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Rinske Drost,
Peter Bouwman,
Sven Rottenberg,
Ute Boon,
Eva Schut,
Sjoerd Klarenbeek,
Christiaan Klijn,
Ingrid van der Heijden,
Hanneke van der Gulden,
Ellen Wientjens,
Mark Pieterse,
Aurelie Catteau,
Pete Green,
Ellen Solomon, Joanna R Morris,
Jos Jonkers
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ABSTRACT: Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.
Cancer cell 12/2011; 20(6):797-809. · 25.29 Impact Factor
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Joanna R Morris
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ABSTRACT: In mammalian cells the accumulation of repair proteins to double-strand breaks is a phosphorylation- and ubiquitylation-regulated process. Some of the genes that encode the kinases and ubiquitin ligases in this pathway are cancer predisposition genes, most prominently the breast cancer predisposition gene BRCA1, which encodes a ubiquitin ligase. How BRCA1 ligase activity was regulated following DNA damage was poorly understood. In this review I summarize new data that show a third post-translational modification, by the small ubiquitin like modifier SUMO, is part of the same cascade, enabling and activating DNA damage-regulated processes, including the BRCA1 ligase activity.
Cancer Research 05/2010; 70(10):3861-3. · 7.86 Impact Factor
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Joanna R Morris
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ABSTRACT: Modification by SUMOs (small ubiquitin-related modifiers) is largely transient and considered to alter protein function through altered protein-protein interactions. These modifications are significant regulators of the response to DNA damage in eukaryotic model organisms and SUMOylation affects a large number of proteins in mammalian cells, including several proteins involved in the response to genomic lesions [Golebiowski, Matic, Tatham, Cole, Yin, Nakamura, Cox, Barton, Mann and Hay (2009) Sci. Signaling 2, ra24]. Furthermore, recent work [Morris, Boutell, Keppler, Densham, Weekes, Alamshah, Butler, Galanty, Pangon, Kiuchi, Ng and Solomon (2009) Nature 462, 886-890; Galanty, Belotserkovskaya, Coates, Polo, Miller and Jackson (2009) Nature 462, 935-939] has revealed the involvement of the SUMO cascade in the BRCA1 (breast-cancer susceptibility gene 1) pathway response after DNA damage. The present review examines roles described for the SUMO pathway in the way mammalian cells respond to genotoxic stress.
Biochemical Society Transactions 02/2010; 38(Pt 1):92-7. · 3.71 Impact Factor
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ABSTRACT: The first breast and ovarian cancer predisposition gene, BRCA1, was cloned over 14 years ago. The features of BRCA1-defective cells and tumours have been well described; these display
genomic instability and multiple centrosomes, are sensitive to DNA-damaging agents that rely on homologous recombination (HR)
for repair and the tumours are of a particular pathological subtype (known as basal). Yet the precise molecular defect downstream
of BRCA1 loss-of-function remains elusive. More than 70 BRCA1-interacting proteins have been described and in this review
we will attempt to draw together recent findings about BRCA1 complexes and activity that inform our current understanding
of the cellular tumour suppressor function of BRCA1.
12/2009: pages 75-92;
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Joanna R Morris,
Chris Boutell,
Melanie Keppler,
Ruth Densham,
Daniel Weekes,
Amin Alamshah,
Laura Butler,
Yaron Galanty,
Laurent Pangon,
Tai Kiuchi,
Tony Ng,
Ellen Solomon
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ABSTRACT: Mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. BRCA1 participates in the DNA damage response and acts as a ubiquitin ligase. However, its regulation remains poorly understood. Here we report that BRCA1 is modified by small ubiquitin-like modifier (SUMO) in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localize with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL). Further, PIAS SUMO ligases are required for complete accumulation of double-stranded DNA (dsDNA) damage-repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. These data demonstrate that the SUMOylation pathway plays a significant role in mammalian DNA damage response.
Nature 12/2009; 462(7275):886-90. · 36.28 Impact Factor
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Julian Barwell,
Laurent Pangon,
Anne Georgiou,
Ian Kesterton,
Caroline Langman,
Audrey Arden-Jones,
Elizabeth Bancroft,
Ashi Salmon,
Imogen Locke,
Zsofia Kote-Jarai, Joanna R Morris,
Ellen Solomon,
Jonathan Berg,
Zoe Docherty,
Richard Camplejohn,
Rosalind Eeles,
Shirley V Hodgson
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ABSTRACT: There is conflicting evidence as to whether individuals who are heterozygous for germ-line BRCA1 or BRCA2 mutations have an altered phenotypic cellular response to irradiation. To investigate this, chromosome breakage and apoptotic response were measured after irradiation in peripheral blood lymphocytes from 26 BRCA1 and 18 BRCA2 mutation carriers without diagnosed breast cancer, and 38 unaffected age, ethnically and sex-matched controls. To assess the role of BRCA1 and BRCA2 in homologous recombination, an S phase enrichment chromosome breakage assay was used. BrdUrd incorporation studies allowed verification of the correct experimental settings. We found that BRCA1 mutation carriers without cancer had increased chromosome breaks as well as breaks and gaps per cell post irradiation using the classical G2 assay (p = 0.01 and 0.004, respectively) and the S phase enrichment assay (p = 0.01 and 0.01, respectively) compared to age-matched unaffected controls. BRCA2 mutation carriers without cancer had increased breaks as well as breaks and gaps per cell post irradiation using the S phase enrichment assay (p = 0.045 and 0.012, respectively). No difference was detected using the G2 assay (p = 0.88 and 0.40 respectively). BRCA1 and BRCA2 mutation carriers had normal cell cycle kinetics and apoptotic response to irradiation compared to age-matched controls. Our results show a demonstrable impairment in irradiation induced DNA repair in women with heterozygous germline BRCA1 and BRCA2 mutations prior to being diagnosed with breast cancer.
International Journal of Cancer 11/2007; 121(7):1631-6. · 5.44 Impact Factor
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ABSTRACT: The N-terminus of the Breast Cancer-1 predisposition protein (BRCA1) associates with the BRCA1-associated RING domain-1 protein (BARD1) to form a heterodimer, which exhibits ubiquitin ligase activity that is abrogated by known cancer-associated BRCA1 missense mutations. The majority of missense substitutions identified in patients with a personal or a family history of disease have not been followed in pedigrees, nor there is a functional understanding of their impact. We have examined, by extensive missense substitution, the interaction of BRCA1 with components that contribute to its ubiquitin ligase activity, BARD1 and the E2 ubiquitin-conjugating enzyme, UbcH5a. Selection from a randomly generated library of BRCA1 missense mutations for variants that inhibit the interaction with these components identified substitutions in residues found altered in patient DNA, indicating a correlation between loss of component-binding and propensity to disease development. We further show that the BRCA1:E2 interaction is sensitive to substitutions in all structural elements of the BRCA1 N-terminus, whereas the BARD1 interaction is sensitive to a subset of BRCA1 substitutions, which also inhibit E2-binding. Patient variants that inhibit the BRCA1:E2 interaction show loss of ubiquitin ligase activity and correlate with disease susceptibility and theoretical predictions of pathogenicity. These data link the loss of ubiquitin ligase activity, through loss of E2-binding, to the majority of non-polymorphic patient variants described within the N-terminus of BRCA1 and illustrate the likely significant role of BRCA1 ubiquitin ligase activity in tumour suppression.
Human Molecular Genetics 03/2006; 15(4):599-606. · 7.64 Impact Factor
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ABSTRACT: Segmental duplications account for up to 6% of the human genome, and the resulting low-copy repeats (LCRs) are known to be associated with more than 20 genomic disorders. Many such duplication events coincided with the burgeoning of the Alu repeat family during the last 50 million years of primate evolution, and it has been suggested that the two phenomena might be causally related. In tracing the evolution of the BRCA1 17q21 region through the primate clade, we discovered the occurrence over the last 40 million years of a complex set of about eight large gene-conversion-mediated rearrangements in the approximately 4 Mb surrounding the BRCA1 gene. These have resulted in the presence of large and probably recombinogenic LCRs across the region, the creation of the NBR2 gene, the duplication of the BRCA1/NBR1 promoter, the bisection of the highly conserved ARF2 gene, and multiple copies of the KIAA0563 gene. The junctions lie within AluS repeats, members of an Alu subfamily which experienced massive expansion during the time that the rearrangements occurred. We present a detailed history of this region over a critical 40 million-year period of genomic upheaval, including circumstantial evidence for a causal link between Alu family expansion and the rearrangement-mediated destruction and creation of transcription units.
Genomics 01/2005; 84(6):1071-82. · 3.02 Impact Factor
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ABSTRACT: The N-terminus of the BRCA1 protein bears a RING finger domain that functions as an E3 ubiquitin ligase in vitro where it is able to catalyse the synthesis of monoubiquitin and polyubiquitin targeted proteins. This activity is greatly increased when BRCA1 is in a complex with its N-terminal binding partner BARD1. In this report we use an immunohistochemical approach to demonstrate the association of cellular BRCA1 with the end product of the ubiquitin conjugation and ligation pathway, conjugated ubiquitin. Association is apparent at DNA replication structures in S-phase and following treatment with hydroxyurea and also at sites of double strand break repair after exposure to ionizing radiation. Down-regulation of endogenous, cellular BRCA1 : BARD1 using siRNA results in abrogation of ubiquitin conjugation in these structures, suggesting that heterodimer activity is required for their formation. Conversely, ectopically expressed full-length BRCA1, but not BRCA1 bearing specific N-terminal amino acid substitutions, is able to cooperate with BARD1 to increase ubiquitin conjugation in cells. Conjugation of ubiquitin in foci is inhibited by the expression of ubiquitin bearing a lysine 6 mutation suggesting that the ubiquitin polymers formed at these sites are dependent on lysine-6 for linkage. Together these data demonstrate that BRCA1 directed ligation of ubiquitin occurs during S-phase and in response to replication stress and DNA damage and is therefore likely to be a significant aspect of BRCA1 cellular activity.
Human Molecular Genetics 05/2004; 13(8):807-17. · 7.64 Impact Factor
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ABSTRACT: Cancer is a multistep process resulting from an accumulation of genetic mutations leading to dysfunction of critical genes, including tumour suppressor genes. Epigenetic changes are now also recognised as an important alternative mechanism of gene inactivation. In particular, aberrant methylation of the promoter region of a gene can lead to silencing ultimately contributing to the initiation or malignant progression of tumours. BRCA1, a breast and ovarian cancer susceptibility gene, is a tumour suppressor gene involved in the maintenance of genome integrity. Recent evidence for BRCA1 hypermethylation corroborates the view that this epigenetic alteration may play a determinant role in tumour suppressor silencing and possibly tumorigenesis. Here, we offer a summary of the data providing evidence for BRCA1 hypermethylation in tumours, and an investigation into the associated mechanism leading to BRCA1 silencing. We also discuss the impact of BRCA1 hypermethylation, as a form of epigenetic change, versus BRCA1 genetic mutations in tumour development.
Seminars in Cancer Biology 11/2002; 12(5):359-371. · 6.47 Impact Factor
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ABSTRACT: The breast and ovarian cancer predisposition gene product BRCA1, binds to BARD1 at its N terminus. In cells BRCA1 is found as a heterodimer with BARD1 and may represent the functionally active form of BRCA1. Using yeast two-hybrid and split-hybrid screens we have identified 16 independent missense mutations which prevent the ability of the BARD1 N terminus to heterodimerize with BRCA1. With reference to the recent structure of the BARD1center dotBRCA1 RING complex (Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M-C., and Klevit, R. E. (2001) Nat. Struct. Biol. 8, 833--837) we note two classes of mutation; those that map to the hydrophobic core forming the BARD1:BRCA1 interface and are substitutions of leucine, and those that map to residues forming intramolecular contacts either in helical packing, or in the conserved zinc chelating cysteine residues of the RING itself. The directed mutation of charged residues predicted to play a role in the interaction could not alone prevent heterodimer formation suggesting that, while polar interactions may participate in the specificity of the interaction, they are not crucial. Together these data provide functional evidence for the requirement of a hydrophobic interface and illustrate that disruption of the tertiary structure by mutations away from the interface itself are able to prevent formation of the heterodimer.
Journal of Biological Chemistry 04/2002; 277(11):9382-6. · 4.77 Impact Factor
