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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.
Nat Commun ; 10(1): 2252, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138797

RESUMO

The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Replicação do DNA , Células HCT116 , Células HEK293 , Células HeLa , Humanos
3.
Proc Natl Acad Sci U S A ; 116(13): 6091-6100, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850524

RESUMO

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinação Homóloga , Nucleossomos/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos
4.
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
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.
Nature ; 557(7703): 57-61, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29670289

RESUMO

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.


Assuntos
Replicação do DNA , Interferon Tipo I/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Citosol/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interferon Tipo I/imunologia , Proteína Homóloga a MRE11/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , RecQ Helicases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/deficiência
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.
DNA Repair (Amst) ; 63: 47-55, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29414053

RESUMO

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1 and Mus81. We suggest these factors comprise a subpathway for channeling repair when Brh2 is compromised in its interplay with DNA.


Assuntos
Replicação do DNA , Reparo de DNA por Recombinação , Ustilago/metabolismo , Proteína BRCA2/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Endonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Resolvases de Junção Holliday/metabolismo , Hidroxiureia/toxicidade , Mutagênicos/toxicidade , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Ustilago/efeitos dos fármacos , Ustilago/enzimologia , Ustilago/genética
12.
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
13.
Nat Commun ; 9(1): 271, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348659

RESUMO

Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes. We propose that BLM protects against genome instability by suppressing recombination at sites of G4 structures, particularly in transcribed regions of the genome.


Assuntos
Síndrome de Bloom/genética , Quadruplex G , Neoplasias/etiologia , RecQ Helicases/metabolismo , Troca de Cromátide Irmã , Animais , Síndrome de Bloom/complicações , Linhagem Celular , Instabilidade Genômica , Humanos , Perda de Heterozigosidade , Camundongos
14.
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
15.
Genetics ; 208(1): 125-138, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29162625

RESUMO

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.


Assuntos
Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Dano ao DNA , Ativação Enzimática , Exodesoxirribonucleases/deficiência , Instabilidade Genômica , Recombinação Homóloga , Mutação , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , RecQ Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/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.
Nat Commun ; 8(1): 2039, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229926

RESUMO

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.


Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Ubiquitinação , Linhagem Celular Tumoral , Proteínas Culina/genética , Proteínas Culina/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , RecQ Helicases/genética
19.
Nucleic Acids Res ; 45(20): 11878-11890, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29059328

RESUMO

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.


Assuntos
DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , RecQ Helicases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Magnetismo , Modelos Moleculares , Conformação de Ácido Nucleico , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , RecQ Helicases/química
20.
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
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