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PubMed1996

HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae.

The high affinity DNA binding factor (HDF) protein of Saccharomyces cerevisiae is composed of two subunits and specifically binds ends of double-stranded DNA. The 70-kDa subunit, HDF1, shows significant homology with the 70-kDa subunit of the human Ku protein. Like the Ku protein, HDF1 has been shown to be involved in recombination and double stranded DNA break repair. We have purified and cloned HDF2, the second subunit of the HDF protein. The amino acid sequence of HDF2 shows a 45.6% homology with the 80-kDa subunit of the Ku protein. HDF1 by itself does not bind DNA, while HDF2 protein on its own seems to displays DNA binding activity. Targeted disruption of the HDF2 gene causes a temperature-sensitive phenotype for growth comparable to the phenotype of hdf1(-) strains. The human Ku protein cannot complement this temperature-sensitive phenotype. hdf2(-) strains are sensitive to bleomycin and methyl methanesulfonate, but this sensitivity is reduced in comparison with hdf1(-) strains.

Antigens, Nuclear↗
PubMed1997

Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.

Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits, p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. In this study, we show that efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrated that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser-33. We also identified Ser-29 as the major Cdc2 kinase phosphorylation site in the p34 subunit.

Amino Acid Sequence↗
PubMed1997

Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit.

The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer. Here we show that a Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002-3850) near the protein kinase domain. Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain. The c-Abl SH3 domain binds to amino acids 3414-3850 of DNA-PKcs. c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414-3850. c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs.Ku complex. Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity.

Animals↗
PubMed1997

Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase.

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein. We report here that HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

Antigens, Nuclear↗
PubMed1997

Defining the minimal domain of Ku80 for interaction with Ku70.

The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.

Antigens, Nuclear↗
PubMed1997

Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor.

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Animals↗
PubMed1998

A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D.

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xalpha receptor (RXRalpha) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

Antigens, Nuclear↗
PubMed1998

Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint.

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12. 2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.

Amino Acid Substitution↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1999

Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end.

Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively.

Antigens, Nuclear↗
PubMed1999

Ku binds telomeric DNA in vitro.

Ku is a heterodimeric protein with high binding affinity for ends, nicks, and gaps in double-stranded DNA. Both in mammalian cells and in budding yeast, Ku plays a role in nonhomologous end joining in the double strand break repair pathway. However, Ku has a more significant role in DNA repair in mammalian cells compared with yeast, in which a homology-dependent pathway is the predominant one. Recently Ku has been shown to be a likely component of the telomeric complex in yeast, suggesting the possibility of a similar role for Ku at mammalian telomeres. However, long single-stranded G-rich overhangs are continuously present at mammalian but not at yeast telomeres. These overhangs have the potential to fold in vitro into G-G base-paired conformations, such as G-quartets, that might prevent Ku from recognizing telomeric ends and thus offer a mechanism to sequester the telomere from the prevalent double strand break repair pathway in mammals. We show here that Ku binds to mammalian telomeric DNA ends in vitro and that G-quartet conformations are unable to prevent Ku from binding with high affinity to the DNA. Our results indicate that the DNA binding characteristics of Ku are consistent with its direct interaction with telomeric DNA in mammalian cells and its proposed role as a telomere end factor.

Antigens, Nuclear↗
PubMed2000

Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins.

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Activation of DNA-dependent protein kinase by single-stranded DNA ends.

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS). To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK(CS). The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS). Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. The structure of DNA-PK(CS) contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.

Antigens, Nuclear↗
PubMed2000

Functional interaction between Ku and the werner syndrome protein in DNA end processing.

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging. The gene responsible for the syndrome was recently cloned and shown to encode a protein with strong homology to DNA/RNA helicases. In addition, the Werner syndrome protein (WRN) possesses an exonuclease activity. Based on the homology to helicases it has been proposed that WRN functions in some aspects of DNA replication, recombination, or repair. However, there is currently no evidence of a role of WRN in any of these processes; therefore, its biological function remains unknown. Using a biochemical approach, we have identified two polypeptides that bind to the WRN protein. Peptide sequence analysis indicates that the two proteins are identical to Ku70 and Ku80, a heterodimer involved in double strand DNA break repair by non-homologous DNA end joining. Protein-protein interaction studies reveal that WRN binds directly to Ku80 and that this interaction is mediated by the amino terminus of WRN. In addition, we show that the binding of Ku alters the specificity of the WRN exonuclease. These results suggest a potential involvement of WRN in the repair of double strand DNA breaks.

Amino Acid Sequence↗
PubMed2002

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

Antigens, Nuclear↗
PubMed2000

A short C-terminal domain of Yku70p is essential for telomere maintenance.

The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70(-) strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku(-) phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of single-stranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ.

Amino Acid Sequence↗
PubMed2000

Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen.

Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis.

Aminopropionitrile↗