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1.  Expression of Ku70 correlates with survival in carcinoma of the cervix 
British Journal of Cancer  2000;83(12):1702-1706.
Cervical carcinoma affects around 3400 women in the UK each year and advanced disease is routinely treated with radiation. As part of a programme to establish rapid and convenient methods of predicting tumour and patient responses to radiotherapy, we have examined the relationship between the pre-treatment expression of the Ku components of the DNA damage recognition complex DNA-PK and patient survival in cervical carcinoma. Using immunohistochemistry of formalin-fixed sections of tumour biopsies, antibodies to Ku70 and Ku80 stained identical regions of tumour and there was a high degree of correlation between the mean number of cells stained positive for the two components in 77 tumours (r = 0.82, P< 0.001). In 53 tumours there was a borderline significant correlation between measurements of tumour radiosensitivity (surviving fraction at 2 gray: SF2) and Ku70 expression (r = 0.26, P = 0.057) and no correlation for Ku80 (r = 0.18, P = 0.19). However, all tumours with a low number of Ku70 or Ku80 positive cells were radiosensitive. Furthermore, using log-rank analysis there was significantly higher survival in the patients whose tumours had a low Ku70 expression (P = 0.046). This difference was also reflected with Ku80, but did not reach statistical significance (P = 0.087). The study suggests that lack of Ku protein leads to radiosensitivity in some tumours and that other factors are responsible for radiosensitive tumours with high Ku expression. It is likely that the most accurate prediction of treatment outcome will lie in assessing the expression of several proteins involved in the recognition and repair of DNA damage, one of which will be Ku. © 2000 Cancer Research Campaign
PMCID: PMC2363444  PMID: 11104569
DNA repair; Ku; radiosensitivity; immunohistochemistry; DNA-PK; predictive assay
2.  Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. 
Nucleic Acids Research  1999;27(17):3494-3502.
In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80.
PMCID: PMC148593  PMID: 10446239
3.  Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80. 
Nucleic Acids Research  1998;26(19):4332-4338.
Ku, a heterodimer of approximately 70 and approximately 80 kDa subunits, is a nuclear protein that binds to double-stranded DNA ends and is a component of the DNA-dependent protein kinase (DNA-PK). Cell lines defective in Ku80 belong to group XRCC5 of ionizing radiation-sensitive mutants. Five new independent Chinese hamster cell mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B, that belong to this group were isolated. To shed light on the nature of the defect in Ku80, the molecular and biochemical characteristics of these mutants were examined. All mutants, except XR-V12B, express Ku80 mRNA, but no Ku80 protein could clearly be detected by immunoblot analysis in any of them. DNA sequence analysis of the Ku80 cDNA from these mutants showed a deletion of 252 bp in XR-V10B; a 6 bp deletion that results in a new amino acid residue at position 107 and the loss of two amino acid residues at positions 108 and 109 in XR-V11B; a missense mutation resulting in a substitution of Cys for Tyr at position 114 in XR-V13B; and two missense mutations in XR-V16B, resulting in a substitution of Met for Val at position 331 and Arg for Gly at position 354. All these mutations cause a similar, 5-7-fold, increase in X-ray sensitivity in comparison to wild-type cells, and a complete lack of DNA-end binding and DNA-PK activities. This indicates that all these mutations lead to loss of the Ku80 function due to instability of the defective protein.
PMCID: PMC147872  PMID: 9742232
4.  XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation. 
Nucleic Acids Research  1998;26(13):3146-3153.
DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.
PMCID: PMC147672  PMID: 9628911
5.  Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20. 
Nucleic Acids Research  1998;26(8):1965-1973.
The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.
PMCID: PMC147487  PMID: 9518490
6.  Molecular and biochemical characterization of xrs mutants defective in Ku80. 
Molecular and Cellular Biology  1997;17(3):1264-1273.
The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.
PMCID: PMC231851  PMID: 9032253
7.  Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. 
Nucleic Acids Research  1996;24(23):4639-4648.
Ku is a heterodimer of polypeptides of approximately 70 and 80 kDa (Ku70 and Ku80, respectively) that binds to DNA ends. Mammalian cells lacking Ku are defective in DNA double-strand break (DSB) repair and in site-specific V(D)J recombination. Here, we describe the identification and characterisation of YKU80, the gene for the Saccharomyces cerevisiae Ku80 homologue. Significantly, we find that YKU80 disruption enhances the radiosensitivity of rad52 mutant strains, suggesting that YKU80 functions in a DNA DSB repair pathway that does not rely on homologous recombination. Indeed, through using an in vivo plasmid rejoining assay, we find that YKU80 plays an essential role in illegitimate recombination events that result in the accurate repair of restriction enzyme generated DSBs. Interestingly, in the absence of YKU80function, residual repair operates through an error-prone pathway that results in recombination between short direct repeat elements. This resembles closely a predominant DSB repair pathway in vertebrates. Together, our data suggest that multiple, evolutionarily conserved mechanisms for DSB repair exist in eukaryotes. Furthermore, they imply that Ku binds to DSBs in vivo and promotes repair both by enhancing accurate DNA end joining and by suppressing alternative error-prone repair pathways. Finally, we report that yku80 mutant yeasts display dramatic telomeric shortening, suggesting that, in addition to recognising DNA damage, Ku also binds to naturally occurring chromosomal ends. These findings raise the possibility that Ku protects chromosomal termini from nucleolytic attack and functions as part of a telomeric length sensing system.
PMCID: PMC146307  PMID: 8972848
8.  Activation domains of transcription factors mediate replication dependent transcription from a minimal HIV-1 promoter. 
Nucleic Acids Research  1996;24(4):549-557.
Transcription from a minimal HIV-1 promoter containing the three Sp1 binding sites and TATA box can be activated without Tat by template DNA replication. Here we show that this activation can also be mediated by recombinant GAL4 fusion proteins containing the activation domains of Sp1, VP16 or CTF (or by full-length GAL4) targeted to the HIV-1 promoter by replacing the Sp1 sites with five GAL4 binding sites. Thus Sp1 is not unique in its ability to mediate replication activated transcription, although the degree of processivity elicited by the different activators varied significantly from strongly processive (GAL4-VP16) to relatively non-processive (GAL4-Sp1 or -CTF). Processive GAL4-VP16-activated transcription, but not efficient initiation, required multiple GAL4 binding sites. In the presence of Tat, transcription with GAL4-SP1 and GAL4-CTF was further activated (principally at the level of processivity) but GAL4-VP16-potentiated transcription was only slightly stimulated. The Tat-dependent switch from non-processive to fully processive transcription was particularly marked for GAL4-Sp1, an effect which may be relevant to the selection of Sp1 binding sites by the HIV-1 promoter.
PMCID: PMC145701  PMID: 8604293
9.  Cell cycle regulation of RNA polymerase III transcription. 
Molecular and Cellular Biology  1995;15(12):6653-6662.
Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.
PMCID: PMC230918  PMID: 8524230
10.  Mitotic regulation of a TATA-binding-protein-containing complex. 
Molecular and Cellular Biology  1995;15(4):1983-1992.
The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.
PMCID: PMC230425  PMID: 7891693
11.  Developmental expression of Sp1 in the mouse. 
Molecular and Cellular Biology  1991;11(4):2189-2199.
The expression of the trans-acting transcription factor Sp1 in mice was defined by a combination of RNA analysis and immunohistochemical localization of the Sp1 protein. Although ubiquitously expressed, there was an unexpected difference of at least 100-fold in the amount of Sp1 message in different cell types. Sp1 protein levels showed corresponding marked differences. Substantial variations in Sp1 expression were also found in some cell types at different stages of development. Sp1 levels appeared to be highest in developing hematopoietic cells, fetal cells, and spermatids, suggesting that an elevated Sp1 level is associated with the differentiation process. These results indicate that Sp1 has a regulatory function in addition to its general role in the transcription of housekeeping genes.
PMCID: PMC359911  PMID: 2005904
12.  Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing. 
Molecular and Cellular Biology  1988;8(3):1067-1075.
Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing.
PMCID: PMC363249  PMID: 2835658
13.  Cloning and functional analysis of the TATA binding protein from Sulfolobus shibatae. 
Nucleic Acids Research  1995;23(10):1775-1781.
Archaea (formerly archaebacteria) comprise a domain of life that is phylogenetically distinct from both Eucarya and Bacteria. Here we report the cloning of a gene from the Archaeon Sulfolobus shibatae that encodes a protein with strong homology to the TATA binding protein (TBP) of eukaryotes. Sulfolobus shibatae TBP is, however, almost as diverged from other archaeal TBPs that have been cloned as it is from eukaryotic TBPs. DNA binding studies indicate that S.shibatae TBP recognizes TATA-like A-box sequences that are present upstream of most archaeal genes. By quantitatively immunodepleting S.shibatae TBP from an in vitro transcription system, we demonstrate that Sulfolobus RNA polymerase is capable of transcribing the 16S/23S rRNA promoter weakly in the absence of TBP. Most significantly, we show that addition of recombinant S.shibatae TBP to this immunodepleted system leads to transcriptional stimulation and that this stimulation is dependent on the A-box sequence of the promoter. Taken together, these findings reveal fundamental similarities between the transcription machineries of Archaea and eukaryotes.
PMCID: PMC306935  PMID: 7784182
14.  c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif. 
Nucleic Acids Research  1993;21(5):1289-1295.
The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-Gln, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.
PMCID: PMC309295  PMID: 8464713

Results 1-14 (14)