|Home | About | Journals | Submit | Contact Us | Français|
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.
In mammalian cells, DNA double-strand breaks (DSBs) are repaired by two main pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) (1–3). Current models predict that NHEJ involves the sequential recruitment of proteins to the DSB in order to juxtapose free double-stranded DNA (dsDNA) ends (synapsis) and seal the phosphodiester backbone (ligation). The core NHEJ apparatus includes the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), the Ku70/Ku80 heterodimer (Ku) and the XRCC4-DNA ligase IV complex [reviewed in (2,4)]. Ku, which has a high affinity for ends of dsDNA, likely binds to the DSB first, followed by the recruitment of DNA-PKcs. The interaction of DNA-PKcs with DNA-end-bound Ku leads to the formation of the DNA-dependent protein kinase holoenzyme, DNA-PK, which is a serine/threonine protein kinase with an in vitro substrate preference for serine or threonine residues followed by glutamine (SQ or TQ motifs) (5,6). Subsequently, the XRCC4-DNA ligase IV complex is recruited via Ku–DNA-PKcs, Ku–DNA ligase IV, and DNA-PKcs–XRCC4 protein–protein interactions [reviewed in (2)]. In addition to the core NHEJ apparatus, additional factors may be required to repair a subset of DSBs, such as those with complex termini. Such factors include Artemis (7), polynucleotide kinase (8), Werner's Syndrome helicase (WRN) (9,10), DNA polymerase μ (11) and DNA polymerase λ (12).
Several studies have shown that the protein kinase activity of DNA-PKcs is essential for NHEJ (13–17); however, the precise role of DNA-PK kinase activity within NHEJ has not been determined. DNA-PKcs is a member of the phosphatidyl inositol 3 kinase-like family of protein kinases (PIKKs) [reviewed in (2)]. Similar to other PIKKs, the in vitro protein kinase activity of DNA-PK is inhibited by wortmannin and LY294002 (18,19), both of which radiosensitize cells and inhibit the repair of DSBs in vivo (17,20,21). In addition, both DNA-PKcs-deficient cells and DNA-PKcs-deficient cells complemented with kinase-dead DNA-PKcs are severely compromised in their ability to repair DSBs (13,14). Consistent with the requirement of DNA-PK kinase activity for NHEJ in vivo, Mg-ATP is required for DNA-PK-dependent end joining in crude extracts from human cells (22).
In vitro, DNA-PK undergoes autophosphorylation that correlates with loss of protein kinase activity (23–25). Some in vitro studies have shown that autophosphorylation promotes dissociation of phosphorylated DNA-PKcs from DNA-bound Ku (24,25), whereas others have failed to see phosphorylation-induced dissociation (26–28). In vitro studies have shown that DNA-PK can protect dsDNA ends from exonuclease digestion and that prior incubation of DNA-PK with ATP prevents dsDNA protection from exonucleases in a wortmannin-inhibitable manner (28). Moreover, ATP and the protein kinase activity of DNA-PK were found to be required to make DNA ends accessible to T4 DNA ligase-mediated end joining (28). In vivo studies also suggest that DNA-PK kinase activity is intimately involved in regulating the accessibility of DNA ends to other proteins. For example, rates of HR were lower in DNA-PKcs-proficient cells that had been treated with the specific DNA-PK inhibitor IC86621 than in cells that lacked DNA-PKcs (29), suggesting that in the absence of DNA-PK kinase activity, the DNA ends are not accessible to alternate DNA repair processes. Together, these data are all consistent with a requirement for autophosphorylation of DNA-PKcs for remodeling of DNA-end-bound DNA-PK prior to ligation (28,30).
We identified previously seven in vitro autophosphorylation sites in DNA-PKcs, six of which are located in the central region of the protein, between amino acids 2609 and 2647 (31). Three of these sites (threonines 2609, 2638 and 2647) were independently identified by other investigators (32,33). Four of the identified sites (threonines 2609, 2638 and 2647, and serine 2612) were phosphorylated in vivo in okadaic-acid-treated cells (31), and DNA-PKcs phosphorylated on threonine 2609 localized to sites of DNA damage in vivo (32). Cells expressing DNA-PKcs containing single mutations at any of the identified phosphorylation sites were not radiosensitive (30,32). In contrast, cells expressing DNA-PKcs containing six serine/threonine to alanine autophosphorylation site mutations (T2609A, S2612A, S2620A, S2624, T2638A and T2647A; referred to here as A6) were more radiosensitive than cells that lacked DNA-PKcs and had a severely impaired ability to repair coding and signal ends in in vivo extrachromosomal V(D)J recombination assays (30,32). Surprisingly, the protein kinase activity of the purified DNA-PKcs A6 mutant protein was indistinguishable from wild-type (wt) DNA-PKcs, including the ability to undergo autophosphorylation-induced inactivation in vitro (30). Cells expressing DNA-PKcs containing six serine/threonine to aspartate mutations as a phosphorylation site mimic (referred to here as D6), were less radiosensitive than cells lacking DNA-PKcs or cells containing the A6 mutant DNA-PKcs, but were significantly more radiosensitive than cells expressing wt DNA-PKcs (30). Also, D6 cells had <10% of the ability of wt cells to rejoin coding ends in extrachromosomal V(D)J recombination assays (30). This suggests that the D6 mutant is significantly compromised with respect to DSB repair and that aspartate is a poor mimic for phosphorylation at these sites. Together these studies suggest a model in which autophosphorylation of DNA-PKcs is required to facilitate DNA end joining, likely by remodeling the DNA-PK holoenzyme, thereby making the DNA ends accessible for ligation.
To characterize the role of DNA-PKcs autophosphorylation in NHEJ, we have examined the repair of DSB in vivo. We found that similar to DNA-PKcs-deficient cells, both A6 and D6 cells were defective in the repair of ionizing radiation (IR)-induced DSBs compared to cells expressing wt DNA-PK. We next sought to rationalize this DSB repair defect based on the biochemical properties of the various purified DNA-PKcs proteins. We confirmed that the phosphorylation mutant forms of DNA-PKcs are catalytically active, and that each mutant form, like wt, undergoes ATP-dependent autophosphorylation and inactivation. However, autophosphorylation mutant forms of DNA-PKcs were defective with respect to the ability to support T4 DNA ligase-mediated DNA end joining. These results demonstrate that DNA-PKcs autophosphorylation on this small cluster of residues mediates the accessibility to DNA-PK-bound DNA ends without otherwise altering the biochemical properties of the protein kinase. We speculate that a remodeling deficiency of DNA-PK holoenzymes containing DNA-PKcs phosphorylation site mutants is responsible for both the radiosensitivity and the defective repair of IR-induced DSBs in vivo observed in the DNA-PK mutant cells.
V3 (DNA-PKcs-deficient) hamster cells expressing vector, wt DNA-PKcs, autophosphorylation mutant A6 (T2609A, S2612A, S2620A, S2624A, T2638A and T2647A) or autophosphorylation site mutant D6 (T2609D, S2612D, S2620D, S2624D, T2638D and T2647D), were as described previously (30). V3 cells expressing the DNA-PKcs A7 (T2609A, S2612A, S2620A, S2624A, T2638A, T2647A and S3205A) mutant were constructed using methods similar to those reported previously (30).
A pulsed-field gel electrophoresis (PFGE) assay for measuring rates of IR-induced DSB repair in cells was as described previously (34). Briefly, cells were embedded in agarose plugs, irradiated with 40 Gy γ radiation and allowed to recover for the indicated times before deproteination and fractionation by PFGE.
Recombinant human DNA-PKcs was purified from 2.0 l of V3 cells expressing wild-type, A6, D6 or A7 DNA-PKcs (30). Cell pellets were washed in ice-cold phosphate-buffered saline (PBS), followed by hypotonic buffer [LSB: 10 mM HEPES, pH 7.2, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 0.2 mM phosphatidyl sulfonyl fluoride (PMSF), 0.2 μg/ml pepstatin A, 0.2 μg/ml aprotinin, 0.2 μg/ml leupeptin and 0.1 mM benzamidine], then frozen in liquid nitrogen. Lysates were thawed, adjusted to contain 350 mM NaCl, 10 mM MgCl2, and 1 mM DTT, and were then centrifuged at 10000 g for 20 min at 4°C. The supernatant was recovered, diluted to contain 75 mM KCl in TB buffer [50 mM Tris–HCl, pH 8.0, 5% (v/v) glycerol, 0.1 mM DTT, 0.2 mM PMSF, 0.1 mM benzamidine, 0.2 μg/ml pepstatin A], and then applied to a pre-equilibrated 25 ml column of DEAE–Sepharose Fast Flow (Amersham Pharmacia, Baie d'Urfe, PQ). The column was washed with TB buffer containing 75 mM KCl and eluted with TB buffer containing 175 mM KCl. DNA-PKcs was detected by immunoblot and the appropriate fractions were pooled and diluted to contain 75 mM KCl in TB buffer, and then applied to a 12.5 ml column of SP–Sepharose (Amersham Biosciences). The column was washed with TB buffer containing 75 mM KCl and eluted with TB buffer containing 175 mM KCl. DNA-PK-containing fractions were pooled and diluted to contain 100 mM KCl in TB buffer and applied to a 1.0 ml column of single-stranded DNA (ssDNA) cellulose (Sigma). The column was washed in TB buffer containing 100 mM KCl and eluted with a linear gradient of TB buffer containing 100–260 mM KCl. DNA-PKcs-containing fractions were diluted to contain 100 mM KCl in TB buffer and applied to a 1 ml Mono Q FPLC column (Amersham Biosciences). The column was washed in TB buffer containing 100 mM KCl and 0.02% (v/v) Tween-20 and eluted with a linear gradient of TB buffer containing 100–350 mM. DNA-PKcs-containing fractions were concentrated and dialyzed to contain 100 mM KCl using a Centricon-50 (Amicon Bioseparations), and then stored in aliquots at −80°C.
DNA-PKcs and Ku were purified from HeLa cells as described previously (35) and stored in 50 mM Tris–HCl, pH 8.0, 5% (v/v) glycerol, 0.2 mM EDTA, 100 mM KCl, 0.1 mM DTT, 0.1 mM benzamidine, 0.5 mM PMSF and 1 μg/ml pepstatin in aliquots at −80°C.
T4 DNA ligase-mediated end joining was modified from a previously published procedure (28). Purified DNA-PKcs and Ku were incubated in ligation buffer containing 50 mM Tris–HCl, pH 7.6, 50 mM NaCl, 10 mM MgCl2, 1 mM ATP, 10 μg BSA, 1 mM DTT, 5% (w/v) polyethylene glycol-8000 and 100 ng of pGEM7Zf(+) plasmid DNA (previously linearized with EcoRI) in a total volume of 10 μl. Where indicated, DNA-PKcs and Ku were pretreated with wortmannin or an equivalent volume of dimethyl sulfoxide (DMSO) prior to addition to the end-joining reactions. Reactions were started by the addition of 0.5 U of T4 DNA ligase (Invitrogen, Carlsbad, CA) and incubated at 37°C. Reactions were stopped by the addition of SDS to 1% (w/v) and EDTA to 10 mM. Samples were deproteinated by the addition of proteinase K (30 μg) and an incubation at 37°C for 30 min. Samples were electrophoresed on 1.2% agarose gels, stained with ethidium bromide, and imaged under ultraviolet light on a GelDoc imager (Bio-Rad).
Cells expressing DNA-PKcs in which six of the previously identified autophosphorylation sites (threonines 2609, 2638 and 2647, and serines 2612, 2620, and 2624) had been mutated to alanine (A6) or aspartate (D6) were extremely sensitive to IR-induced cell killing. In in vivo extrachromosomal V(D)J recombination assays, D6 and A6 cells exhibited profound defects in the rejoining of coding ends or both coding and signal ends, respectively (30). However, the ability of these cell lines to repair DNA damage in the context of chromatin has not been examined. To determine whether cells expressing the A6 or D6 mutant DNA-PKcs proteins were also defective in repairing IR-induced DSBs, V3 cells (that lack DNA-PKcs) or V3 cells expressing wild-type, A6 or D6 human DNA-PKcs were irradiated and the rate of DSB repair was monitored using pulsed-field electrophoresis (Figure (Figure1).1). Cells expressing either the A6 or D6 mutant of DNA-PKcs were significantly impaired in their ability to repair DSBs. In fact, the A6 cells were slightly more impaired at repairing DSBs than the DNA-PKcs-deficient cells (Figure (Figure1),1), which is consistent with the higher radiosensitivity of A6 cells in clonogenic survival assays relative to DNA-PKcs-deficient cells (30). Thus, we surmise that phosphorylation at this cluster of sites is required for the repair of IR-induced DSBs in vivo.
To further probe the mechanism for defective DSB repair and radiation sensitivity in these cells, we examined the biochemical properties of the purified DNA-PKcs proteins. Wild-type DNA-PKcs and the phosphorylation site mutants A6 and D6 were purified from V3 cells to ~95% homogeneity (Figure (Figure2A).2A). Each purification yielded between 200 and 350 μg of the various DNA-PKcs proteins per 2.0 l of cell culture. An A7 DNA-PKcs phosphorylation site mutant was also purified which contains the same mutations as A6 DNA-PKcs but with an additional S to A mutation at S3205 (Figure (Figure2A).2A). Serine 3205 was previously identified as an in vitro DNA-PKcs autophosphorylation site (31). Substitution of this site to either alanine or aspartic acid, either alone or in combination with the A6/D6 mutations, does not appear to alter DNA-PK function in vitro or in living cells (data not shown).
In the absence of added Ku, the protein kinase activity of each of the purified DNA-PKcs proteins was very low, indicating that the purified DNA-PKcs proteins were not contaminated with rodent Ku (Figure (Figure2B).2B). When purified human Ku was titrated into the reactions, the activity of both wt and mutant DNA-PKcs proteins were stimulated equally up to an ~10-fold stimulation compared to when no Ku was present (Figure (Figure2B),2B), suggesting that all mutant proteins can productively interact with Ku to activate DNA-PK kinase activity. In addition, the A6, D6 and A7 mutant proteins underwent ATP-induced loss of protein kinase activity to an extent similar to that of wt (Figure (Figure2C),2C), indicating that phosphorylation at these sites is not required for inactivation of DNA-PK under in vitro conditions. These results corroborate our previous results using DNA–cellulose pull-down assays and DNA-PKcs purified using a Ku-affinity column (30). Given that the mutant proteins were all Ku-stimulated and underwent ATP-dependent inactivation, we assessed the relative decrease in autophosphorylation of the mutant proteins relative to wt DNA-PKcs. In each case, there was a 20–25% reduction in the autophosphorylation of the A6, D6 and A7 DNA-PKcs proteins (Figure (Figure2D),2D), potentially indicating that a large number of additional in vitro DNA-PKcs autophosphorylation sites remain to be identified.
In order to determine if the mutant DNA-PKcs proteins were able to support DNA end joining, we utilized an elegant method devised by van Gent and colleagues (28) in which T4 DNA ligase was used to join DNA ends of a linear dsDNA substrate in the presence or absence of DNA-PK. In this assay, ATP was shown to be required for T4 DNA ligase-mediated DNA end joining in the presence of purified DNA-PK, and this ATP-dependent T4 DNA ligase-mediated DNA end joining in the presence of DNA-PK was inhibited by wortmannin (28). As shown previously (28), addition of purified wt DNA-PK to reactions containing T4 DNA ligase promoted intermolecular DNA end joining (Figure (Figure3).3). Addition of wortmannin had no effect on end joining either by T4 DNA ligase alone or T4 DNA ligase in the presence of Ku, whereas wortmannin inhibited T4 DNA ligase-mediated ligation in the presence of DNA-PKcs or DNA-PKcs plus Ku (Figure (Figure3).3). Therefore, as shown previously (28) and under the assay conditions used in this paper, T4 DNA ligase-mediated intermolecular joining was dependent on the protein kinase activity of DNA-PK. We next assayed for the ability of autophosphorylation mutant proteins A6 and D6 to support end joining in this assay at various incubation times. Significantly, the ability of A6 and D6 mutant DNA-PKcs proteins to support T4-mediated end joining (in the presence of Ku) was substantially reduced relative to wt DNA-PKcs, particularly at early time points (Figure (Figure4A).4A). Quantitation of the DNA ligation product intensity showed that after 5 min, there was at least 12-fold more DNA-end ligation by T4 DNA ligase in the presence of wt DNA-PKcs as compared to A6/D6 mutant DNA-PKcs (Figure (Figure4B).4B). The A7 mutant DNA-PKcs also showed a reduced ability to support T4-mediated end joining (data not shown).
NHEJ is the major mechanism for the repair of IR-induced DSBs in mammalian cells (2,4). Although the protein kinase activity of DNA-PK is required for NHEJ, the precise role of DNA-PK kinase activity within NHEJ was unknown. Here, we demonstrate that autophosphorylation of a cluster of sites in the central region of DNA-PKcs (amino acids 2609–2648) is required for efficient repair of IR-induced DSBs in vivo. Purified DNA-PKcs proteins containing mutations of serine/threonine to alanine or aspartate at this cluster of sites did not affect the catalytic activity of DNA-PKcs. However, autophosphorylation-defective DNA-PKcs proteins were dramatically compromised in their ability to support T4 DNA ligase-mediated end joining. These data provide strong evidence that autophosphorylation of DNA-PKcs at this cluster of sites is required to remodel DNA-end-bound DNA-PK complexes prior to ligation of DNA ends, as postulated previously (28,30) (Figure (Figure5).5). In the context of DSBs in chromatin, this autophosphorylation-dependent remodeling may release DNA-PK-bound DNA ends to either XRCC4/DNA ligase IV for ligation, various NHEJ factors for processing or for alternating the DSB repair processes. In fact, a recent study using the XRCC4-DNA ligase IV complex also found that the DNA-PKcs A6 mutant (also called ABCDE) failed to support in vitro DNA end joining (37). Similarly, the D6 mutant (in which the phosphorylation sites are replaced with aspartic acid, a phosphorylation mimic) also has reduced end-joining activity in XRCC4-DNA ligase IV assays. We believe these studies to be the first assignment of a defined biochemical/molecular function to specific phosphorylation sites within DNA-PKcs.
As found previously for DNA-PKcs purified by a Ku80 pull-down procedure, DNA-PKcs containing alanine in place of threonines 2609, 2638 and 2647, and serines 2612, 2620 and 2624 still lost protein kinase activity when pre-incubated in the presence of ATP and DNA (30), suggesting that these sites are not required for autophosphorylation-induced loss of DNA-PK activity in vitro. Indeed, the A6 mutant DNA-PKcs was phosphorylated to ~75–80% of wild-type in vitro (Figure (Figure2D),2D), indicating that DNA-PKcs is autophosphorylated at additional sites in vitro. One such site is serine 2056 [(38); Y. Yu and S. P. Lees-Miller, unpublished data]. Data presented here also exclude a previously identified in vitro site (serine 3205) from involvement in DNA-PK autophosphorylation-dependent inactivation (Figure (Figure2C)2C) and suggest serine 3205 is unlikely to even be a pronounced in vitro phosphorylation site (Figure (Figure2D).2D). Further studies will be required to identify additional in vitro and in vivo autophosphorylation sites within DNA-PKcs and to determine the role these sites play in the regulation of NHEJ.
Thanks to Katarzyna Kycia and Shujuan Fang for their support, Dr N. Torben Bech-Hansen for use of the CHEF-DRIII apparatus and members of the Lees-Miller lab for help with the preparation of this paper. This work was supported by grant #13639 from the Canadian Institutes for Health Research (CIHR). W.D.B. is supported by graduate studentships and awards from the Alberta Heritage Foundation for Medical Research (AHFMR), Alberta Scholarship Programs and the Natural Sciences and Engineering Research Council of Canada (NSERC). D.M. is supported by a graduate studentship from the Alberta Cancer Board. J.G. was supported by a summer studentship from the AHFMR. S.P.L.M. is a Scientist of the AHFMR, an Investigator of the CIHR and holds the Alberta Cancer Foundation/Engineered Air Chair in Cancer Research.