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PubMed2001

Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection.

Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal.

3T3 Cells↗
PubMed2001

Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration.

Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.

Agrobacterium tumefaciens↗
PubMed2002

Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70.

The Ku70/80 heterodimer is a critical component of the non-homologous end-joining (NHEJ) pathway and of the telomere cap in yeast and mammals. We report the molecular characterization of the KU70 and KU80 genes in Arabidopsis and describe the consequences of a Ku70 deficiency. Arabidopsis KU70/80 genes are ubiquitously expressed and their products form stable heterodimers in vitro. Plants harboring a T-DNA insertion in KU70 exhibit no growth or developmental defects under standard growth conditions. However, mutant seedlings are hypersensitive to gamma-irradiation-induced double-strand breaks. Unexpectedly, we found that mutants are hypersensitive to methyl methanosulfonate during seed germination, but lose this sensitivity in seedlings, implying that the requirement for NHEJ varies during plant development. Lack of Ku70 results in a dramatic deregulation of telomere length control, with mutant telomeres expanding to more than twice the size of wild type by the second generation. Furthermore, in contrast to the situation in mammals, chromosome fusions are not associated with a Ku deficiency in Arabidopsis. These findings imply that Ku may play a different role in capping plant and animal telomeres.

Alleles↗
PubMed2002

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.

Antigens, Nuclear↗
PubMed2002

Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK.

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.

Antigens, Nuclear↗
PubMed2002

Mammalian Ku86 mediates chromosomal fusions and apoptosis caused by critically short telomeres.

Here we analyze the functional interaction between Ku86 and telomerase at the mammalian telomere by studying mice deficient for both proteins. We show that absence of Ku86 prevents the end-to-end chromosomal fusions that result from critical telomere shortening in telomerase-deficient mice. In addition, Ku86 deficiency rescues the male early germ cell apoptosis triggered by short telomeres in these mice. Together, these findings define a role for Ku86 in mediating chromosomal instability and apoptosis triggered by short telomeres. In addition, we show here that Ku86 deficiency results in telomerase-dependent telomere elongation and in the fusion of random pairs of chromosomes in telomerase-proficient cells, suggesting a model in which Ku86 keeps normal-length telomeres less accessible to telomerase-mediated telomere lengthening and to DNA repair activities.

Animals↗
PubMed2002

Synapsis of DNA ends by DNA-dependent protein kinase.

The catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) is required for a non-homologous end-joining pathway that repairs DNA double-strand breaks produced by ionizing radiation or V(D)J recombination; however, its role in this pathway has remained obscure. Using a neutravidin pull-down assay, we found that DNA-PK(CS) mediates formation of a synaptic complex containing two DNA molecules. Furthermore, kinase activity was cooperative with respect to DNA concentration, suggesting that activation of the kinase occurs only after DNA synapsis. Electron microscopy revealed complexes of two DNA ends brought together by two DNA-PK(CS) molecules. Our results suggest that DNA-PK(CS) brings DNA ends together and then undergoes activation of its kinase, presumably to regulate subsequent steps for processing and ligation of the ends.

Antigens, Nuclear↗
PubMed2000

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster.

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.

Animals↗
PubMed2001

Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms.

DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ in yeast chromosomes has been observed only when HR is blocked, as in rad52 mutants or in the absence of a homologous repair template. We detected yKu70p-dependent imprecise NHEJ at a frequency of approximately 0.1% in HR-competent Rad+ haploid cells. Interestingly, yku70 mutation increased DSB-induced HR between direct repeats by 1.3-fold in a haploid strain and by 1.5-fold in a MAT homozygous (a/a) diploid, but yku70 had no effect on HR in a MAT heterozygous (a/alpha) diploid. yku70 might increase HR because it eliminates the competing precise NHEJ (religation) pathway and/or because yKu70p interferes directly or indirectly with HR. Despite the yku70-dependent increase in a/a cells, HR remained 2-fold lower than in a/alpha cells. Cell survival was also lower in a/a cells and correlated with the reduction in HR. These results indicate that MAT heterozygosity enhances DSB-induced HR by yKu-dependent and -independent mechanisms, with the latter mechanism promoting cell survival. Surprisingly, yku70 strains survived a DSB slightly better than wild type. We propose that this reflects enhanced HR, not by elimination of precise NHEJ since this pathway produces viable products, but by elimination of yKu-dependent interference of HR.

Alleles↗
PubMed2002

Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.

Antigens, Nuclear↗
PubMed2003

Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae.

Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements. We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene. Approximately 1% of successfully repaired cells have accompanying chromosomal rearrangements consisting of large insertions, deletions, aberrant gene conversions, or other more complex changes. We have analyzed rearrangements in isogenic wild-type, rad52, yku80, and rad52 yku80 strains, to determine the types of events that occur in the presence or absence of these key repair proteins. Deletions were found in all strain backgrounds, but insertions were dependent upon the presence of Yku80p. A rare RAD52- and YKU80-independent form of deletion was present in all strains. These events were characterized by long one-sided deletions (up to 13 kb) and extensive imperfect overlapping sequences (7-22 bp) at the junctions. Our results demonstrate that the frequency and types of repair events depend on the specific genetic context. This approach can be applied to a number of problems associated with chromosome stability.

Antigens, Nuclear↗
PubMed2001

Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells.

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

Adult↗
PubMed1996

The V(D)J recombination defect in the xrs-6 cell line results from a point mutation in the Ku80 gene.

Defective expression of the Ku80 gene has been implicated as underlying the V(D)J recombination and DNA double-strand break repair defects in the xrs-6 Chinese hamster ovary cell line. We now show that the mutation in the Ku80 gene involves a G to A transition 15 bp upstream of exon 2. This mutation creates a new splice acceptor site which results in the generation of Ku80 transcript that cannot encode a functional product due a 13 nucleotide insertion and a resulting frameshift.

Amino Acid Sequence↗
PubMed2001

In vitro evidence for homologous recombinational repair in resistance to melphalan.

BACKGROUND: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. METHODS: Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. RESULTS: Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. CONCLUSION: Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.

Antigens, Nuclear↗
PubMed2002

Ligation of a primer at a mutation: a method to detect low level mutations in DNA.

Detection of low frequency mutations following exposure to mutagens or during the early stages of cancer development is instrumental for risk assessment and molecular diagnosis. We present a sensitive new method to detect trace levels of DNA mutations induced within a large excess of wild-type sequences. The method is based on mutation-induced generation of new restriction enzyme recognition sites. A DNA sequence is amplified from genomic DNA or cDNA using a high fidelity polymerase. The purified PCR product is digested with a restriction enzyme that recognizes the newly generated restriction site, partially dephosphorylated and ligated with an oligonucleotide at the position of the mutation. The ligated oligonucleotide is then utilized in two rounds of PCR to amplify the mutated DNA but not the wild-type allele that contains no restriction site. An A-->T polymorphism in mRNA (tenascin gene, A(2366)-->T, Asn-->Ile) and a G-->A polymorphism in genomic DNA (Ku gene, G(74582)-->A, Val-->Ile), both of which generate a restriction site for the enzyme SAU3A1, demonstrate the application. Eleven patient samples pre-characterized for the G(74582)-->A polymorphism in the repair gene Ku are used to demonstrate the reliability of this approach. This technique quantitatively detects the Ku G-->A polymorphism at a mutant frequency of 1.6x10(-6) relative to the wild-type allele. Mutations in p53 that are frequently induced by mutagens can readily be detected using the present method. As an example, using a second enzyme BbvI, a mutation frequently encountered in human cancers (G(14154)-->A mutation, p53 codon 245, Arg-->Gln) was detected in patient samples. The process does not require radioactivity, utilizes established procedures and overcomes several factors known to produce false positives in RFLP-based assays. The present amplification via primer ligation at the mutation (APRIL-ATM) has potential applications in the detection of mutagen-generated genetic alterations, early detection of tumor marker mutations in bodily discharges and the diagnosis of minimal residual disease.

Adenocarcinoma↗
PubMed2005

Interaction of radiation- and bleomycin-induced lesions and influence of glutathione level on the interaction.

Radiation-induced exchange aberrations are thought to arise as a consequence of misrejoining of free ends of DNA double strand breaks (dsbs). In quiescent mammalian cells this process of misrejoining is prevalently taken up by the non-homologous end joining (NHEJ) process. In order to investigate the role of glutathione (GSH) in DNA dsb rejoining, the interaction of the lesions induced by bleomycin (Blem) and by radiation was studied since the lesions caused by both have similar and apparent rapid rates of repair. Endogenous GSH was depleted by buthionine sulfoximine (BSO) and chromosome aberrations (CAs) of human lymphocytes were scored from first cycle metaphases. Gamma radiation was administered 2 h after Blem treatment in combined studies. In the case of BSO, the treatment was given 3 h before Blem treatment. The BSO-treated samples showed higher sensitivity to radiation than BSO-untreated ones. Combined treatment of Blem and radiation induced higher frequency of CAs, in particular the exchange aberrations and interstitial deletions. However, such increased frequency of exchange aberrations was reduced drastically and the frequency of terminal deletions was increased significantly when combined treatment was given to BSO-pretreated cells. The consistent level of Ku70 protein in all the treated samples, with undetectable level of Rad51 in the G0-lymphocytes indicates the involvement of NHEJ pathway in misrejoining of DNA dsbs. It may be hypothesized that reduction in the frequency of exchange aberrations as induced by Blem + radiation combined treatment in BSO-treated samples could be because of reduced NHEJ pathway.

Antigens, Nuclear↗
PubMed1993

Interactions of USF and Ku antigen with a human DNA region containing a replication origin.

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

Amino Acid Sequence↗
PubMed1996

Extrachromosomal recombination occurs efficiently in cells defective in various DNA repair systems.

A series of different frameshift mutations of a firefly luciferase reporter plasmid was created so that no activity was obtained when they were transfected into mammalian cells. Co-transfection of these constructs with short fragments of the original sequence resulted in luciferase activity in different cell lines (A-549, NIH 3T3 and Jurkat). The level of this activity was dependent on the length of the fragment, regardless of cell line examined. Two different transfection techniques (lipofection and adenovirus-enhanced gene transfer) gave similar results. It was shown by polymerase chain reaction that expression of detectable luciferase required recombination of the transfected molecules. Cells with defined defects in DNA repair pathways were examined for their ability to perform this extrachromosomal recombination. Cells lacking normal Ku p80, (ADP-ribosyl)transferase, MLH1 or XP-C were all capable of restoring expression to the frameshifted constructs. Given the pivotal roles of the above molecules in the pathways of DNA repair, it seems that this recombination derives from a different activity.

3T3 Cells↗