increased DNA concentrations of the overhang eﬀectors were required to reach maximal activity suggests a decrease in aﬃnity of the DNA–PK complex for these eﬀectors compared to full duplex (data not shown). Influence of DNA effectors on autophosphorylation of DNA-PK It has been shown that DNA-PK activity is modulated by autophosphorylation. Upon autophosphorylation, the kinase is believed to dissociate from the DNA eﬀector, thus lowering overall kinase activity of DNA-PK (25). To investigate if the level of DNA-PK autophosphory- lation was responsible for the diﬀerential peptide phos- phorylation observed in Figure 2, kinase assays were performed as described above, with streptavidin bound eﬀectors having either 30 or 50 overhang ends, Y-shaped termini, or blunt termini accessible for DNA-PK binding. Following incubation, the samples were separated on