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1.
Nat Commun ; 10(1): 2253, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138795

RESUMO

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Homeostase do Telômero/genética , Telômero/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , RecQ Helicases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
2.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
3.
PLoS Genet ; 15(2): e1007942, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735491

RESUMO

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.


Assuntos
Dano ao DNA , Ligases/metabolismo , Mitose , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Epistasia Genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Ligases/deficiência , Ligases/genética , Modelos Biológicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/deficiência , RecQ Helicases/genética , RecQ Helicases/metabolismo , Troca de Cromátide Irmã/efeitos dos fármacos , Sumoilação
4.
Breast Cancer Res Treat ; 174(3): 553-560, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30610487

RESUMO

BACKGROUND: The identification of new hereditary breast cancer genes is an area of highly active research. In 2015, two independent studies provided initial evidence for a novel breast cancer susceptibility gene, RECQL, a DNA helicase which plays an important role in the DNA damage response. Several subsequent studies in independent patient cohorts have provided further data on RECQL variant frequency in additional populations, some of which have brought in to question the increased breast cancer risk associated with RECQL mutations. RESULTS: The initial reports present findings from whole exome sequencing of high-risk familial breast cancer cases in the French-Canadian, Polish and Han Chinese populations and estimate the carrier frequency of pathogenic RECQL mutations in high-risk breast cancer patients who have previously tested negative for BRCA1 and BRCA2 mutations to be approximately 1-2%. Proposed founder mutations were identified in French-Canadian and Polish populations. Functional studies support loss of function of the helicase activity of RECQL for some of the reported pathogenic mutations. An additional study in a cohort of Southern Chinese high-risk breast cancer patients estimated the frequency of pathogenic RECQL mutations to be 0.54%. A possible Chinese founder mutation was identified, but only a small number of controls were sequenced. Subsequent case-control studies screening for the Polish founder mutation in patients from Germany and Belarus did not find any evidence for increased breast cancer risk for this variant. An Australian case-control study also failed to identify an increased risk of breast cancer associated with RECQL loss of function variants. CONCLUSIONS: RECQL plays an important role in DNA repair, and is a plausible candidate breast cancer susceptibility gene. Initial studies showed evidence of an association between variants in this gene and an increased breast cancer risk in three separate populations, and identified founder mutations with significantly increased odds ratios. However, several subsequent studies have failed to support the association. With the limited and conflicting evidence available, there remains debate as to whether there is an increased breast cancer risk in individuals carrying RECQL loss of function variants. Further studies are required to better quantify the risks associated with RECQL variants and the current evidence base is not sufficient to justify routine inclusion of RECQL on breast cancer gene panels in clinical use. Management of patients in whom RECQL variants have been identified should be based on clinician assessment, in the context of the family history. Further studies are required to better quantify the risks to RECQL mutation carriers and may also guide management and potential therapeutic targeting for patients.


Assuntos
Neoplasias da Mama/genética , Mutação em Linhagem Germinativa , RecQ Helicases/genética , Neoplasias da Mama/etnologia , Canadá/etnologia , Estudos de Casos e Controles , China/etnologia , Feminino , Predisposição Genética para Doença , Humanos , Linhagem , Penetrância , Polônia , Sequenciamento Completo do Exoma
5.
J Biol Chem ; 294(8): 2690-2699, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30591583

RESUMO

The DNA damage response relies on protein modifications to elicit physiological changes required for coping with genotoxic conditions. Besides canonical DNA damage checkpoint-mediated phosphorylation, DNA damage-induced sumoylation has recently been shown to promote genotoxin survival. Cross-talk between these two pathways exists in both yeast and human cells. In particular, sumoylation is required for optimal checkpoint function, but the underlying mechanisms are not well-understood. To address this question, we examined the sumoylation of the first responder to DNA lesions, the ssDNA-binding protein complex replication protein A (RPA) in budding yeast (Saccharomyces cerevisiae). We delineated the sumoylation sites of the RPA large subunit, Rfa1 on the basis of previous and new mapping data. Findings using a sumoylation-defective Rfa1 mutant suggested that Rfa1 sumoylation acts in parallel with the 9-1-1 checkpoint complex to enhance the DNA damage checkpoint response. Mechanistically, sumoylated Rfa1 fostered an interaction with a checkpoint adaptor protein, Sgs1, and contributed to checkpoint kinase activation. Our results suggest that SUMO-based modulation of a DNA damage sensor positively influences the checkpoint response.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Quebras de DNA de Cadeia Simples , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sumoilação , Reparo do DNA , Fosforilação , Conformação Proteica , RecQ Helicases/genética , Proteína de Replicação A/química , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
6.
Diabetes ; 67(8): 1673-1683, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29752424

RESUMO

Maternal hyperglycemia during pregnancy is associated with excess fetal growth and adverse perinatal and developmental outcomes. Placental epigenetic maladaptation may underlie these associations. We performed an epigenome-wide association study (>850,000 CpG sites) of term placentas and prenatal maternal glycemic response 2-h post oral glucose challenge at 24-30 weeks of gestation among 448 mother-infant pairs. Maternal 2-h glycemia postload was strongly associated with lower DNA methylation of four CpG sites (false discovery rate [FDR] q <0.05) within the phosphodiesterase 4B gene (PDE4B). Additionally, three other individual CpG sites were differentially methylated relative to maternal glucose response within the TNFRSF1B, LDLR, and BLM genes (FDR q <0.05). DNA methylation correlated with expression of its respective genes in placental tissue at three out of four independent identified loci: PDE4B (r = 0.31, P < 0.01), TNFRSF1B (r = -0.24, P = 0.013), and LDLR (r = 0.32, P < 0.001). In an independent replication cohort (N = 65-108 samples), results were consistent in direction but not significantly replicated among tested CpG sites in PDE4B and TNFRSF1B Our study provides evidence that maternal glycemic response during pregnancy is associated with placental DNA methylation of key inflammatory genes whose expression levels are partially under epigenetic control.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Metilação de DNA , Epigênese Genética , Resistência à Insulina , Placenta/metabolismo , Receptores de LDL/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Peso ao Nascer , Estudos de Coortes , Ilhas de CpG , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Teste de Tolerância a Glucose , Hemoglobina A Glicada/análise , Humanos , Recém-Nascido , Placenta/enzimologia , Placentação , Gravidez , Estudos Prospectivos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Receptores de LDL/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Nascimento a Termo , Adulto Jovem
7.
Int J Mol Sci ; 19(4)2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29642415

RESUMO

Biallelic mutations in RECQL4 gene, a caretaker of the genome, cause Rothmund-Thomson type-II syndrome (RTS-II) and confer increased cancer risk if they damage the helicase domain. We describe five families exemplifying clinical and allelic heterogeneity of RTS-II, and report the effect of pathogenic RECQL4 variants by in silico predictions and transcripts analyses. Complete phenotype of patients #39 and #42 whose affected siblings developed osteosarcoma correlates with their c.[1048_1049del], c.[1878+32_1878+55del] and c.[1568G>C;1573delT], c.[3021_3022del] variants which damage the helicase domain. Literature survey highlights enrichment of these variants affecting the helicase domain in patients with cancer outcome raising the issue of strict oncological surveillance. Conversely, patients #29 and #19 have a mild phenotype and carry, respectively, the unreported homozygous c.3265G>T and c.3054A>G variants, both sparing the helicase domain. Finally, despite matching several criteria for RTS clinical diagnosis, patient #38 is heterozygous for c.2412_2414del; no pathogenic CNVs out of those evidenced by high-resolution CGH-array, emerged as contributors to her phenotype.


Assuntos
Mutação , Fenótipo , Síndrome de Rothmund-Thomson/genética , Adolescente , Adulto , Linhagem Celular Tumoral , Criança , Feminino , Homozigoto , Humanos , Masculino , Linhagem , RecQ Helicases/genética , RecQ Helicases/metabolismo , Síndrome de Rothmund-Thomson/patologia
8.
PLoS Genet ; 14(4): e1007355, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29659569

RESUMO

Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including ATM and CHEK2, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely RAD51C, FANCD2, FANCI, CEP57 and RECQL4. Furthermore, using in silico pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified "likely pathogenic" missense variants in ATM, BRIP1, CHEK2 and TP53. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study.


Assuntos
Mutação em Linhagem Germinativa , Neoplasias da Próstata/genética , Adulto , Idade de Início , Proteínas Mutadas de Ataxia Telangiectasia/genética , Neoplasias da Mama/genética , Estudos de Casos e Controles , Quinase do Ponto de Checagem 2/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Simulação por Computador , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Genes p53 , Predisposição Genética para Doença , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Linhagem , RNA Helicases/genética , RecQ Helicases/genética , Análise de Sequência de DNA
9.
Genetics ; 209(2): 439-456, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29654124

RESUMO

Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes MSH2 and MSH6 are overexpressed more frequently than MSH3, and that MSH2 and MSH6 are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.


Assuntos
Ciclo Celular , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Proteína 2 Homóloga a MutS/genética , Proteínas de Saccharomyces cerevisiae/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para Cima
10.
PLoS Genet ; 14(3): e1007250, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29505562

RESUMO

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.


Assuntos
Dano ao DNA/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Epistasia Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genoma Fúngico , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Gene ; 654: 110-115, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29462647

RESUMO

BACKGROUND: Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder mainly characterized by cutaneous poikiloderma, sparse hair, short stature and skeletal defects. Deleterious mutations in the RecQ-like DNA helicase type 4 (RECQL4) gene have been detected in approximately two-thirds of RTS cases. METHODS: Three Chinese patients from two unrelated families were enrolled for clinical evaluation. Targeted next-generation sequencing (NGS) using a custom panel consisting of 705 short-stature-related genes was performed for the probands. Variants detected by NGS were confirmed by Sanger sequencing and examined in family members. RESULTS: The probands presented with characteristic features of severe growth delay, poikiloderma mostly on the face, buttocks and extremities, sparse or absent hair, eyelashes, and eyebrows, forearm reduction defects, small hands with hypoplasia of the middle phalanx (little finger) in one of the probands, epicanthus, hypertelorism, and dental abnormalities. In addition, novel auricle features and other rare facial features, including narrow palpebral fissure, depressed nasal bridge, and small chin were exhibited. Four novel RECQL4 variants were identified, including three pathogenic frameshift variants, c.1724_1725delAC, p.His575fs*7; c.2421dupT, p.Asp808*; c.1770_1807del, p.Pro591fs*2, and one likely pathogenic missense variant, c.691G>A, p.Gly231Ser. CONCLUSION: Our study expands the mutational spectrum of RECQL4 gene and reveals novel phenotypes observed in Chinese RTS patients.


Assuntos
Mutação , RecQ Helicases/genética , Síndrome de Rothmund-Thomson/etnologia , Síndrome de Rothmund-Thomson/genética , Criança , Pré-Escolar , China , Análise Mutacional de DNA , Saúde da Família , Feminino , Genes Recessivos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo
12.
PLoS One ; 13(2): e0192483, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29470542

RESUMO

Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.


Assuntos
DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Exodesoxirribonuclease V/genética , Controle de Qualidade , RecQ Helicases/genética , Recombinação Genética , Divisão Celular , Reparo do DNA , Escherichia coli/genética , Mutação , Estresse Fisiológico , Raios Ultravioleta
13.
Oncol Res ; 26(7): 1113-1121, 2018 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-29386092

RESUMO

miR-522-3p is known to degrade bloom syndrome protein (BLM) and enhance expression of other proto-oncogenes, leading to tumorigenesis. This study aimed to investigate the molecular mechanisms of miR-522-3p in human colorectal cancer (CRC) cells. Expressions of miR-522-3p in CRC and adjacent tissues, as well as in normal human colon epithelial cell line (FHC) and five CRC cell lines, were detected. Human CRC cell lines, HCT-116 and HT29, were transfected with miR-522-3p mimic, inhibitor, or scrambled controls. Then cell viability, apoptosis, cell cycle progression, and the expressions of c-myc, cyclin E, CDK2, and BLM were assessed. It was found that miR-522-3p was highly expressed in CRC tissues when compared to adjacent nontumor tissues and was highly expressed in CRC cell lines when compared to FHC cells. miR-522-3p overexpression promoted cell viability, reduced apoptotic cell rate, arrested more cells in the S phase, and upregulated c-myc, cyclin E, and CDK2 expression. BLM was a target gene of miR-522-3p, and miR-522-3p suppression did not exert antiproliferative and proapoptotic activities when BLM was silenced. These findings demonstrate that miR-522-3p upregulation negatively regulates the expression of BLM, with upregulation of c-myc, CDK2, and cyclin E, and thereby promoting the proliferation of human CRC cells.


Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , MicroRNAs/genética , RecQ Helicases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , RecQ Helicases/genética , Células Tumorais Cultivadas
14.
BMJ Case Rep ; 20182018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29367366

RESUMO

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder with clinical features consisting of poikiloderma, skeletal abnormalities, sparse hair, absent or scanty eyelashes and eyebrows and short stature. Patients with RTS due to genetic mutations of RECQL4 genes carry a high risk of developing osteosarcoma during childhood. Because of this, early genetic diagnosis is important. Here, we describe a 14-year-old white boy who developed an erythematous rash on both cheeks before the age of 3 months and was noted to have absent eyelashes and scanty eyebrows. He was found to have compound heterozygous mutations of the RECQL4 gene alleles at the age of 6 months and was diagnosed to have RTS type II. He subsequently developed osteosarcoma at age 10 which was successfully treated, and currently he has been tumour free for over 3 years.


Assuntos
Mutação , Osteossarcoma/genética , RecQ Helicases/genética , Síndrome de Rothmund-Thomson/genética , Adolescente , Humanos , Masculino
15.
Cancer Lett ; 413: 1-10, 2018 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-29080750

RESUMO

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy.


Assuntos
Canal Anal/anormalidades , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/metabolismo , Craniossinostoses/enzimologia , Nanismo/enzimologia , Instabilidade Genômica , Comunicação Interatrial/enzimologia , Deformidades Congênitas dos Membros/enzimologia , Neoplasias/enzimologia , Patela/anormalidades , Rádio (Anatomia)/anormalidades , RecQ Helicases/metabolismo , Síndrome de Rothmund-Thomson/enzimologia , Canal Anal/enzimologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Craniossinostoses/genética , Reparo do DNA , Replicação do DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Nanismo/genética , Inibidores Enzimáticos/uso terapêutico , Predisposição Genética para Doença , Comunicação Interatrial/genética , Humanos , Deformidades Congênitas dos Membros/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Patela/enzimologia , Fenótipo , Rádio (Anatomia)/enzimologia , RecQ Helicases/antagonistas & inibidores , RecQ Helicases/genética , Síndrome de Rothmund-Thomson/genética
16.
G3 (Bethesda) ; 8(2): 737-752, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29279302

RESUMO

Paused or stalled replication forks are major threats to genome integrity; unraveling the complex pathways that contribute to fork stability and restart is crucial. Experimentally, fork stalling is induced by growing the cells in presence of hydroxyurea (HU), which depletes the pool of deoxynucleotide triphosphates (dNTPs) and slows down replication progression in yeast. Here, I report an epistasis analysis, based on sensitivity to HU, between CLB2, the principal mitotic cyclin gene in Saccharomyces cerevisiae, and genes involved in fork stability and recombination. clb2Δ cells are not sensitive to HU, but the strong synergistic effect of clb2Δ with most genes tested indicates, unexpectedly, that CLB2 has an important role in DNA replication, in the stability and restart of stalled forks, and in pathways dependent on and independent of homologous recombination. Results indicate that CLB2 functions in parallel with the SGS1 helicase and EXO1 exonuclease to allow proper Rad51 recombination, but also regulates a combined Sgs1-Exo1 activity in a pathway dependent on Mec1 and Rad53 checkpoint protein kinases. The data argue that Mec1 regulates Clb2 to prevent a deleterious Sgs1-Exo1 activity at paused or stalled forks, whereas Rad53 checkpoint activation regulates Clb2 to allow a necessary Sgs1-Exo1 activity at stalled or collapsed forks. Altogether, this study indicates that Clb2 regulates the activity of numerous nucleases at single-stranded gaps created by DNA replication. A model is proposed for the function and regulation of Clb2 at stalled forks. These data provide new perspectives on the role of mitotic cyclins at the end of S phase.


Assuntos
Ciclina B/genética , Dano ao DNA , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Ciclina B/metabolismo , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Genéticos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Fase S/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Biochem Soc Trans ; 46(1): 77-95, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29273621

RESUMO

Helicases are molecular motors that play central roles in nucleic acid metabolism. Mutations in genes encoding DNA helicases of the RecQ and iron-sulfur (Fe-S) helicase families are linked to hereditary disorders characterized by chromosomal instabilities, highlighting the importance of these enzymes. Moreover, mono-allelic RecQ and Fe-S helicase mutations are associated with a broad spectrum of cancers. This review will discuss and contrast the specialized molecular functions and biological roles of RecQ and Fe-S helicases in DNA repair, the replication stress response, and the regulation of gene expression, laying a foundation for continued research in these important areas of study.


Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , RecQ Helicases/metabolismo , Aberrações Cromossômicas , DNA Helicases/genética , Humanos , Proteínas com Ferro-Enxofre/genética , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , RecQ Helicases/genética , Especificidade por Substrato
18.
Mol Microbiol ; 107(1): 81-93, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29052918

RESUMO

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Telômero/fisiologia , Ustilago/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico/fisiologia , Rad51 Recombinase/genética , RecQ Helicases/genética , Recombinação Genética/genética , Recombinação Genética/fisiologia , Telomerase/metabolismo , Telômero/metabolismo , Ustilago/metabolismo
19.
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
20.
Curr Genet ; 64(2): 459-468, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28918480

RESUMO

DNA-damage tolerance (DDT) is employed by eukaryotes to deal with replication blocks on the template strand, and is divided into two parallel pathways that are activated by sequential ubiquitination of proliferating cell nuclear antigen (PCNA) at the Lys164 residue. Rad6-Rad18-mediated PCNA monoubiquitination promotes translesion DNA synthesis (TLS) and the monoubiquitinated PCNA can be further polyubiquitinated by an Mms2-Ubc13-Rad5 complex, leading to error-free lesion bypass. We previously reported that the DNA helicase Sgs1 is required for error-free lesion bypass, probably through the double-Holliday junction migration and subsequent resolution. Surprisingly, a synthetic genetic array (SGA) screen using rev1 and rev3 as baits did not reveal an anticipated synthetic effect with sgs1, indicating a possible involvement of Sgs1 in TLS. Here, we report detailed genetic analyses demonstrating that Sgs1 plays a key role in efficient TLS and that it is probably required for the signaling of DNA damage leading to PCNA monoubiquitination. These studies collectively illustrate that Sgs1 participates in both branches of DDT and possibly plays a role in pathway choice.


Assuntos
Dano ao DNA/genética , DNA/biossíntese , RecQ Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA , Complexos Multiproteicos/genética , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
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