Synapsis of DNA ends by DNA-dependent protein kinase

Abstract

The catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) is required for a non-homologous end-joining pathway that repairs DNA double-strand breaks produced by ionizing radiation or V(D)J recombination; however, its role in this pathway has remained obscure. Using a neutravidin pull-down assay, we found that DNA-PK(CS) mediates formation of a synaptic complex containing two DNA molecules. Furthermore, kinase activity was cooperative with respect to DNA concentration, suggesting that activation of the kinase occurs only after DNA synapsis. Electron microscopy revealed complexes of two DNA ends brought together by two DNA-PK(CS) molecules. Our results suggest that DNA-PK(CS) brings DNA ends together and then undergoes activation of its kinase, presumably to regulate subsequent steps for processing and ligation of the ends.

Fig. 1. DNA-PK_CS mediates synapsis of DNA ends. (A) DNA synapsis versus DNA-PK_CS concentration. In the pull-down assay, DNA-PK_CS was incubated with neutravidin–agarose beads in binding buffer with two types of 42 bp DNA fragments: radiolabeled f42* (0.4 nM) and biotinylated f42B (4 nM). ‘Radioactivity pulled down’ is the fraction of total f42* that remained attached to the beads after washing, and signifies the amount of synapsis, which increased with increasing DNA-PK_CS concentration. (B) Specificity of the pull-down assay. The pull-down assay was performed in the presence or absence of DNA-PK_CS (14.4 nM), f42* (0.4 nM), f42B (4 nM) and unlabeled f42 (4 nM). The signal required formation of a complex containing DNA-PK_CS, f42* and f42B. The complex was non-covalent, since it was disrupted by high salt concentration. (C) Effect of salt concentration on DNA synapsis. DNA-PK_CS (14.4 nM), Ku (14.4 nM) or both were incubated with f42B (4 nM) and f42* (0.4 nM) in binding buffer at 30 or 150 mM NaCl. Synapsis occurred at both salt concentrations, but was not stimulated by Ku. (D) Effect of Ku on DNA synapsis. DNA-PK_CS (14.4 nM), Ku (14.4 nM) or both were incubated with the longer DNA fragments f181B (4 nM) and f181* (0.4 nM) in a buffer of 10 mM HEPES pH 7.4, 20% glycerol and 80 mM NaCl. Under these modified conditions, Ku was able to stimulate DNA synapsis weakly.

Fig. 2. Electron microscopic visualization of DNA-PK_CS and Ku associated with DNA. Linearized plasmid DNA (100 ng, 2 nM) terminating in 5′ non-complementary, single-stranded overhangs was incubated with either DNA-PK_CS (22 nM), Ku (22 nM) or both proteins, and visualized by electron microscopy. DNA–protein complexes are displayed in order of observed abundance from top to bottom for each of the reactions. Bar = 1 kb DNA. For quantification of the distribution of DNA molecules, see Table I. (A–E) Linearized plasmid DNA incubated with DNA-PK_CS. (A) DNA with DNA-PK_CS bound to one end. (B) Unbound DNA. (C and D) DNA circularized by DNA-PK_CS. (E) DNA with DNA-PK_CS bound to both ends. (F–J) Linearized plasmid DNA incubated with Ku. (F) Unbound DNA. (G) DNA with Ku bound to one end. (H and I) DNA with Ku translocated to internal sites. (J) DNA with Ku bound to both ends. (K–O) Linearized plasmid DNA incubated with DNA-PK_CS and Ku. DNA-PK_CS is much larger than Ku; therefore, we are unable to determine by this technique whether the protein represents DNA-PK_CS alone or DNA-PK_CS plus Ku. (K) DNA with protein bound to both ends. (L and M) DNA circularized by protein. (N) DNA with protein bound to one end. (O) Unbound DNA.

Fig. 3. Electron microscopic image of DNA synapsis mediated by DNA-PK. Linearized plasmid DNA (100 ng, 2 nM) was incubated with DNA-PK_CS (22 nM) and Ku (22 nM) and visualized by electron microscopy. Each end of the DNA molecule in the upper left is bound to a single protein complex. The DNA molecule in the lower right has been circularized with what appears to be two protein complexes bound to the ends of the DNA. Bar = 1 kb DNA.

Fig. 4. Distribution of projected surface areas for protein complexes bound to DNA. (A) DNA incubated with DNA-PK_CS. (B) DNA incubated with DNA-PK_CS and Ku. The top histograms display the distribution of projected surface area for end-bound protein, and the bottom histograms display the distribution for circle-bound protein. Surface area measurements are in the same relative units for both panels. Note the difference in scale on the y-axis for (A) and (B). The mean projected surface areas of circle-bound protein are 1.8- and 1.7-fold larger than the projected surface areas of end-bound protein in (A) and (B), respectively, indicating that DNA synapsis involves two DNA-PK_CS molecules.

Fig. 5. Role of DNA-PK_CS kinase activity in synapsis. (A) DNA-PK_CS kinase activity is not required for DNA synapsis. DNA-PK_CS (14.4 nM) was incubated with f42* and f42B in kinase buffer plus ATP and peptide substrate in the presence or absence of wortmannin (10 µM). The upper panel shows the pull-down assay, in which DNA synapsis was unaffected by wortmannin. The lower panel shows the gel-based kinase assay, in which phosphorylation of the peptide substrate was completely inhibited by wortmannin. (B) Activation of DNA-PK_CS by DNA is cooperative. The upper panel shows the gel-based kinase assay, in which DNA-PK_CS (5.8 nM) was incubated with f42 (0.04–2.05 nM, increasing in 1.3-fold increments) and synthetic peptide (285 µM) in kinase buffer. In the lower panel, kinase activation was plotted as a function of DNA concentration. The inset shows kinase activity for the 11 subsaturating DNA concentrations in a log–log plot. The arrows represent the highest DNA concentration used in the linear and log–log plots. The slope of 1.7 demonstrates significant cooperativity in the activation of DNA-PK_CS by DNA.

Fig. 6. Model for DNA-PK_CS binding and kinase activation. Ku binding: the Ku heterodimer binds to DNA ends, forming a ring structure (depicted in cross-section) that encircles the DNA (Walker et al., 2001). DNA-PK_CS binding: DNA-PK_CS is recruited to the DNA end by Ku, which then translocates inward. Double-stranded DNA binds to an open channel in DNA-PK_CS. Synapsis: two DNA-PK_CS molecules bring two DNA ends together to form a synaptic complex. Kinase activation: one single-stranded DNA end is inserted into an opening to the enclosed cavity adjacent to the open channel in DNA-PK_CS. The other single-stranded end is inserted into an opening to the enclosed cavity on the opposing DNA-PK_CS molecule. The interactions between DNA-PK_CS and single-stranded DNA ends stabilize the synaptic complex and fully activate the kinase.