A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining

Abstract

DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-PK activation with DNA effectors containing two unannealed ends was identical to activation observed with fully duplex DNA effectors of the same length. The presence of a 6-base single-stranded extension resulted in decreased activation compared to the fully duplex DNA. DNA-PK activation using DNA effectors with compatible termini displayed increased activity compared to effectors with noncompatible termini. A strand orientation preference was observed in these reactions and suggests a model where the 3' strand of the terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ.

Figure 1.

DNA effectors used in this study. Duplex DNA effectors were prepared from gel purified single strand oligonucleotides. The addition of streptavidin, as indicated by the oval, results in blocking of the 5′ biotinylated terminus, indicated by the B. The effectors generated have either a 3′ overhang, 5′ overhang or Y-shaped structure at the DNA-PK accessible terminus.

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, 3′ and 5′ 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.

Figure 3.

Titration of DNA effectors containing 3′ and 5′ overhangs. DNA with a 3′ or 5′ 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, 3′ and 5′ overhang effectors and Y-shaped effectors. Reactions were performed as described in Figure 1, initiated with 1.0 μCi [γ-³²P] ATP, and stopped with SDS. Reaction products were separated by 8% SDS–PAGE. The gel was dried and exposed to a PhosphorImager.

Figure 5.

Dimeric activation of DNA-PK from effectors containing compatible homopolymeric overhang ends. (A) Effectors with either a 6-base poly-T 3′ overhang or a 6-base poly-A 3′ 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 5′ overhang or a 6-base poly-A 5′ 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.

Figure 6.

Activation of DNA-PK with DNA effectors containing compatible mixed sequence overhang ends. Effectors with 6-bp 3′ 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.

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.

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 5′ end can insert itself into an active site on the periphery of the kinase. (B) A 5′ overhang containing effector can activate in the absence of dimerization while a 3′ overhang (C) displays less activation. When the 3′ overhang possesses a complementary sequence to the second DNA molecule with a 3′ 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 5′ strand accessible for kinase activation. This activation could then occur in either a cis (D) or trans (E) fashion.