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DNA Repair

The p53 response to DNA damage

By | DNA Repair

The p53 response to DNA damage

David W. Meek

The p53 tumour suppressor protein is a highly potent transcription factor which, under normal circumstances, is maintained at low levels through the action of MDM2, an E3 ubiquitin ligase which directs p53 ubiquitylation and degradation. Expression of the mdm2 gene is stimulated by p53 and this reciprocal relationship forms the basis of a negative feedback loop. Both genotoxic and non-genotoxic stresses that induce p53 focus principally on interruption of the p53-MDM2 loop with the consequence that p53 becomes stabilised, leading to changes in the expression of p53-responsive genes. The biological outcome of inducing this pathway can be either growth arrest or apoptosis: factors affecting the functioning of the loop, the biochemical activity of p53 itself and the cellular environment govern the choice between these outcomes in a cell type- and stress-specific manner.

 

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p53-Mediated DNA Repair Responses to UV Radiation: Studies of Mouse Cells Lacking p53, p21, and/or gadd45 Genes

By | DNA Repair

p53-Mediated DNA Repair Responses to UV Radiation: Studies of Mouse Cells Lacking p53, p21, and/or gadd45 Genes

Martin L. Smith, James M. Ford, M. Christine Hollander, Rachel A. Bortnick, Sally A. Amundson, Young R. Seo, Chu-xia Deng, Philip C. Hanawalt, and Albert J. Fornace, Jr.

Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse em- bryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21 (Cdkn1a), as well as MEFs lacking both gadd45 and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss of p53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wild- type MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.

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Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development

By | DNA Repair

Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development

Yijie Gao, David O. Ferguson, Wei Xie, John P. Manis, JoAnn Sekiguchi, Karen M. Frank, Jayanta Chaudhuri, James Horner, Ronald A. DePinho & Frederick W. Alt

XRCC4 is a non-homologous end-joining protein employed in

DNA double strand break repair and in V(D)J recombination1,2. In mice, XRCC4-de®ciency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis2. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis3. Here we show that p53-de®ciency rescues several aspects of the XRCC4-de®cient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no sig- ni®cant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-de®ciency allowed postnatal survival of XRCC4-de®cient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking ampli®ed c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-de®cient embryonic ®broblasts exhibited marked genomic instability including chromosomal translocations. Our ®ndings support a crucial role for the non-homologous end- joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.

DNA repair ability is reduced in a variety of pathologic conditions. In addition, in some of these diseases a disturbance in cellular Ca homeostasis occurs or cytosolic [Ca2÷] responses to various stimuli are impaired. The leading environ- mental cause for genomal DNA damage is ultraviolet (UV) irradiation. The aims of the present study were (I) to evaluate a possible dependence of UV-induced DNA repair ability on cytosolic Ca2÷ in human lymphocytes and (2) to assess the direct effect of UV irradiation on Ca 2÷ homeostasis in these cells. UV-induced DNA repair ability in lymphocytes was maximal at I mmol/L CaCI2 in the me- dium. Suppression of DNA repair ability occurred after elevation or reduction of cellular [Ca2÷] when various methods were used, including changes in Ca2÷ concentration in the medium, cellular Ca2÷ depletion by ethyleneglycol-bis- (l~aminoethylether)-N,N,N’,N’-tetraacetic acid, excessive Ca2+ concentration induced by ionophore, and shortening of Ca2÷ presence time during repair synthesis. UV irradiation caused an immediate and significant rise in cytosolic [Ca2÷] that was the result of both enhanced Ca2÷ uptake and inhibition of plasma membrane Ca-adenosine triphosphatase activity. The tyrosine kinase inhibitor genistein inhibited both UV-induced DNA repair and UV-induced cyto- solic [Ca2÷] elevation. These results emphasize the importance of a precise cellular Ca 2÷ level regulation for the optimal DNA repair process. UV irradiation, by inducing cellular Ca2÷ rise, may activate DNA repair as soon as DNA is damaged. (J Lab Clin Med 1997;130:33-41)

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The role of calcium in human lymphocyte DNA repair ability

By | DNA Repair

The role of calcium in human lymphocyte DNA repair ability

Uzi Gafter,Tsipora Malachi, Yaacov Ori, and Haim Breitbart

DNA repair ability is reduced in a variety of pathologic conditions. In addition, in some of these diseases a disturbance in cellular Ca homeostasis occurs or cytosolic [Ca2÷] responses to various stimuli are impaired. The leading environ- mental cause for genomal DNA damage is ultraviolet (UV) irradiation. The aims of the present study were (I) to evaluate a possible dependence of UV-induced DNA repair ability on cytosolic Ca2÷ in human lymphocytes and (2) to assess the direct effect of UV irradiation on Ca 2÷ homeostasis in these cells. UV-induced DNA repair ability in lymphocytes was maximal at I mmol/L CaCI2 in the me- dium. Suppression of DNA repair ability occurred after elevation or reduction of cellular [Ca2÷] when various methods were used, including changes in Ca2÷ concentration in the medium, cellular Ca2÷ depletion by ethyleneglycol-bis- (l~aminoethylether)-N,N,N’,N’-tetraacetic acid, excessive Ca2+ concentration induced by ionophore, and shortening of Ca2÷ presence time during repair synthesis. UV irradiation caused an immediate and significant rise in cytosolic [Ca2÷] that was the result of both enhanced Ca2÷ uptake and inhibition of plasma membrane Ca-adenosine triphosphatase activity. The tyrosine kinase inhibitor genistein inhibited both UV-induced DNA repair and UV-induced cyto- solic [Ca2÷] elevation. These results emphasize the importance of a precise cellular Ca 2÷ level regulation for the optimal DNA repair process. UV irradiation, by inducing cellular Ca2÷ rise, may activate DNA repair as soon as DNA is damaged. (J Lab Clin Med 1997;130:33-41)

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