from full duplex DNA eﬀectors. However, the presence of single-strand overhangs results in signiﬁcantly less activity. This ﬁnding is contrary to results obtained in a Ku-independent DNA-PKcs activation assay where a signiﬁcant increase in activity was observed when DNA with a single-strand overhang were used as eﬀectors (21). While important mechanistically, elegant in vivo studies show that DNA-PKcs recruitment to the site of a DSB is dependent on Ku, highlighting the importance of study- ing the mechanism of holoenzyme activity (27). The diﬀerential activation observed with unannealed ends may be explained in part by an energetic argument. The presence of Ku at the termini alleviates the need for preseparated strands. Thus, Ku may promote strand opening in conjunction with DNA-PKcs. While Ku has been reported to possess strand separation activity (28) kinetic analysis brings the catalytic nature of this activity into question (29). In any event, Ku facilitation of unwinding in conjunction with DNA-PKcs to generate the required single-strand ends could explain the diﬀer- ential activation as a function of single strand ends. The inhibition of DNA-PK activity resulting from a 30 overhang compared to a 50 overhang DNA eﬀector, combined with the synergistic activity exhibited from complementary overhangs suggests a unique role for each single-strand terminus in DNA-PK activation. These results suggest that the 50 end is more important for contact with the kinase, thus resulting in a greater degree of activation than the 30 end. However, each of these strands on their own does not result in maximal activation of DNA-PK, indicating that while each strand plays an important role, both strands are necessary for maximal DNA-PK activation. We have constructed a model for DNA-PK activation, based on existing biochemical and structural analysis of the kinase (17,22,23) in conjunction with the results presented in this paper (Figure 8). Following Ku binding and recruitment of DNA-PKcs to the site of the break, the kinase binds to the DNA terminus. The DNA strand is postulated to protrude through the kinase, with the terminus appearing on the opposite opening of the kinase. Following insertion through the kinase, evidence points to fraying or unwind- ing of the DNA ends (22). Our data show that a DNA fragment with a 50 overhang induces greater DNA-PK activation than a DNA fragment with a 30 overhang. Yet, when two complementary 30 overhang DNA eﬀectors are incubated together, a synergistic level of DNA-PK activation is observed. When two pieces of DNA with 50 complementary overhangs are incubated together, the synergistic level of activation is signiﬁcantly lower. Combined, this data suggest that the 50 strand is important for activation, while the 30 strand may be important in scanning for regions of microhomology. The potential role of the 30 strand in microhomology searching is also consistent with the 50 to 30 exonuclease activity of Artemis degrading the 50 strand. This activity would generate a 30 single strand at the terminus, which displays relatively low activity until a complementary sequence is identiﬁed. Previous data suggest the possibility that following fraying of the DNA ends after DNA-PK binding, the strands may insert themselves into active sites on the Nucleic Acids Research, 2008, Vol. 36, No. 12 4029 Figure 8. Model for activation of DNA-PK. DNA, depicted in black and orange, threads through DNA-PK, depicted in yellow. (A) When DNA of adequate length threads through the kinase, the ends separate and the 50 end can insert itself into an active site on the periphery of the kinase. (B) A 50 overhang containing eﬀector can activate in the absence of dimerization while a 30 overhang (C) displays less activa- tion. When the 30 overhang possesses a complementary sequence to the second DNA molecule with a 30 overhang, the two DNA ends can anneal, thus orienting the two DNA-PK molecules into a synaptic complex. The annealing may allow for more DNA to thread through the kinase, potentially making more of the 50 strand accessible for kinase activation. This activation could then occur in either a cis (D) or trans (E) fashion. perimeter of the kinase (30). Our data suggest that each DNA termini is bound by DNA-PK in a dimeric complex independent of homology. The DNA is scanned for regions of microhomology, which potentially pulls the DNA further through the kinase upon which once homol- ogous regions are identiﬁed, increased kinase activity results. The model predicts that this activation is accom- panied by insertion of the 50 end into the active pocket on the periphery of the kinase (Figure 8). This dimeric activation is expressed as a synergistic level of activity when the two complementary eﬀectors are simultaneously incubated in an assay for DNA-PK activation. Whether the insertion of the 50 single strand occurs in a cis or trans fashion is a question that still needs to be answered with future experiments. The single strand ends with complementary areas have the potential to promote activation of the DNA-PK bound ends. DNA-PKcs has been found to exist in mainly in a monomeric state (31). However, it is thought that when DNA-PKcs is bound to the ends of two DNA molecules at the site of a DSB, the protein kinase is responsible for bringing the two DNA ends into proximity of each other in the form of a synaptic complex. The formation of this synaptic complex is thought to corre- spond to the dimerization of DNA-PKcs. Furthermore, the dimerization of DNA-PKcs and subsequent formation