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1.
Eur J Med Genet ; 61(2): 94-97, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29056561

RESUMO

Bloom syndrome is an autosomal recessive condition characterized by severe pre- and postnatal growth deficiency, immunodeficiency, an increased risk for malignancies, craniofacial dysmorphisms, and "typical" erythematous sun-sensitive skin lesions of the face. This facial rash has a butterfly-shaped distribution around the nose and is usually observed for the first time during the early years of life. Though reported as being a main feature of Bloom syndrome, there seems to be phenotypic variability regarding this facial skin rash among patients. It has been previously reported that in some individuals with Bloom syndrome these sun-sensitive lesions are less prominent or even absent. In this report we describe a 36 year old woman with short stature, microcephaly, several dysmorphisms, congenital hypothyroidism and premature ovarian failure. She was diagnosed with nasopharyngeal carcinoma at 36 years of age, only a few months after her consultation at the department of Clinical Genetics. Whole Exome Sequencing demonstrated that she had Bloom syndrome caused by a compound heterozygous mutation in BLM (c.2207_2212delinsTAGATTC; p.(Tyr736Leufs*5) and c.3681del; p.(Lys1227Asnfs*52)). She did not have facial sun-sensitive erythematous rash during childhood nor adulthood. We conclude that Bloom syndrome does not always present with erythematous sun-sensitive skin lesions of the face. We would like to underline that phenotypic variation regarding this "hallmark" feature of Bloom syndrome exists. Being aware of this might prevent a delay in diagnosing this rare short-stature syndrome and, subsequently, its potential clinical implications.


Assuntos
Síndrome de Bloom/patologia , Eritema/patologia , Fenótipo , Adulto , Síndrome de Bloom/genética , Diagnóstico Diferencial , Eritema/etiologia , Eritema/genética , Feminino , Humanos , RecQ Helicases/genética , Luz Solar/efeitos adversos
2.
J Cell Biol ; 216(12): 3991-4005, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29042409

RESUMO

Sgs1, the orthologue of human Bloom's syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription-replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop-forming potential. Analysis of BLM in Bloom's syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop-associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop-mediated genome instability.


Assuntos
Síndrome de Bloom/genética , DNA/química , Instabilidade Genômica , RecQ Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Fibroblastos/metabolismo , Fibroblastos/patologia , Dosagem de Genes , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , RNA/genética , RNA/metabolismo , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Nat Commun ; 8(1): 693, 2017 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-28947735

RESUMO

Cells from Bloom's syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.Cytidine deaminase (CDA) deficiency leads to genome instability. Here the authors find a synthetic lethal interaction between CDA and the microtubule-associated protein Tau deficiencies, and report that Tau depletion affects rRNA synthesis, ribonucleotide pool balance, and rDNA stability.


Assuntos
Síndrome de Bloom/genética , DNA Ribossômico/metabolismo , Proteínas tau/fisiologia , Síndrome de Bloom/patologia , Sobrevivência Celular , Citidina Desaminase/deficiência , Regulação para Baixo , Instabilidade Genômica , Células HeLa , Humanos , RecQ Helicases/genética , Regulação para Cima , Proteínas tau/genética , Proteínas tau/metabolismo
4.
Annu Rev Biochem ; 86: 461-484, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28654322

RESUMO

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.


Assuntos
Adenina/metabolismo , Síndrome de Bloom/genética , DNA Catalítico/genética , Guanina/metabolismo , Polimorfismo Genético , Síndrome de Werner/genética , Evolução Biológica , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Catálise , Reparo do DNA , DNA Catalítico/metabolismo , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Hidrólise , Sequências Repetidas Invertidas , Mutação , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Globinas beta/genética , Globinas beta/metabolismo
5.
Redox Biol ; 11: 375-383, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28063379

RESUMO

Rare pleiotropic genetic disorders, Ataxia-telangiectasia (A-T), Bloom syndrome (BS) and Nijmegen breakage syndrome (NBS) are characterised by immunodeficiency, extreme radiosensitivity, higher cancer susceptibility, premature aging, neurodegeneration and insulin resistance. Some of these functional abnormalities can be explained by aberrant DNA damage response and chromosomal instability. It has been suggested that one possible common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Recent studies indicate new, alternative sources of oxidative stress in A-T, BS and NBS cells, including NADPH oxidase 4 (NOX4), oxidised low-density lipoprotein (ox-LDL) or Poly (ADP-ribose) polymerases (PARP). Mitochondrial abnormalities such as changes in the ultrastructure and function of mitochondria, excess mROS production as well as mitochondrial damage have also been reported in A-T, BS and NBS cells. A-T, BS and NBS cells are inextricably linked to high levels of reactive oxygen species (ROS), and thereby, chronic oxidative stress may be a major phenotypic hallmark in these diseases. Due to the presence of mitochondrial disturbances, A-T, BS and NBS may be considered mitochondrial diseases. Excess activity of antioxidant enzymes and an insufficient amount of low molecular weight antioxidants indicate new pharmacological strategies for patients suffering from the aforementioned diseases. However, at the current stage of research we are unable to ascertain if antioxidants and free radical scavengers can improve the condition or prolong the survival time of A-T, BS and NBS patients. Therefore, it is necessary to conduct experimental studies in a human model.


Assuntos
Ataxia Telangiectasia/genética , Síndrome de Bloom/genética , Reparo do DNA , Mitocôndrias/metabolismo , Síndrome de Quebra de Nijmegen/genética , Estresse Oxidativo/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patologia , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Mitocôndrias/patologia , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
6.
PLoS Genet ; 12(12): e1006483, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27977684

RESUMO

Bloom syndrome is a recessive human genetic disorder with features of genome instability, growth deficiency and predisposition to cancer. The only known causative gene is the BLM helicase that is a member of a protein complex along with topoisomerase III alpha, RMI1 and 2, which maintains replication fork stability and dissolves double Holliday junctions to prevent genome instability. Here we report the identification of a second gene, RMI2, that is deleted in affected siblings with Bloom-like features. Cells from homozygous individuals exhibit elevated rates of sister chromatid exchange, anaphase DNA bridges and micronuclei. Similar genome and chromosome instability phenotypes are observed in independently derived RMI2 knockout cells. In both patient and knockout cell lines reduced localisation of BLM to ultra fine DNA bridges and FANCD2 at foci linking bridges are observed. Overall, loss of RMI2 produces a partially active BLM complex with mild features of Bloom syndrome.


Assuntos
Síndrome de Bloom/genética , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Neoplasias/genética , Proteínas Nucleares/genética , Síndrome de Bloom/complicações , Síndrome de Bloom/patologia , Instabilidade Cromossômica/genética , DNA Helicases/genética , DNA Cruciforme/genética , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , Complexos Multiproteicos/genética , Neoplasias/complicações , Neoplasias/patologia , Troca de Cromátide Irmã/genética
7.
Nucleic Acids Res ; 44(14): 6787-93, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27185886

RESUMO

Sister chromatid exchanges (SCEs) are considered sensitive indicators of genome instability. Detection of SCEs typically requires cells to incorporate bromodeoxyuridine (BrdU) during two rounds of DNA synthesis. Previous studies have suggested that SCEs are induced by DNA replication over BrdU-substituted DNA and that BrdU incorporation alone could be responsible for the high number of SCE events observed in cells from patients with Bloom syndrome (BS), a rare genetic disorder characterized by marked genome instability and high SCE frequency. Here we show using Strand-seq, a single cell DNA template strand sequencing technique, that the presence of variable BrdU concentrations in the cell culture medium and in DNA template strands has no effect on SCE frequency in either normal or BS cells. We conclude that BrdU does not induce SCEs and that SCEs detected in either normal or BS cells reflect DNA repair events that occur spontaneously.


Assuntos
Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Bromodesoxiuridina/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Moldes Genéticos
8.
Oncotarget ; 7(22): 32351-61, 2016 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-27083049

RESUMO

Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.


Assuntos
Síndrome de Bloom/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Anemia de Fanconi/enzimologia , Neoplasias/enzimologia , RecQ Helicases/metabolismo , Transdução de Sinais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Reagentes para Ligações Cruzadas/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RecQ Helicases/química , RecQ Helicases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Transfecção , Raios Ultravioleta
9.
DNA Repair (Amst) ; 41: 73-84, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27100209

RESUMO

Mutation of Bloom helicase (BLM) causes Bloom syndrome (BS), a rare human genetic disorder associated with genome instability, elevation of sister chromatid exchanges, and predisposition to cancer. Deficiency in BLM homologs in Drosophila and yeast brings about significantly increased rates of recombination between imperfectly matched sequences ("homeologous recombination," or HeR). To assess whether BLM deficiency provokes an increase in HeR in human cells, we transfected an HeR substrate into a BLM-null cell line derived from a BS patient. The substrate contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI, as well as a functional tk gene to serve as a potential recombination partner for the tk-neo gene. The two tk sequences on the substrate displayed 19% divergence. A double-strand break was introduced by expression of I-SceI and repair events were recovered by selection for G418-resistant clones. Among 181 events recovered, 30 were accomplished via HeR with the balance accomplished by nonhomologous end-joining. The frequency of HeR events in the BS cells was elevated significantly compared to that seen in normal human fibroblasts or in BS cells complemented for BLM expression. We conclude that BLM deficiency enables HeR in human cells.


Assuntos
Cromossomos Humanos/genética , Recombinação Homóloga , RecQ Helicases/deficiência , RecQ Helicases/genética , Sequência de Bases , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Mutação
10.
Cutis ; 97(2): E10-3, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26919505

RESUMO

Bloom syndrome, a rare autosomal-recessive disorder, characteristically presents with photosensitivity, telangiectatic facial erythema, and growth deficiency. We present a case of Bloom syndrome with uncommon clinical manifestations including alopecia areata, eyebrow hair loss, flat nose, reticular pigmentation, and short sharpened distal phalanges with fingernails that were wider than they were long. We detected the Bloom syndrome gene, BLM, which is one of the members of the RecQ family of DNA helicases, and found changes in 2 heterozygous nucleotide sites in the patient as well as her father and mother.


Assuntos
Síndrome de Bloom/diagnóstico , Dermatoses Faciais/etiologia , Testes Genéticos/métodos , Vesícula/etiologia , Vesícula/patologia , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Criança , Eritema/etiologia , Eritema/patologia , Dermatoses Faciais/patologia , Feminino , Humanos
11.
PLoS Genet ; 11(7): e1005384, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26181065

RESUMO

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.


Assuntos
Síndrome de Bloom/genética , Citidina Desaminase/genética , Poli(ADP-Ribose) Polimerases/genética , Pirimidinas/metabolismo , Síndrome de Bloom/patologia , Linhagem Celular , Centrômero/genética , Sítios Frágeis do Cromossomo/genética , Segregação de Cromossomos/genética , Citidina Desaminase/deficiência , Replicação do DNA/genética , Instabilidade Genômica , Humanos , Mitose/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/biossíntese , RecQ Helicases/genética , Troca de Cromátide Irmã/genética
12.
Pediatr Hematol Oncol ; 30(6): 544-53, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23647505

RESUMO

BACKGROUND: An increased incidence of non-Hodgkin lymphoma (NHL) has been seen in various primary immune deficiency (PID) cases. The present study aimed to evaluate the clinical characteristics and treatment outcomes of five cases with NHL associated with primary immunodeficiency. METHODS: We retrospectively evaluated five patients with primary immunodeficiency who developed NHL. Two patients had ataxia-telangiectasia (A-T), one patient had common variable immunodeficiency (CVID), one patient had Bloom's Syndrome, and one patient had Wiskott-Aldrich syndrome (WAS). RESULTS: All patients were male (median age, 8 years). Stage distribution was stage III in three patients and stage IV in two patients. Three patients had B-cell lymphoma and two had T-cell lymphoma. Reduced doses of Berlin-Frankfurt-Münster (BFM) and French Society of Pediatric Oncology (SFOP) regimens were used in four patients according to histopathological subtype. The two patients with ataxia and one patient with Bloom's Syndrome died of progressive/relapsed disease at months 5, 19, and 6, respectively. The patient with CVID associated with T-cell lymphoma has been in remission for 7 years. A full-dosage regimen of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was successfully used in the patient with WAS and B-cell lymphoma; he was still in remission after 3 years. CONCLUSION: Primary immunodeficiency diseases are one of the strongest known risk factors for the development of NHL. Management of these patients remains problematic. There is a great need to develop new therapeutic approaches in this group. The use of rituximab in combination with CHOP may provide a promising treatment option for B-cell lymphomas associated with immunodeficiency.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Imunodeficiência de Variável Comum , Linfoma não Hodgkin , Síndrome de Wiskott-Aldrich , Adolescente , Adulto , Anticorpos Monoclonais Murinos/administração & dosagem , Ataxia Telangiectasia/complicações , Ataxia Telangiectasia/tratamento farmacológico , Ataxia Telangiectasia/patologia , Síndrome de Bloom/complicações , Síndrome de Bloom/tratamento farmacológico , Síndrome de Bloom/patologia , Criança , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunodeficiência de Variável Comum/patologia , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Lactente , Recém-Nascido , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/etiologia , Linfoma não Hodgkin/patologia , Masculino , Prednisona/administração & dosagem , Estudos Retrospectivos , Fatores de Risco , Vincristina/administração & dosagem , Síndrome de Wiskott-Aldrich/complicações , Síndrome de Wiskott-Aldrich/tratamento farmacológico , Síndrome de Wiskott-Aldrich/patologia
13.
Mutat Res ; 752(2): 138-52, 2013 Apr-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23276657

RESUMO

Helicases have important roles in nucleic acid metabolism, and their prominence is marked by the discovery of genetic disorders arising from disease-causing mutations. Missense mutations can yield unique insight to molecular functions and basis for disease pathology. XPB or XPD missense mutations lead to Xeroderma pigmentosum, Cockayne's syndrome, Trichothiodystrophy, or COFS syndrome, suggesting that DNA repair and transcription defects are responsible for clinical heterogeneity. Complex phenotypes are also observed for RECQL4 helicase mutations responsible for Rothmund-Thomson syndrome, Baller-Gerold syndrome, or RAPADILINO. Bloom's syndrome causing missense mutations are found in the conserved helicase and RecQ C-terminal domain of BLM that interfere with helicase function. Although rare, patient-derived missense mutations in the exonuclease or helicase domain of Werner syndrome protein exist. Characterization of WRN separation-of-function mutants may provide insight to catalytic requirements for suppression of phenotypes associated with the premature aging disorder. Characterized FANCJ missense mutations associated with breast cancer or Fanconi anemia interfere with FANCJ helicase activity required for DNA repair and the replication stress response. For example, a FA patient-derived mutation in the FANCJ Iron-Sulfur domain was shown to uncouple its ATPase and translocase activity from DNA unwinding. Mutations in DDX11 (ChlR1) are responsible for Warsaw Breakage syndrome, a recently discovered autosomal recessive cohesinopathy. Ongoing and future studies will address clinically relevant helicase mutations and polymorphisms, including those that interfere with key protein interactions or exert dominant negative phenotypes (e.g., certain mutant alleles of Twinkle mitochondrial DNA helicase). Chemical rescue may be an approach to restore helicase activity in loss-of-function helicase disorders. Genetic and biochemical analyses of disease-causing missense mutations in human helicase disorders have led to new insights to the molecular defects underlying aberrant cellular and clinical phenotypes.


Assuntos
Síndrome de Bloom/genética , Síndrome de Cockayne/genética , DNA Helicases/genética , Anemia de Fanconi/genética , Mutação de Sentido Incorreto/genética , Xeroderma Pigmentoso/genética , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Síndrome de Cockayne/enzimologia , Síndrome de Cockayne/patologia , Anemia de Fanconi/enzimologia , Anemia de Fanconi/patologia , Humanos , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/patologia
15.
BMC Mol Biol ; 13: 33, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23110454

RESUMO

Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RecQ Helicases/metabolismo , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Linhagem Celular , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Família Multigênica , Fenótipo , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
16.
Mol Med Rep ; 4(4): 607-9, 2011 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21567087

RESUMO

Bloom syndrome (BS) is a rare autosomal genetic disorder characterized by lupus-like erythematous telangi-ectasias of the face, sun sensitivity, infertility, stunted growth, upper respiratory infection, and gastrointestinal infections commonly associated with decreased immuno-globulin levels. The syndrome is associated with immuno-deficiency of a generalized type, ranging from mild and essentially asympto-matic to severe. Chromosomal abnormalities are hallmarks of the disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BS is caused by mutations in BLM, a member of the RecQ helicase family. We determined whether BLM deficiency has any effects on cell growth and death in BLM-deficient cells and mice. BLM-deficient EB-virus-transformed cell lines from BS patients and embryonic fibroblasts from BLM-/- mice showed slower growth than wild-type cells. BLM-deficient cells showed abnormal p53 protein expression after irradiation. In BLM-/- mice, small body size, reduced number of fetal liver cells and increased cell death were observed. BLM deficiency causes the up-regulation of p53, double-strand break and apoptosis, which are likely observed in irradiated control cells. Slow cell growth and increased cell death may be one of the causes of the small body size associated with BS patients.


Assuntos
Apoptose , Síndrome de Bloom/metabolismo , RecQ Helicases/deficiência , Animais , Síndrome de Bloom/patologia , Tamanho Corporal , Linhagem Celular Transformada , Quebras de DNA de Cadeia Dupla , Humanos , Camundongos , Camundongos Knockout , Radiação Ionizante , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteína Supressora de Tumor p53/metabolismo
17.
PLoS One ; 6(4): e18189, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21533166

RESUMO

Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology.


Assuntos
Cromossomos Artificiais Bacterianos , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Fases de Leitura Aberta , Células-Tronco Pluripotentes/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Nature ; 471(7340): 642-6, 2011 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-21399624

RESUMO

In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.


Assuntos
Síndrome de Bloom/genética , Aberrações Cromossômicas , Cromossomos Humanos , DNA Cruciforme , Troca de Cromátide Irmã , Idade de Início , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Cromátides/genética , Cromátides/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/deficiência , Endonucleases/genética , Endonucleases/metabolismo , Instabilidade Genômica/genética , Resolvases de Junção Holliday/deficiência , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Humanos , Metáfase , Neoplasias/genética , Neoplasias/patologia , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RecQ Helicases/deficiência , RecQ Helicases/genética , Recombinases/deficiência , Recombinases/genética , Recombinases/metabolismo , Troca de Cromátide Irmã/genética
19.
Mol Cancer Res ; 8(9): 1234-47, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20719863

RESUMO

BLM helicase, the protein mutated in Bloom syndrome, is involved in signal transduction cascades after DNA damage. BLM is phosphorylated on multiple residues by different kinases either after stress induction or during mitosis. Here, we have provided evidence that both Chk1 and Chk2 phosphorylated the NH(2)-terminal 660 amino acids of BLM. An internal region within the DExH motif of BLM negatively regulated the Chk1/Chk2-dependent NH(2)-terminal phosphorylation event. Using in silico analysis involving the Chk1 structure and its known substrate specificity, we predicted that Chk1 should preferentially phosphorylate BLM on serine 646 (Ser(646)). The prediction was validated in vitro by phosphopeptide analysis on BLM mutants and in vivo by usage of a newly generated phosphospecific polyclonal antibody. We showed that the phosphorylation at Ser(646) on BLM was constitutive and decreased rapidly after exposure to DNA damage. This resulted in the diminished interaction of BLM with nucleolin and PML isoforms, and consequently decreased BLM accumulation in the nucleolus and PML nuclear bodies. Instead, BLM relocalized to the sites of DNA damage and bound with the damage sensor protein, Nbs1. Mutant analysis confirmed that the binding to nucleolin and PML isoforms required Ser(646) phosphorylation. These results indicated that Chk1-mediated phosphorylation on BLM at Ser(646) might be a determinant for regulating subnuclear localization and could act as a marker for the activation status of BLM in response to DNA damage.


Assuntos
Dano ao DNA , Fosfosserina/metabolismo , Proteínas Quinases/metabolismo , RecQ Helicases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Humanos , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Fosfotreonina/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , RecQ Helicases/química
20.
Am J Dermatopathol ; 31(8): 786-91, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19820394

RESUMO

Bloom syndrome is a rare genodermatosis of autosomal recessive inheritance. Although lupus-like skin lesions characterize this disorder, mechanisms of photosensitivity are poorly understood. In this case presentation, the authors report a patient with Bloom syndrome whose lupus-like facial rash revealed striking histopathologic similarities to cutaneous lupus erythematosus.


Assuntos
Síndrome de Bloom/patologia , Lúpus Eritematoso Cutâneo/patologia , Transtornos de Fotossensibilidade/patologia , Dermatopatias/patologia , Adolescente , Síndrome de Bloom/complicações , Síndrome de Bloom/genética , Diagnóstico Diferencial , Humanos , Masculino , Transtornos de Fotossensibilidade/complicações , Transtornos de Fotossensibilidade/genética , Dermatopatias/etiologia , Dermatopatias/genética
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