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DNA Damage: Air-breaks?

2002; Elsevier BV; Volume: 12; Issue: 7 Linguagem: Inglês

10.1016/s0960-9822(02)00788-1

1879-0445

Deborah E. Barnes,

Cancer-related Molecular Pathways

Cells deficient in repairing DNA double-strand breaks have an increased level of spontaneous chromosomal aberrations. Modulating the level ofmolecular oxygen and its reactive metabolites demonstrates that oxygen metabolism is a major source of genomic instability. Cells deficient in repairing DNA double-strand breaks have an increased level of spontaneous chromosomal aberrations. Modulating the level ofmolecular oxygen and its reactive metabolites demonstrates that oxygen metabolism is a major source of genomic instability. Organisms have evolved to utilise oxygen, in the very air that we breathe, to drive the energy production that sustains life. However, there is a price to be paid — reactive oxygen species are generated as by-products of aerobic metabolism and can damage bio-macromolecules, including DNA. For this reason, oxidative metabolism has been confined to the mitochondria, and cells use a battery of enzymatic defences to detoxify reactive oxygen species or, as a last resort, repair any damage done. Reactive oxygen species have been trumpeted as major players in the aetiology of degenerative diseases, ageing and cancer; hence exhortations to eat more fruit and vegetables, rich in antioxidants such as vitamin C [1.Ames B.N. Shigenaga M.K. Hagen T.M. Oxidants, antioxidants, and the degenerative diseases of aging.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 7915-7922Crossref PubMed Scopus (5241) Google Scholar]. The effect of oxidative damage on proteins or lipids is limited temporally by continual biosynthesis and turnover but in DNA it can lead to 'fixed' mutations if polymerases copy damaged templates during DNA replication. Lieber and colleagues, whose work was published recently in Current Biology[2.Karanjawala Z.E. Murphy N. Hinton D.R. Hsieh C.-L. Lieber M.R. Oxygen metabolism causes chromosome breaks and is associated with the neuronal apoptosis observed in DNA double-strand break repair mutants.Curr. Biol. 2002; 12: 397-402Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar], demonstrate a direct link between oxygen metabolism and genomic instability. They experimentally altered levels of oxygen and/or reactive oxygen species in both cultured cells and a transgenic mouse model deficient in the repair of DNA double-strand breaks, and show that oxygen metabolism is a major source of the observed genomic instability. Moreover, as well as base damage and single-strand breaks, this implies that oxidative metabolism is a significant source of endogenous double-strand breaks. Non-homologous end joining is a mechanism for repairing DNA double-strand breaks [3.Barnes D.E. Non-homologous end joining as a mechanism of DNA repair.Curr. Biol. 2001; 11: R455-R457Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar], and cells deficient in any component of this pathway are sensitive to agents, like ionizing radiation, that generate such damage. Key components include the Ku70–Ku86 complex involved in binding or bridging broken DNA ends, and the DNA ligase IV–XRCC4 heterodimer, which ligates the re-aligned strands (Fig. 1). Lieber and colleagues had previously reported elevated levels of chromosomal breaks (assayed by cytogenetic abnormalities of metaphase spreads) in primary fibroblast cell cultures from gene-targeted mice homozygous or heterozygous for null alleles of Ku86 or DNA ligase IV. This observation was subsequently correlated with oncogenic changes in repair-deficient mouse models in work from other laboratories, adding the non-homologous end joining proteins to the swelling ranks of genome 'guardians' [4.Roth D.B. Gellert M. New guardians of the genome.Nature. 2000; 404: 823-825Crossref PubMed Scopus (37) Google Scholar], and firmly establishing the connection between double-strand breaks and chromosomal instability [5.Van Gent D.C. Hoeijmakers J.H.J. Kanaar R. Chromosomal stability and the DNA double-stranded break connection.Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (922) Google Scholar]. The source of these chromosome breaks remained undefined, however, and it is this question that the authors sought to answer in the present work. They found that culturing end-joining-deficient cells at lowered oxygen tension reduced chromosomal aberrations to a level seen in repair-proficient cells. This residual level presumably reflects other endogenous sources of strand breaks, such as stalled replication forks, which are repaired by homologous recombination, a mechanistically different pathway, in dividing cells [3.Barnes D.E. Non-homologous end joining as a mechanism of DNA repair.Curr. Biol. 2001; 11: R455-R457Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Despite the routine use of artificially high levels of oxygen as standard cell culture practice, the authors report no alteration in cell growth at physiological oxygen tension that might affect their assay. Pioneering work from the Ames laboratory hypothesised reduced oxidative DNA damage in primary fibroblasts cultured at physiological oxygen concentration, assessed by delayed onset of senescence [6.Chen Q. Fischer A. Reagan J.D. Yan L.J. Ames B.N. Oxidative DNA damage and the senescence of human diploid fibroblast cells.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 4337-4341Crossref PubMed Scopus (538) Google Scholar]. The present data show oxidative damage is a major source of the genomic instability in end-joining-deficient cells, and thus of endogenous DNA double-strand breaks. So is this the case? Oxygen chemistry and its biological effects are complex (for reviews see [7.Disdaroglu, M. and Karakaya, A.E., eds. (1999). Advances in DNA damage and repair: oxygen radical effects, cellular protection, and biological consequences. NATO ASI Series, Life Sciences, Vol. 302 (New York: Kluwer Academic/Plenum).Google Scholar]), but the villain of the piece is the hydroxyl radical (Fig. 2), which can give rise to modified bases, base loss and single-strand breaks in DNA. Superoxide dismutase (or to be exact, Cu/Zn superoxide dismutase, encoded by the SOD1 gene) acts as an antioxidant, reducing the superoxide anion radical. But, in terms of DNA damage, the latter is relatively innocuous, and overexpression of superoxide dismutase, rather perversely, increases levels of the much more reactive hydroxyl radical. Superoxide dismutase has been shown to exacerbate radiosensitivity when expressed from a transgene in the Atm-deficient mouse, a model of the human disease ataxia telangiectasia where oxidative stress may cause some clinical symptoms [8.Peter Y. Rotman G. Lotem J. Elson A. Shiloh Y. Groner Y. Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice.EMBO J. 2001; 20: 1538-1546Crossref PubMed Scopus (47) Google Scholar]. In the present work, Lieber and colleagues show that expression of the SOD1 transgene increases chromosome instability in their fibroblast cell cultures and this effect can be modulated by oxygen tension. However, expression of the transgene does not significantly stimulate chromosome aberrations in Ku86-deficient cells. At first glance, this might appear to indicate that damage induced by hydroxyl radicals does not require functional end-joining for its repair, i.e. does not produce double-strand breaks. Indeed the received wisdom is that the latter arise as staggered single-strand interruptions or via simultaneous repair of clustered lesions on both DNA strands — a so-called 'multiply damaged site' [9.Dianov G.L. O'Neill P. Goodhead D.T. Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA.Bioessays. 2001; 23: 745-749Crossref PubMed Scopus (100) Google Scholar]. This is more likely to occur with ionizing radiation where there is a 'track' of energy giving rise to a chain reaction of both direct and indirect (via the aqueous milieu) assaults on the DNA; hence the radiosensitivity of end-joining-deficient cells. In contrast, endogenous oxidative damage would occur randomly along DNA — as shown to be the case here but as single-strand not double-strand breaks. It seems plausible that increased damage in Ku86−/−SOD1 fibroblasts is obscured by the operation of checkpoints, leading to a greater proportion of arrested cells, not scored in the assay. But whether oxygen metabolism generates DNA double-strand breaks directly, or these arise in repair-deficient cells secondarily to oxidative insult, clearly merits further investigation. Another intriguing facet of this work is the observed increase in neuronal apoptosis in Ku86-deficient mouse embryos expressing the SOD1 transgene. Neuronal apoptosis due to deficient end joining was first observed in DNA ligase IV null and XRCC4 null mice, which die mid-term in utero[10.Barnes D.E. Stamp G. Rosewell I. Denzel A. Lindahl T. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice.Curr. Biol. 1998; 8: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 11.Gao Y. Sun Y. Frank K.M. Dikkes P. Fujiwara Y. Seidl K.J. Sekiguchi J.M. Rathbun G.A. Swat W. Wang J. et al.A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.Cell. 1998; 95: 891-902Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar]. Neuronal cells are exquisitely sensitive to DNA damage; blocking the apoptotic pathway, by ablation of the Atm or p53 genes, allowed viable mice to be born, ostensibly free of gross neural defects [12.Lee Y. Barnes D.E. Lindahl T. McKinnon P.J. Defective neurogenesis resulting from DNA ligase IV deficiency requires Atm.Genes Dev. 2000; 14: 2576-2580Crossref PubMed Scopus (113) Google Scholar, 13.Frank K.M. Sharpless N.E. Gao Y. Sekiguchi J.M. Ferguson D.O. Zhu C. Manis J.P. Horner J. DePinho R.R. Alt F.W. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.Mol. Cell. 2000; 5: 993-1002Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. A qualitatively similar but less severe neural phenotype was later reported in Ku-deficient embryos [14.Gu Y. Sekiguchi J. Gao Y. Dikkes P. Frank K. Ferguson D. Hasty P. Chun J. Alt F.W. Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2668-2673Crossref PubMed Scopus (162) Google Scholar]. The link between increased chromosomal breaks in cultured cells and exacerbated neuronal apoptosis in Ku86−/−SOD1 mice is at best a correlation but provides the first clues that the source of DNA damage, at least in Ku-deficient embryos, might be the high oxygen metabolism in the brain. This being so, it should be possible to conduct reciprocal experiments to see if neuronal apoptosis is ameliorated by intervention with antioxidants in the diet or in utero[1.Ames B.N. Shigenaga M.K. Hagen T.M. Oxidants, antioxidants, and the degenerative diseases of aging.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 7915-7922Crossref PubMed Scopus (5241) Google Scholar, 15.Eriksson U.J. Oxidative DNA damage and embryo development.Nat. Med. 1999; 5: 715Crossref PubMed Scopus (25) Google Scholar]. However, apoptosis occurs at a discrete stage of development [10.Barnes D.E. Stamp G. Rosewell I. Denzel A. Lindahl T. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice.Curr. Biol. 1998; 8: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 11.Gao Y. Sun Y. Frank K.M. Dikkes P. Fujiwara Y. Seidl K.J. Sekiguchi J.M. Rathbun G.A. Swat W. Wang J. et al.A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.Cell. 1998; 95: 891-902Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar], as rapidly dividing neural cells exit mitosis and start to differentiate and migrate; it is unclear how this would fit with the very generalised oxidative stress modelled by the SOD1 transgene. Are newly differentiated neurons particularly sensitive because homologous recombination is compromised in non-dividing cells? These dilemmas would pertain equally to both Ku86 deficiency and DNA ligase IV/XRCC4 deficiency. But more perplexing are the striking differences between DNA ligase IV/XRCC4 null and Ku null mice. The latter do not die in utero but develop to adulthood, while additional loss of Atm, which rescues the embryonic lethality of DNA ligase IV/XRCC4 null alleles, actually causes embryonic death, and much earlier, in Ku-deficient or DNA-PKcs-deficient mice [16.Sekiguchi J. Ferguson D.O. Chen H.T. Yang E.M. Earle J. Frank K. Whitlow S. Gu Y. Xu Y. Nussenweig A. Alt F.W. Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 3243-3248Crossref PubMed Scopus (129) Google Scholar]. Clearly DNA-PK has another role(s), which overlaps with a function of the pleiotropic Atm protein in early mouse development; this could relate to telomere function [17.di Fagagna FdA Hande M.P. Tong W-M Roth D. Lansdorp P.M. Wang Z-Q. Jackson S.P. Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells.Curr. Biol. 2001; 11: 1192-1196Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar]. So why is the neural phenotype more severe in DNA ligase IV/XRCC4-deficient mice, when the incidence of endogenous oxidative damage should be the same as in the Ku null mouse? Does end-binding/activation of Ku/DNA-PKcs block an alternative repair pathway [18.Adachi N. Ishino T. Ishii Y. Takeda S. Koyama H. DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: implications for DNA double-strand break repair.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12109-12113Crossref PubMed Scopus (115) Google Scholar]? What is the contribution of a DNA ligase IV-independent end joining activity [19.Landberg R. Mavinakere M. Campbell C. Deficient DNA end joining activity in extracts from Fanconi anemia fibroblasts.J. Biol Chem. 2001; 276: 9543-9549Crossref PubMed Scopus (53) Google Scholar]? These putative relationships are shown schematically in Fig. 3. Whether these data can be extrapolated to DNA ligase IV/XRCC4 null mice remains to be seen. The emerging human syndrome resulting from DNA ligase IV mutations is equivocal about the link between the end joining defect and chromosomal instability/cancer predisposition [20.O'Driscoll M. Cerosaletti K.M. Girard P.-M. Dai Y. Stumm M. Kysela B. Hirsch B. Gennery A. Palmer S.E. Seidel J. et al.DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.Mol. Cell. 2001; 8: 1175-1185Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar], while an experimentally tractable chicken B-cell line has not always proved a good model for mammalian systems [5.Van Gent D.C. Hoeijmakers J.H.J. Kanaar R. Chromosomal stability and the DNA double-stranded break connection.Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (922) Google Scholar, 18.Adachi N. Ishino T. Ishii Y. Takeda S. Koyama H. DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: implications for DNA double-strand break repair.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12109-12113Crossref PubMed Scopus (115) Google Scholar]. As with all good science, this work provokes as many questions as it answers. The availability of null mouse models, and relevant double mutants, should help elucidate the complex associations between oxygen metabolism, endogenous DNA double-strand breaks, embryonic development and the genomic stability of proliferating cells that is necessary for the avoidance of cancer.