Structure of DNA-dependent protein kinase: implications for its regulation by DNA.
(25/14812)
DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described. (+info)
Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH.
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As part of TFIIH, XPB and XPD helicases have been shown to play a role in nucleotide excision repair (NER). Mutations in these subunits are associated with three genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The strong heterogeneous clinical features observed in these patients cannot be explained by defects in NER alone. We decided to look at the transcriptional activity of TFIIH from cell lines of XP individuals. We set up an immunopurification procedure to isolate purified TFIIH from patient cell extracts. We demonstrated that mutations in two XP-B/CS patients decrease the transcriptional activity of the corresponding TFIIH by preventing promoter opening. The defect of XPB in transcription can be circumvented by artificial opening of the promoter. Western blot analysis and enzymatic assays indicate that XPD mutations affect the stoichiometric composition of TFIIH due to a weakness in the interaction between XPD-CAK complex and the core TFIIH, resulting in a partial reduction of transcription activity. This work, in addition to clarifying the role of the various TFIIH subunits, supports the current hypothesis that XP-B/D patients are more likely to suffer from transcription repair syndromes rather than DNA repair disorders alone. (+info)
Tight correlation between inhibition of DNA repair in vitro and transcription factor IIIA binding in a 5S ribosomal RNA gene.
(27/14812)
UV-induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)-5S rDNA complex and Xenopus oocyte nuclear extracts. TFIIIA alters CPD formation primarily in the transcribed strand of the 50 bp internal control region (ICR) of 5S rDNA. During NER in vitro, CPD removal is reduced at most sites in both strands of the ICR when TFIIIA is bound. Efficient repair occurs just outside the TFIIIA-binding site (within 10 bp), and in the absence of 5S rRNA transcription. Interestingly, three CPD sites within the ICR [+56, +75 (transcribed strand) and +73 (non-transcribed strand)] are repaired rapidly when TFIIIA is bound, while CPDs within approximately 5 bases of these sites are repaired much more slowly. CPDs at these three sites may partially displace TFIIIA, thereby enabling rapid repair. However, TFIIIA is not completely displaced during NER, at least at sites outside the ICR, even though the NER complex could be sterically hindered by TFIIIA. Such inefficient repair of transcription factor binding sites could increase mutation frequency in regulatory regions of genes. (+info)
Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes.
(28/14812)
Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. KEYWORDS: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation (+info)
The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin.
(29/14812)
Camptothecin is an antitumor agent that kills cells by converting DNA topoisomerase I into a DNA-damaging poison. Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood. We report here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair camptothecin-mediated damage to DNA. The hypersensitivity to camptothecin in the trf4 mutant does not result from elevated expression of DNA topoisomerase I. We show that Trf4 is a nuclear protein whose expression is cell cycle-regulated at a post-transcriptional level. Suppression of camptothecin hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may influence higher order chromosome structure, ZDS1 and ZDS2. We have isolated and sequenced two human TRF4 family members, hTRF4-1 and hTRF4-2. The hTRF4-1 gene maps to chromosome 5p15, a region of frequent copy number alteration in several tumor types. The evolutionary conservation of TRF4 suggests that it may also influence mammalian cell sensitivity to camptothecin. (+info)
Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5' single-strand break and is independent of the mismatch repair system.
(30/14812)
The repair of 12-, 27-, 62-, and 216-nucleotide unpaired insertion/deletion heterologies has been demonstrated in nuclear extracts of human cells. When present in covalently closed circular heteroduplexes or heteroduplexes containing a single-strand break 3' to the heterology, such structures are subject to a low level repair reaction that occurs with little strand bias. However, the presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs repair to the incised DNA strand. Because nick direction of repair is independent of the strand in which a particular heterology is placed, the observed strand bias is not due to asymmetry imposed on the heteroduplex by the extrahelical DNA segment. Strand-specific repair by this system requires ATP and the four dNTPs and is inhibited by aphidicolin. Repair is independent of the mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 and occurs by a mechanism that is distinct from that of the conventional mismatch repair system. Large heterology repair in nuclear extracts of human cells is also independent of the XPF gene product, and extracts of Chinese hamster ovary cells deficient in the ERCC1 and ERCC4 gene products also support the reaction. (+info)
Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage.
(31/14812)
DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 microM) or NU1025 (300 microM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 +/- 0.6 and 1.7 +/- 0.2, respectively. When used in combination, a DEF10 of 7.8 +/- 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 +/- 0.04 and 1.20 +/- 0.05, respectively, compared with 1.01 +/- 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 +/- 0.05 with NU1025 compared with IR alone, 1.02 +/- 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 microM WM and 300 microM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential. (+info)
Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.
(32/14812)
The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage. (+info)