Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

Abstract

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.

Figure 1

Defective repair of IR-induced DSBs in cells expressing DNA-PKcs autophosphorylation mutants. DNA-PKcs-deficient V3 cells (solid circles), V3 cells expressing full-length wild-type human DNA-PKcs (PRKDC, solid triangles), or V3 cells expressing DNA-PKcs containing either serine/threonine to alanine (A6 PRKDC, open circles) or serine/threonine to aspartic acid (D6 PRKDC, open triangles) were irradiated in agarose plugs and analyzed by PFGE as described in Materials and Methods.

Figure 2

Autophosphorylation mutant DNA-PKcs proteins purified from V3 cells are indistinguishable from wild-type with respect to biochemical activity. (A) Purified DNA-PKcs (0.5 μg of WT, A6, D6 or A7) was fractionated using SDS–PAGE and either stained with Coomassie blue or transferred to nitrocellulose and detected by immunoblot (αDNA-PKcs). (B) Purified DNA-PKcs (20 ng of WT, A6, D6 or A7) was assayed in the presence of 0, 7.5, 15, 30 or 60 ng purified Ku for kinase activity towards a peptide substrate. (C) Purified DNA-PKcs (WT, A6, D6 or A7) and Ku were pre-incubated for 0–10 min either in the absence (solid symbols) or presence (open symbols) of ATP, and then assayed in the presence of a full complement of ATP, essential co-factors and peptide substrate as described previously (39). Each point represents an average of duplicates. (D) Purified DNA-PKcs (WT, A6, D6 or A7) and Ku were incubated in the presence of [γ-³²P]ATP under conditions that support DNA-PK autophosphorylation [as described in (31)] and fractionated using SDS–PAGE. DNA-PKcs proteins were excised from the gel and ³²P incorporation was quantitated by Cerenkov counting. Each point represents an average of duplicates.

Figure 3

DNA-PKcs kinase activity regulates T4 DNA ligase-mediated DNA end joining. Purified DNA-PKcs (1 μg, lanes 10–15) and/or Ku (0.5 μg, lanes 6–8 and 13–15) were incubated in the absence (lanes 5 and 9) or presence (lanes 6–8 and 10–15) of T4-DNA ligase. Lanes 2–4 contained T4 DNA ligase alone (no Ku or DNA-PKcs). Lane 1 contained linearized ds plasmid DNA alone. Samples in lanes 4, 8, 12 and 15 were pre-incubated with wortmannin (W) (1 μM) or an equivalent volume of DMSO (D) (lanes 3, 7, 11 and 14). Reactions were stopped after 30 min and analyzed as described in Materials and Methods.

Figure 4

Autophosphorylation defective DNA-PKcs does not support T4-mediated ligation of DNA ends. (A) Conditions were as in Figure 3, except that reactions contained T4 DNA ligase, purified Ku and wt, A6, D6 or A7 DNA-PKcs proteins, as indicated. Reactions were terminated after 5, 10, 20 or 30 min, as indicated. (B) The total intensity of DNA ligation products in (A) was quantitated using ImageQuant (v.5.2) and expressed relative to maximal observed intensity as percent ligation.

Figure 5

Model for the role of DNA-PKcs autophosphorylation in DNA end ligation. In the first step on NHEJ, Ku (indicated by the small, closed circles) binds to the ends of the DSB. DNA-PKcs (large gray ellipses) is recruited to form the active DNA-PK complex. Upon synapsis, DNA-PK undergoes autophosphorylation, which causes a conformational change that renders the DNA ends accessible to end joining by T4 DNA ligase or the XRCC4/DNA-ligase IV complex.