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PubMed2002

Regulation of osteocalcin gene expression by a novel Ku antigen transcription factor complex.

We previously described an osteocalcin (OC) fibroblast growth factor (FGF) response element (FRE) DNA binding activity as a target of Msx2 transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a variant of Tubedown-1, as constituents of the purified OCFRE-binding complex. Northern and Western blot analyses demonstrate expression of Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment regulates Ku, but not Tbdn100, protein accumulation. Gel supershift studies confirm sequence-specific DNA binding of Ku in the OCFRE complex; chromatin immunoprecipitation assays confirm association of Ku and Tbdn100 with the endogenous OC promoter. In the promoter region -154 to -113, the OCFRE is juxtaposed to OSE2, an osteoblast-specific element that binds Runx2 (Osf2, Cbfa1). Expression of the Ku.Tbdn100 complex up-regulates both the basal and Runx2-dependent transcription driven by this 42-bp OC promoter element, reconstituted in CV-1 cells. Synergistic transactivation occurs in the presence of activated FGF receptor 2 signaling. Msx2 suppresses Ku- and Runx2-dependent transcription; suppression is dependent upon the Msx2 homeodomain NH(2)-terminal arm and extension. Pull-down assays confirm physical interactions between Ku and these co-regulatory transcription factors, consistent with the functional interactions identified. Finally, cultured Ku70 -/- calvarial cells exhibit a profound, selective deficiency in OC expression as compared with wild-type calvarial cells, confirming the biochemical data showing a role for Ku in OC transcription. In toto, these data indicate that a novel Ku antigen complex assembles on the OC promoter, functioning in concert with Msx2 and Runx2 to regulate OC gene expression.

3T3 Cells↗
PubMed2002

Expression of Ku70 and Ku80 mediated by NF-kappa B and cyclooxygenase-2 is related to proliferation of human gastric cancer cells.

Cyclooxygenase-2 (COX-2) expression is mediated by constitutive NF-kappaB and regulates human gastric cancer cell growth and proliferation. Inactivating Ku70 or Ku80 suppresses cell growth and induces apoptosis. It has been hypothesized that Ku70 and Ku80 expression may be associated with NF-kappaB activation and COX-2 expression and is involved in cell proliferation. In this study, we found that inhibition of constitutive NF-kappaB (by transfecting a mutated IkappaBalpha gene) and of COX-2 (by treatment with indomethacin and NS-398) suppressed Ku70 and Ku80 expression in cells. Treatment with prostaglandin E(2) adenocarcinoma gastric (AGS) increased expression of these Ku proteins in cells with low constitutive NF-kappaB levels. Inhibition of the Ku DNA end-binding activity by transfection with the C-terminal Ku80 expression gene suppressed cell proliferation. Ku70 or Ku80 overexpression by transfection with the Ku70 or Ku80 expression gene, respectively, enhanced proliferation of cells with low NF-kappaB levels. These results demonstrate that Ku70 and Ku80 expression is mediated by constitutively activated NF-kappaB and constitutively expressed COX-2 in gastric cancer cells and that the high Ku DNA end-binding activity contributes to cell proliferation. Ku70 and Ku80 expression may be related to gastric cell proliferation and carcinogenesis.

Antigens, Nuclear↗
PubMed2002

Human Ku70/80 associates physically with telomerase through interaction with hTERT.

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.

Antigens, Nuclear↗
PubMed2002

Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast.

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80(+), the gene encoding Ku80. Both pku70(+) and pku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.

Antigens, Nuclear↗
PubMed2003

Selective inhibition of class switching to IgG and IgE by recruitment of the HoxC4 and Oct-1 homeodomain proteins and Ku70/Ku86 to newly identified ATTT cis-elements.

Immunoglobulin (Ig) class switching is central to the maturation of the antibody response as IgG, IgA, and IgE are endowed with more diverse biological effector functions than IgM. It is induced upon engagement of CD40 on B lymphocytes by CD40L expressed by activated CD4+ T cells and exposure of B cells to T cell-secreted cytokines including interleukin-4 and transforming growth factor-beta. It begins with germ line IH-CH transcription and unfolds through class switch DNA recombination (CSR). We show here that the HoxC4 and Oct-1 homeodomain proteins together with the Ku70/Ku86 heterodimer bind as a complex to newly identified switch (S) regulatory ATTT elements (SREs) in the Igamma and Iepsilon promoters and downstream regions to dampen basal germ line Igamma-Cgamma and Iepsilon-Cepsilon transcriptions and repress CSR to Cgamma and Cepsilon. This mechanism is inactive in the Calpha1/Calpha2 loci because of the lack of SREs in the Ialpha1/Ialpha2 promoters. Accordingly, in resting human IgM+IgD+ B cells, HoxC4, Oct-1, and Ku70/Ku86 can be readily identified as bound to the Igamma and Iepsilon promoters but not the Ialpha1/Ialpha2 promoters. CD40 signaling dissociates the HoxC4.Oct-1. Ku complex from the Igamma and Iepsilon promoter SREs, thereby relieving the IH-CH transcriptional repression and allowing CSR to unfold. Dissociation of HoxC4.Oct-1. Ku from DNA is hampered by CD153 engagement, a CD40-signaling inhibitor. Thus, these findings outline a HoxC4.Oct-1. Ku-dependent mechanism of selective regulation of class switching to IgG and IgE and further suggest distinct co-evolution and shared CSR activation pathways in the Cgamma and Cepsilon as opposed to the Calpha1/Calpha2 loci.

Antigens, Nuclear↗
PubMed2003

Novel localization of the DNA-PK complex in lipid rafts: a putative role in the signal transduction pathway of the ionizing radiation response.

Increased sensitivity to ionizing radiation (IR) has been shown to be due to defects in DNA double-strand break repair machinery. The major pathway in mammalian cells dedicated to the repair of DNA double-strand breaks is by the nonhomologous end-joining machinery. Six components function in this pathway, of which three (Ku70, Ku86, and DNA-PKcs) constitute a protein complex known as DNA-dependent protein kinase (DNA-PK). However, it is now recognized that the cellular radiation response is complex, and radiosensitivity may be also regulated at different levels in the radiation signal transduction pathway. In addition to DNA damage, exposure to IR triggers intracellular signaling cascades that overlap with pathways initiated by ligand engagement to a receptor. In this study, we provide evidence for the novel localization of the DNA-PK complex in lipid rafts. We also show this property is not a generalized characteristic of all DNA repair proteins. Furthermore, we have detected Ku86 in yeast lipid rafts. Our results suggest that the components of this complex might be recruited separately to the plasma membrane by tethering with raft-resident proteins. In addition, we found an irradiation-induced differential protein phosphorylation pattern dependent upon DNA-PKcs in lipid rafts. Thus, we speculate that another role for the DNA-PKcs subunit and perhaps for the holoenzyme is in the signal transduction of IR response.

Animals↗
PubMed2003

Regulatory interactions between the checkpoint kinase Chk1 and the proteins of the DNA-dependent protein kinase complex.

Checkpoints are biochemical pathways that provide cells a mechanism to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase, Chk1, regulates mitotic progression in response to DNA damage by blocking the activation of Cdk1/cyclin B. In this study, we investigate the regulatory interaction between Chk1 and members of the Atm family of kinases and the functional role of the C-terminal non-catalytic domains of Chk1. Chk1 stimulates the kinase activity of DNA-PK (protein kinase) complexes, which leads to increased phosphorylation of p53 on Ser-15 and Ser-37. In addition, Chk1 stimulates DNA-PK-dependent end-joining reactions in vitro. We also show that Chk1 protein complexes bind to single-stranded DNA and DNA ends. These results indicate a connection between components that regulate the checkpoint pathways and DNA-PK complex proteins, which have a role in the repair of double strand breaks.

Antigens, Nuclear↗
PubMed2003

Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule.

The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

Antigens, Nuclear↗
PubMed2003

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells.

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.

Antigens, Nuclear↗
PubMed2004

Characterization of proteins binding to E-box/Ku86 sites and function of Ku86 in transcriptional regulation of the human xanthine oxidoreductase gene.

We reported previously that E-box and TATA-like elements repress human xanthine oxidoreductase gene (hXOR) expression. In the present investigation, we determined the means by which the E-box site functions in this basal repression. DNA affinity purification demonstrated that at least five proteins are involved in the nuclear protein complex binding to the E-box and adjacent Ku86-binding sites. Amino acid sequence analysis demonstrated that three proteins, DNA-PK catalytic subunit, Ku86, and Ku70 are components of DNA-dependent protein kinase (DNA-PK). By electrophoretic mobility shift assays, gel-shift, and site-directed mutagenesis, we confirmed Ku86 binding to the Ku86 site. Studies indicated that the other two proteins of the complex are AREB6-like proteins binding to the E-box. Pull-down and immunoprecipitation analyses demonstrated the binding of Ku86 to AREB6-like proteins. The functional loss of Ku86 increases hXOR promoter activity and transcript expression. Based on the findings, we propose that DNA-PK/AREB6-like proteins play a central role in repression of basal hXOR activity. AREB6-like proteins specifically bind to the E-box, whereas Ku86 binds an adjacent site and recruits DNA-PK catalytic subunit and Ku70 proteins. A working model is presented to account for the role of DNA-PK and AREB6-like proteins in regulating hXOR activity.

Antigens, Nuclear↗
PubMed2003

Separation-of-function mutants of yeast Ku80 reveal a Yku80p-Sir4p interaction involved in telomeric silencing.

The Saccharomyces cerevisiae Ku heterodimer comprising Yku70p and Yku80p is involved in telomere maintenance and DNA repair by the pathway of non-homologous end joining. It is also a key regulator of transcriptional silencing of genes placed in close proximity to telomeres. Here, we describe the identification of separation-of-function mutants of Yku80p that exhibit defects in silencing but not DNA repair and show that these mutations map to an evolutionarily conserved domain within Yku80p. Furthermore, we reveal that Yku80p interacts with the silent information regulator protein Sir4p and that this interaction is mediated by the N-terminal 200 amino acid residues of Sir4p. Notably, this interaction also requires the region of Yku80p that contains the sites of the silencing defective mutations. Finally, we show that these mutations impair the Yku80p-Sir4p interaction and recruitment of Sir3p to telomeric regions in vivo. Taken together with other data, these findings indicate that the Yku80p-Sir4p interaction plays a vital role in the assembly of telomeric heterochromatin.

Amino Acid Sequence↗
PubMed2003

The Ku antigen-recombination signal-binding protein Jkappa complex binds to the nuclear factor-kappaB p50 promoter and acts as a positive regulator of p50 expression in human gastric cancer cells.

The p50 subunit of NF-kappaB is a transcription factor that regulates the expression of a variety of genes. Previously, we showed that the expression of Ku antigen, a DNA repair protein, is mediated by NF-kappaB in gastric cancer AGS cells (Lim, J. W., Kim, H., and Kim, K. H. (2002) J. Biol. Chem. 277, 46093-46100). In this study, we report that the inhibition of Ku activity reduced both p50 expression and nuclear NF-kappaB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jkappa (RBP-Jkappa), a DNA-binding protein. Ku antigen, RBP-Jkappa, and p50 were found to bind to the DNA region containing the kappaB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jkappa bound sequence-specifically to downstream elements of kappaB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IkappaBalpha, Ku80, and RBP-Jkappa. A reporter assay demonstrated that p50 transcription was positively mediated by NF-kappaB, Ku antigen, and RBP-Jkappa and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jkappa and NF-kappaB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.

Antigens, Nuclear↗
PubMed2003

A positive role for the Ku complex in DNA replication following strand break damage in mammals.

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.

Animals↗
PubMed2004

Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1.

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN(H), a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRN-associated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD(+), PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.

Antigens, Nuclear↗
PubMed2004

The human Rap1 protein complex and modulation of telomere length.

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.

Antigens, Nuclear↗
PubMed2004

Positive and negative modulation of the transcriptional activity of the ETS factor ESE-1 through interaction with p300, CREB-binding protein, and Ku 70/86.

Epithelium-specific ETS (ESE)-1 is a prototypic member of a novel subset of the ETS transcription factor family that is predominantly expressed in cells of epithelial origin but can also be induced in other cell types including vascular endothelial and smooth muscle cells in response to inflammatory stimuli. To further define the molecular mechanisms by which the transcriptional activity of ESE-1 is regulated, we have focused our attention on identifying proteins that interact with ESE-1. We have determined that Ku70, Ku86, p300, and CREB-binding protein (CBP) are ESE-1 interacting proteins. The Ku proteins have previously been shown to bind to breaks in DNA where they function to recruit additional proteins that promote DNA repair. Interestingly, Ku70 and Ku 86 negatively regulate the transcriptional activity of ESE-1. Using a series of deletion constructs, we have determined that the Ku proteins bind to the DNA-binding domain of ESE-1. The Ku proteins inhibit the ability of ESE-1 to bind to oligonucleotide probes in gel mobility shift assays. The finding that Ku proteins can interact with other transcription factors and block their function has not been previously demonstrated. In contrast, co-transfection of p300 and CBP with ESE-1 enhances the transcriptional activity of ESE-1. Moreover, the induction of ESE-1 in response to inflammatory cytokine interleukin-1 is associated with a parallel increase of the expression of p300 in vascular endothelial cells, suggesting that in the setting of inflammation, the transcriptional activity of ESE-1 is positively modulated by interaction with the transcriptional co-activator p300. In summary, our results demonstrated that the activity of ESE-1 is positively and negatively modulated by other interacting proteins including Ku70, Ku86, p300, and CBP.

Antigens, Nuclear↗
PubMed2004

Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit modulate RAG-mediated cleavage: implications for the enforcement of the 12/23 rule.

The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.

Antigens, Nuclear↗
PubMed2004

Non-homologous end joining requires that the DNA-PK complex undergo an autophosphorylation-dependent rearrangement at DNA ends.

Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate.

Carrier Proteins↗