Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends

Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs.Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.     

Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have now generated an approximately 13 A three-dimensional map of DNA-PKcs, revealing the overall architecture and topology of the 4128 residue polypeptide chain and allowing location of domains. The highly conserved C-terminal PIKK catalytic domain forms a central structure from which FAT and FATC domains protrude. Conformational changes observed in these domains on DNA binding suggest that they transduce DNA-induced conformational changes to the catalytic core and regulate kinase activity. The N-terminal segments form long curved tubular-shaped domains based on helical repeats to create interacting surfaces required for macromolecular assembly. Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family.     

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

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.     

DNA-dependent protein kinase catalytic subunit. Structural requirements for kinase activation by DNA ends.

DNA-dependent protein kinase (DNA-PK) is a DNA end-activated protein kinase composed of a catalytic subunit, DNA-PKcs, and a DNA binding subunit, Ku, that is involved in repair of DNA double-stranded breaks (DSBs). We have previously shown that DNA-PKcs interacts with single-stranded DNA (ssDNA) ends with a separate ssDNA binding site to be activated for its kinase activity. Here, the properties of the ssDNA binding site were examined by using DNA fragments with modified ssDNA extensions. DNA fragments with a wide range of ssDNA modifictations activated DNA-PKcs, indicating a relaxed specificity for the chemical structure of terminal nucleotides of a DSB. Methyl substitution of the phosphate backbone impaired kinase activation but not binding, indicating that interaction with the DNA backbone was involved in kinase activation. Experiments with RNA and RNA/DNA hybrid fragments suggested that the discrimination between RNA and DNA ends resides in the double-stranded DNA binding function of DNA-PKcs. DNA fragments exposing only one ssDNA end activated DNA-PKcs poorly, suggesting that DNA-PKcs distinguishes between DSBs and ssDNA breaks by simultaneous interaction with two ssDNA ends. These properties potentially explain how DNA-PKcs can be specifically activated by DSBs but still recognize the diverse chemical structures exposed when DSBs are introduced by ionizing radiation.     

Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.     

Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.     

Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs.     

N-terminal constraint activates the catalytic subunit of the DNA-dependent protein kinase in the absence of DNA or Ku

The DNA-dependent protein kinase (DNA-PK) was identified as an activity and as its three component polypeptides 25 and 15 years ago, respectively. It has been exhaustively characterized as being absolutely dependent on free double stranded DNA ends (to which it is directed by its regulatory subunit, Ku) for its activation as a robust nuclear serine/threonine protein kinase. Here, we report the unexpected finding of robust DNA-PKcs activation by N-terminal constraint, independent of either DNA or its regulatory subunit Ku. These data suggest that an N-terminal conformational change (likely induced by DNA binding) induces enzymatic activation.     

The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.     

Terminal DNA structure and ATP influence binding parameters of the DNA-dependent protein kinase at an early step prior to DNA synapsis

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the non-homologous end-joining pathway of DNA double-strand break repair in mammalian cells. The ability of DNA-PKcs to sense and respond to different terminal DNA structures is postulated to be important for its regulatory function. It is unclear whether discrimination occurs at the time of formation of the initial protein–DNA complex or later, at the time of formation of a paired, or synaptic complex between opposing DNA ends. To gain further insight into the mechanism of regulation, we characterized the binding of DNA-PKcs to immobilized DNA fragments that cannot undergo synapsis. Results showed that DNA-PKcs strongly discriminates between different terminal structures at the time of initial complex formation. Although Ku protein stabilizes DNA-PKcs binding overall, it is not required for discrimination between terminal structures. Base mispairing, temperature and the presence of an interstrand linkage influence the stability of the initial complex in a manner that suggests a requirement for DNA unwinding, reminiscent of the ‘open complex’ model of RNA polymerase–promoter DNA interaction. ATP and a nonhydrolyzable ATP analog also influence the stability of the DNA-PKcs•DNA complex, apparently by an allosteric mechanism that does not require DNA-PKcs autophosphorylation.     

The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA

The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells. DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460). Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process. It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity. Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA. We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini. When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation. The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini.     

The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication

Exposure of cells to ionizing radiation inhibits DNA replication in a dose-dependent manner. The dose response is biphasic and the initial steep component reflects inhibition of replicon initiation thought to be mediated by activation of the S-phase checkpoint. In mammalian cells, inhibition of replicon initiation requires the ataxia telagiectasia mutated (ATM) gene, a member of the phosphatidyl inositol kinase-like (PIKL) family of protein kinases. We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation. Exposure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibited replicon initiation in a time-dependent manner. Inhibition was at a maximum 1 h after irradiation and recovered at later times. Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for recovery, or only limited recovery, was observed for up to 8 h after irradiation. In addition a defect was observed in the maturation of nascent DNA. Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h. Normal kinetics of recovery were observed in xrs-5 cells, deficient in Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these cell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells may not be attributed entirely to the prolonged presence of unrepaired DNA dsb. We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in normal cells facilitates recovery of DNA replication after irradiation.     

Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution.

CK2alpha is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 A resolution, of recombinant maize CK2alpha (rmCK2alpha) in the presence of ATP and Mg2+, shows the enzyme in an active conformation stabilized by interactions of the N-terminal region with the activation segment and with a cluster of basic residues known as the substrate recognition site. The close interaction between the N-terminal region and the activation segment is unique among known protein kinase structures and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP and GTP as phosphorylating agents.     

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.     

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites

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DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25 bp dsDNA or 25 bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35 bp blunt ended dsDNA) or 25 bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes.     

Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.     

Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.