Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.     

A structural model for regulation of NHEJ by DNA-PKcs autophosphorylation

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer together form the biologically critical DNA-PK complex that plays key roles in the repair of ionizing radiation-induced DNA double-strand breaks through the non-homologous end-joining (NHEJ) pathway. Despite elegant and informative electron microscopy studies, the mechanism by which DNA-PK co-ordinates the initiation of NHEJ has been enigmatic due to limited structural information. Here, we discuss how the recently described small angle X-ray scattering structures of full-length Ku heterodimer and DNA-PKcs in solution, combined with a breakthrough DNA-PKcs crystal structure, provide significant insights into the early stages of NHEJ. Dynamic structural changes associated with a functionally important cluster of autophosphorylation sites play a significant role in regulating the dissociation of DNA-PKcs from Ku and DNA. These new structural insights have implications for understanding the formation and control of the DNA-PK synaptic complex, DNA-PKcs activation and initiation of NHEJ. More generally, they provide prototypic information for the phosphatidylinositol-3 kinase-like (PIKK) family of serine/threonine protein kinases that includes Ataxia Telangiectasia-Mutated (ATM) and ATM-, Rad3-related (ATR) as well as DNA-PKcs.     

The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM

Members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including the ATM, DNA-PKcs, Atr, and Trrap proteins, function in signal transduction pathways that activate the DNA damage response. PIKK proteins contain a conserved C-terminal FAT/kinase domain/FATC domain structure. The FATC domain of ATM mediates the interaction between ATM and Tip60, a histone acetyltransferase that regulates activation of ATM. Here, we examined whether the FATC domains of DNA-PKcs, Atr, and Trrap were also able to interact with Tip60. Deletion of the FATC domain of ATM blocked the interaction between ATM and Tip60 and suppressed the activation of ATM kinase activity by DNA damage. Replacement of the FATC domain of ATM with the FATC domains of DNA-PKcs, Atr, or Trrap restored the activation of ATM and its association with Tip60. These results indicate that the FATC domains of DNA-PKcs, Atr, Trrap, and ATM are functionally equivalent. Immunoprecipitation experiments demonstrated that Tip60 is constitutively associated with DNA-PKcs and that the histone acetyltransferase activity associated with DNA-PKcs is up-regulated by DNA damage. When Tip60 expression was suppressed by small interfering RNA, the activation of DNA-PKcs (measured by autophosphorylation of DNA-PKcs at serine 2056 and threonine 2609) was inhibited, demonstrating a key role for Tip60 in the activation of DNA-PKcs by DNA damage. The conserved FATC domain of PIKK proteins may therefore function as a binding domain for the Tip60 histone acetyltransferase. Further, the ability of Tip60 to regulate the activation of both ATM and DNA-PKcs in response to DNA damage demonstrates that Tip60 is a key component of the DNA damage-signaling network.     

Common mechanisms of PIKK regulation

Kinases in the phosphoinositide three-kinase-related kinase (PIKK) family include ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PKcs (DNA-dependent protein kinase catalytic subunit), mTOR (mammalian target of rapamycin), and SMG1 (suppressor with morphological effect on genitalia family member). These atypical protein kinases regulate DNA damage responses, nutrient-dependent signaling, and nonsense-mediated mRNA decay. This review focuses on the mechanisms regulating the PIKK family with a strong emphasis on the DNA damage regulated kinases. We outline common regulatory themes and suggest how discoveries about the regulation of one PIKK can be informative for the other family members.     

Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is essential for the repair of double-stranded DNA breaks (DSBs) in non- homologous end joining (NHEJ) and during V(D)J recombination. DNA-PKcs binds single- and double-stranded DNA in vitro, and in vivo the Ku heterodimer probably helps recruit it to DSBs with high affinity. Once loaded onto DNA, DNA-PKcs acts as a scaffold for other repair factors to generate a multiprotein complex that brings the two DNA ends together. Human DNA-PKcs has been analysed by electron microscopy in the absence and presence of double-stranded DNA, and the three-dimensional reconstruction of DNA-bound DNA-PKcs displays large conformational changes when compared with the unbound protein. DNA-PKcs seems to use a palm-like domain to clip onto the DNA, and this new conformation correlates with the activation of the kinase. We suggest that the observed domain movements might help the binding and maintenance of DNA-PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase.     

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.     

Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs

Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.     

Structural analysis of the basal state of the Artemis:DNA-PKcs complex

Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.     

Identification of DNA-PK in the arthropods. Evidence for the ancient ancestry of vertebrate non-homologous end-joining

Cellular life depends upon the preservation and transmission of genetic material. Double stranded DNA breaks (DSBs) cause catastrophic gene loss in cell division and must be promptly and accurately repaired. In eukaryotes DSBs may be repaired by either non-homologous end-joining (NHEJ), single strand annealing or homologous recombination (HR). Vertebrate NHEJ has been shown to depend upon the DNA-dependent protein kinase (DNA-PK) consisting of the phosphatidylinositol 3 (PI 3)-kinase like (PIKK) catalytic sub-unit (DNA-PKcs) and the DNA targeting factor Ku. Our analysis of recently completed genomes found several novel PIKKs in Anopheles gambiae and Drosophila melanogaster including a novel mosquito DNA-PKcs orthologue, the first non-vertebrate DNA-PKcs described to date. We also detected a DNA-PKcs fragment in the high quality EST set of Apis mellifera ligustica (honey bee) suggesting that DNA-PK is a far older and more important eukaryotic complex than previously thought.     

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.     

The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs(-/-) cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.     

DNA-PKcs基因沉默抑制人乳腺上皮细胞对低剂量辐射损伤的修复

DNA-PKcs作为DNA依赖性蛋白激酶(DNA-PK)的催化亚基在DNA双链断裂(DSBs)的非同源末端重组(NHEJ)通路中起重要的作用。本实验以人乳腺上皮细胞株MCF10F为研究对象,通过siRNA技术抑制细胞内DNA-PKcs的表达,用50cGy137CS照射细胞,测定细胞生长曲线以确定细胞对低剂量辐射(LDR)的敏感性,同时检测DNA修复相关蛋白表达的变化,旨在探讨DNA依赖蛋白激酶(DNA-PKcs)基因沉默对人乳腺上皮细胞株MCF10F低剂量辐射敏感性的影响及机制。结果显示:转染特异性siRNA可使人乳腺上皮细胞(MCF10F)DNA-PKcs基因沉默,增殖受到明显的抑制;50cGyγ射线辐射可使乳腺细胞内DNA-PKcs、Ku80、ATM、P53等DNA修复相关蛋白表达增多,但DNA-PKcs基因沉默细胞(MCF10Fpk)中,这些蛋白表达显著低于对照组(MCF10Fmock)。以上结果提示,DNA-PKcs基因沉默可引起乳腺细胞对低剂量辐射敏感性增加,其原因可能与相关DNA修复蛋白表达减少有关。     

The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.     

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.     

细胞周期监控点蛋白Rad9与非同源末端连接修复蛋白Ku70的相互作用研究

Rad9是一种重要的细胞周期监控点调控蛋白．越来越多的证据显示,Rad9也可与多种DNA损伤修复通路中的蛋白质相互作用,并调节其功能,在DNA损伤修复中发挥重要作用．非同源末端连接修复是DNA双链断裂的一条重要修复途径．Ku70、Ku80和DNA依赖的蛋白激酶催化亚基(DNA-PKcs)共同组成DNA依赖的蛋白激酶复合物(DNA-PK),在非同源末端修复连接中起重要作用．本研究中,检测到Rad9与Ku70有直接的物理相互作用和功能相互作用．我们在不同的细胞模型中发现,Rad9基因敲除、Rad9蛋白去除或Rad9表达降低会导致非同源末端连接效率明显下降．已有的研究表明,DNA损伤可导致细胞中Ku70与染色质结合增加及DNA-PKcs激酶活性增强．我们的结果显示,与野生小鼠细胞相比,Rad9基因敲除的小鼠细胞中, DNA损伤诱导的上述效应均减弱．综上所述,我们的研究首次报道了Rad9与非同源末端连接修复蛋白Ku70间有相互作用,并提示Rad9可通过调节Ku70/Ku80/DNA-PKcs复合物功能参与非同源末端连接修复．     

Mutual regulation between DNA-PKcs and snail1 leads to increased genomic instability and aggressive tumor characteristics

Although the roles of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) in the non-homologous end joining (NHEJ) of DNA repair are well-recognized, the biological mechanisms and regulators by DNA-PKcs besides DNA repair, have not been clearly described. Here, we show that active DNA-PKcs caused by ionizing radiation, phosphorylated Snail1 at serine (Ser) 100, led to increased Snail1 stability. Furthermore, phosphorylated Snail1 at Ser100 reciprocally inhibited the kinase activity of DNA-PKcs, resulting in an inhibition of DNA repair activity. Moreover, Snail1 phosphorylation by DNA-PKcs was involved in genomic instability and aggressive tumor characteristics. Our results describe novel cellular mechanisms that affect genomic instability, sensitivity to DNA-damaging agents, and the migration of tumor cells by reciprocal regulation between DNA-PKcs and Snail1.     

Cryo-EM structure of the DNA-dependent protein kinase catalytic subunit at subnanometer resolution reveals alpha helices and insight into DNA binding

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the nonhomologous end joining pathway for repair of double-stranded DNA (dsDNA) breaks. Here, we present a 7A resolution structure of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction. This structure is composed of density rods throughout the molecule that are indicative of alpha helices and reveals structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within the density. Surprisingly, models for the kinase domain can be docked into either the crown or base of the molecule at this resolution, although real space refinement suggests that the base location is the best fit. We propose a model for the interaction of DNA with DNA-PKcs in which one turn of dsDNA enters the central channel and interacts with a resolved alpha-helical protrusion.     

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.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

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.