Publications (8)92.89 Total impact
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Article: Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair.
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ABSTRACT: Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.Nucleic Acids Research 05/2011; 39(16):6986-97. · 8.03 Impact Factor -
Article: An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity.
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ABSTRACT: Artemis is required for V(D)J recombination and the repair of a subset of radiation-induced DNA double strand breaks (DSBs). Artemis-null patients display radiosensitivity (RS) and severe combined immunodeficiency (SCID), classified as RS-SCID. Strongly impacting hypomorphic Artemis mutations confer marked infant immunodeficiency and a predisposition for EBV-associated lymphomas. Here, we provide evidence that a polymorphic Artemis variant (c.512C > G: p.171P > R), which has a world-wide prevalence of 15%, is functionally impacting. The c.512C > G mutation causes an approximately 3-fold decrease in Artemis endonuclease activity in vitro. Cells derived from a patient who expressed a single Artemis allele with the polymorphic mutational change, showed radiosensitivity and a DSB repair defect in G2 phase, with Artemis cDNA expression rescuing both phenotypes. The c.512C > G change has an additive impact on Artemis function when combined with a novel C-terminal truncating mutation (p.436C > X), which also partially inactivates Artemis activity. Collectively, our findings provide strong evidence that monoallelic expression of the c.512C > G variant impairs Artemis function causing significant radiosensitivity and a G2 phase DSB repair defect. The patient exhibiting monoallelic c.512C > G-Artemis expression showed immunodeficiency only in adulthood, developed bilateral carcinoma of the nipple and myelodysplasia raising the possibility that modestly decreased Artemis function can impact clinically.DNA repair 09/2010; 9(9):1003-10. · 4.20 Impact Factor -
Article: Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome.
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ABSTRACT: Hypomorphic mutations in DNA ligase IV (LIG4) cause a human syndrome of immunodeficiency, radiosensitivity, and growth retardation due to defective DNA repair by the nonhomologous end-joining (NHEJ) pathway. Lig4-null mice are embryonic lethal, and better mouse models are needed to study human LigIV syndrome. We recently identified a viable mouse strain with a Y288C hypomorphic mutation in the Lig4 gene. Lig4Y288C mice exhibit a greater than 10-fold reduction of LigIV activity in vivo and recapitulate the immunodeficiency and growth retardation seen in human patients. Here, we have demonstrated that the Lig4Y288C mutation leads to multiple defects in lymphocyte development and function, including impaired V(D)J recombination, peripheral lymphocyte survival and proliferation, and B cell class switch recombination. We also highlight a high incidence of thymic tumors in the Lig4Y288C mice, suggesting that wild-type LigIV protects against malignant transformation. These findings provide explanations for the complex lymphoid phenotype of human LigIV syndrome.The Journal of clinical investigation 06/2009; 119(6):1696-705. · 15.39 Impact Factor -
Article: XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.
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ABSTRACT: XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.Nucleic Acids Research 01/2009; 37(2):482-92. · 8.03 Impact Factor -
Article: Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.
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ABSTRACT: DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.DNA Repair 07/2007; 6(6):712-22. · 4.14 Impact Factor -
Article: DNA repair is limiting for haematopoietic stem cells during ageing.
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ABSTRACT: Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.Nature 07/2007; 447(7145):686-90. · 36.28 Impact Factor -
Article: Radiation-induced delayed cell death in a hypomorphic Artemis cell line.
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ABSTRACT: Null mutations in Artemis confer a condition described as RS-SCID, in which patients display radiosensitivity combined with severe combined immunodeficiency. Here, we characterize the defect in Artemis in a patient who displayed progressive combined immunodeficiency (CID) and elevated lymphocyte apoptosis. The patient is a compound heterozygote with novel mutations in both alleles, resulting in Artemis proteins with either L70 deletion or G126D substitution. Both mutational changes impact upon Artemis function and a fibroblast cell line derived from the patient (F96-224) has greatly reduced Artemis protein. In contrast to Artemis null cell lines, which fail to repair a subset of DNA double strand breaks (DSBs) induced by ionizing radiation, F96-224 cells show slow but residual DSB rejoining. Despite showing intermediate cellular and clinical features, F96-224 cells are as radiosensitive as Artemis null cell lines. We developed a FACS-based assay to examine cell division and cellular characteristics for 10 days following exposure to ionizing radiation (2 and 4 Gy). This analysis demonstrated that F96-224 cells show delayed cell death when compared with rapid growth arrest of an Artemis null cell line, and the emergence of a cycling population shown by a control line. F96-224 cells also display elevated chromosome aberrations when compared with control cells. F96-224 therefore represents a novel phenotype for a hypomorphic cell line. We suggest that delayed cell death contributes to the progressive CID phenotype of the Artemis patient.Human Molecular Genetics 05/2006; 15(8):1303-11. · 7.64 Impact Factor -
Article: Nbs1 is required for ATR-dependent phosphorylation events.
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ABSTRACT: Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.The EMBO Journal 02/2005; 24(1):199-208. · 9.20 Impact Factor
Top co-authors
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Institutions
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2005–2011
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University of Sussex
- Genome Damage and Stability Centre
Brighton, ENG, United Kingdom
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2006
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University College London
- Institute of Child Health
London, ENG, United Kingdom
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