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1.
Nature ; 571(7766): 521-527, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270457

RESUMO

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.


Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Isomerismo , Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo
2.
Nat Cell Biol ; 21(6): 743-754, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160708

RESUMO

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.


Assuntos
Proteína Centromérica A/genética , Centrômero/genética , Cromossomos Humanos/genética , Replicação do DNA/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Fase G1/genética , Células HeLa , Histonas/genética , Humanos , Nucleossomos/genética
3.
Nat Commun ; 10(1): 2313, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127121

RESUMO

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.


Assuntos
Biologia Computacional/métodos , Quebras de DNA de Cadeia Dupla , Sequenciamento Completo do Genoma/métodos , Linhagem Celular Tumoral , Replicação do DNA/genética , DNA Topoisomerases Tipo I/metabolismo , Genoma Fúngico/genética , Genoma Humano/genética , Humanos , Saccharomycetales/genética
4.
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
5.
Biomed Res Int ; 2019: 3842312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058188

RESUMO

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.


Assuntos
Envelhecimento/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , RNA/genética , Animais , Reparo do DNA/genética , Replicação do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Menopausa/genética , Camundongos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Ovulação/genética , RNA/biossíntese , Transcriptoma/genética
6.
Arch Virol ; 164(8): 2091-2106, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31139938

RESUMO

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.


Assuntos
Circovirus/genética , Replicação do DNA/genética , Endonucleases/genética , Replicação Viral/genética , Adenosina Trifosfatases/genética , Infecções por Circoviridae/virologia , DNA Helicases/genética , DNA Viral/genética , GTP Fosfo-Hidrolases/genética , Origem de Replicação/genética , Transativadores/genética
7.
Life Sci ; 223: 1-8, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30862568

RESUMO

AIMS: The aim of this study was to investigate the relationship between anti-HBV treatment and the regulation of HDACs during HBV DNA replication. METHODS: HDAC activities and HBV DNA levels in CHB patients' sera were measured and correlation analysis was made. The changes of HDAC2, HDAC6, AH3 and histone H3 levels in normal control and 4 CHB patient liver tissue samples before and after antiviral treatment were examined. The HDAC inhibitor, TSA, anti-HBV agents, ETV and IFN-α were used to stimulate HepG2.2.15 cells. The levels of HBV DNA, pgRNA in supernatants, and cccDNA in the cells were determined by PCR. The HDAC activity, HDAC6, HDAC2, AH3 and H3 protein levels in cells were tested at days 3, 6, and 9 after treatments. KEY FINDINGS: HDAC activity was positively correlated with HBV DNA in the HBV patients' sera. The levels of HDAC2, HDAC6 and AH3 were notably decreased after antiviral treatment. When compared with antiviral treatment group, the normal liver tissue showed obviously decreased HDAC2, HDAC6 and AH3 protein levels. In vitro study, the level of HBV DNA, the HDAC activity, and the HDAC2, HDAC6 and AH3 protein levels decreased in the ETV, IFN-α and TSA groups compared with the control group. The pgRNA level in supernatants was declined in the IFN-α group and increased in the ETV and TSA groups. cccDNA expression was suppressed by IFN-α. SIGNIFICANCE: The changes of HBV replicative products during antiviral treatment are associated with histone deacetylation. Acetylated histone H3 is involved in the process of hepatitis B virus DNA replication.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Histona Desacetilases/metabolismo , Histonas/genética , Adulto , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Técnicas de Cultura de Células , Replicação do DNA/genética , Feminino , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/enzimologia , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
8.
Neoplasia ; 21(4): 353-362, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30856376

RESUMO

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer.


Assuntos
CDC2-CDC28 Quinases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Resistencia a Medicamentos Antineoplásicos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Tolerância a Radiação , Neoplasias do Colo do Útero/metabolismo , Biomarcadores Tumorais , CDC2-CDC28 Quinases/genética , Proteínas de Transporte/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Mitocôndrias/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Prognóstico , Tolerância a Radiação/genética , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/terapia
9.
Proc Natl Acad Sci U S A ; 116(9): 3734-3739, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808759

RESUMO

In herpes simplex virus type 1 (HSV-1) infection, the coupling of genome replication and transcription regulation has been known for many years; however, the underlying mechanism has not been elucidated. We performed a comprehensive transcriptomic assessment and factor-binding analysis for Pol II, TBP, TAF1, and Sp1 to assess the effect genome replication has on viral transcription initiation and elongation. The onset of genome replication resulted in the binding of TBP, TAF1, and Pol II to previously silent late promoters. The viral transcription factor, ICP4, was continuously needed in addition to DNA replication for activation of late gene transcription initiation. Furthermore, late promoters contain a motif that closely matches the consensus initiator element (Inr), which robustly bound TAF1 postreplication. Continued DNA replication resulted in reduced binding of Sp1, TBP, and Pol II to early promoters. Therefore, the initiation of early gene transcription is attenuated following DNA replication. Herein, we propose a model for how viral DNA replication results in the differential utilization of cellular factors that function in transcription initiation, leading to the delineation of kinetic class in HSV-productive infection.


Assuntos
Proteínas Imediatamente Precoces/genética , RNA Polimerase II/genética , Simplexvirus/genética , Transcrição Genética , Animais , Cercopithecus aethiops , Replicação do DNA/genética , Genoma Viral/genética , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , TATA Box/genética , Fatores de Transcrição/genética , Células Vero , Replicação Viral/genética
10.
Nat Genet ; 51(3): 529-540, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804559

RESUMO

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.


Assuntos
Replicação do DNA/genética , DNA/genética , Mamíferos/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Variações do Número de Cópias de DNA/genética , Período de Replicação do DNA/genética , Células-Tronco Embrionárias/fisiologia , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Instabilidade Genômica/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Fase S/genética , Cromossomo X/genética
11.
Proc Natl Acad Sci U S A ; 116(8): 3221-3228, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718423

RESUMO

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Ciclina D1/genética , Interações Hospedeiro-Patógeno/genética , Legionella pneumophila/genética , Replicação do DNA/genética , Humanos , Imunidade Inata/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/genética , Doença dos Legionários/microbiologia , Macrófagos/metabolismo , Translocação Genética/genética
12.
Virus Genes ; 55(3): 274-279, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30767118

RESUMO

The temporal coordination of viral gene expression is imperative for the regulation of the herpesvirus replication cycle. While the main factors of this transcriptional coordination are known, the subtler control mechanisms of gene expression remain elusive. Recent long read sequencing-based approached have revealed an intricate meshwork of overlaps between the herpesvirus transcripts and the overlap of the replication origins with noncoding RNAs. It has been shown that the transcriptional apparatuses can physically interfere with one another while transcribing overlapping regions. We hypothesize that transcriptional interference regulates the global gene expression across the herpesvirus genome. Additionally, an overall decrease in transcriptional activity in individual viral genes has been observed following the onset of DNA replication. An overlap of the replication origins with specific transcripts has also been described in several herpesviruses. The genome-wide interactions between the transcriptional apparatuses and between the replication and transcriptional machineries suggest the existence of novel layers of genetic regulation.


Assuntos
DNA Viral/biossíntese , Herpesviridae/genética , RNA Viral/biossíntese , Origem de Replicação/genética , Replicação do DNA/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes/genética , Genoma Viral/genética , RNA Viral/genética
13.
Nucleic Acids Res ; 47(7): 3680-3698, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30715486

RESUMO

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs.


Assuntos
DNA Helicases/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/genética , Proteínas de Ligação a Poli(A)/genética , Replicação do DNA/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mitocondrial/química , RNA Mitocondrial/genética
14.
Nucleic Acids Res ; 47(7): 3619-3630, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30767012

RESUMO

Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'→3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.


Assuntos
Bacteriófagos/enzimologia , DNA Polimerase Dirigida por RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taq Polimerase/genética , Replicação do DNA/genética , Variação Genética/genética , Metagenoma/genética , Engenharia de Proteínas , RNA Viral/genética , DNA Polimerase Dirigida por RNA/química , Taq Polimerase/química
15.
Genes Dev ; 33(5-6): 282-287, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30808656

RESUMO

Here we show that translesion synthesis (TLS) opposite 1,N6-ethenodeoxyadenosine (εdA), which disrupts Watson-Crick base pairing, occurs via Polι/Polζ-, Rev1-, and Polθ-dependent pathways. The requirement of Polι/Polζ is consistent with the ability of Polι to incorporate nucleotide opposite εdA by Hoogsteen base pairing and of Polζ to extend synthesis. Rev1 polymerase and Polθ conduct TLS opposite εdA via alternative error-prone pathways. Strikingly, in contrast to extremely error-prone TLS opposite εdA by purified Polθ, it performs predominantly error-free TLS in human cells. Reconfiguration of the active site opposite εdA would provide Polθ the proficiency for error-free TLS in human cells.


Assuntos
Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/metabolismo , Domínio Catalítico , Adutos de DNA/metabolismo , Humanos
16.
Proc Natl Acad Sci U S A ; 116(8): 3062-3071, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718408

RESUMO

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.


Assuntos
Envelhecimento/genética , Reparo do DNA/genética , Longevidade/genética , Neoplasias/genética , Envelhecimento/patologia , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Genótipo , Humanos , Mutação/genética , Acúmulo de Mutações , Taxa de Mutação , Neoplasias/patologia , Fenótipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
17.
PLoS Pathog ; 15(2): e1007609, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30779786

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The IFIT family of proteins inhibits replication of some viruses, but their effects on KSHV lytic replication was unknown. Here we show that KSHV lytic replication induces IFIT expression in epithelial cells. Depletion of IFIT1, IFIT2 and IFIT3 (IFITs) increased infectious KSHV virion production 25-32-fold compared to that in control cells. KSHV lytic gene expression was upregulated broadly with preferential activation of several genes involved in lytic viral replication. Intracellular KSHV genome numbers were also increased by IFIT knockdown, consistent with inhibition of KSHV DNA replication by IFITs. RNA seq demonstrated that IFIT depletion also led to downregulation of IFN ß and several interferon-stimulated genes (ISGs), especially OAS proteins. OAS down-regulation led to decreased RNase L activity and slightly increased total RNA yield. IFIT immunoprecipitation also showed that IFIT1 bound to viral mRNAs and cellular capped mRNAs but not to uncapped RNA or trimethylated RNAs, suggesting that IFIT1 may also inhibit viral mRNA expression through direct binding. In summary, IFIT inhibits KSHV lytic replication through positively regulating the IFN ß and OAS RNase L pathway to degrade RNA in addition to possibly directly targeting viral mRNAs.


Assuntos
Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Herpesvirus Humano 8/metabolismo , Replicação do DNA/genética , Regulação para Baixo , Células Epiteliais , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Herpesvirus Humano 8/patogenicidade , Humanos , Imunidade Inata/fisiologia , Interferon beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/imunologia , Proteínas/metabolismo , RNA , RNA Mensageiro , Ativação Viral , Latência Viral/genética , Replicação Viral/genética
18.
Nat Commun ; 10(1): 759, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770810

RESUMO

Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.


Assuntos
DNA Mitocondrial/genética , Deleção de Sequência/genética , Southern Blotting , Replicação do DNA/genética , Humanos , Proteínas Mitocondriais/genética , Mutação/genética
19.
PLoS Genet ; 15(2): e1007925, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716077

RESUMO

Telomerase-independent ALT (alternative lengthening of telomeres) cells are characterized by high frequency of telomeric homologous recombination (HR), C-rich extrachromosomal circles (C-circles) and C-rich terminal 5' overhangs (C-overhangs). However, underlying mechanism is poorly understood. Here, we show that both C-circle and C-overhang form when replication fork collapse is induced by strand break at telomeres. We find that endogenous DNA break predominantly occur on C-rich strand of telomeres in ALT cells, resulting in high frequency of replication fork collapse. While collapsed forks could be rescued by replication fork regression leading to telomeric homologous recombination, those unresolved are converted to C-circles and C-overhang at lagging and leading synthesized strand, respectively. Meanwhile, multiple hallmarks of ALT are provoked, suggesting that strand break-induced replication stress underlies ALT. These findings provide a molecular basis underlying telomeric HR and biogenesis of C-circle and C-overhang, thus implicating the specific mechanism to resolve strand break-induced replication defect at telomeres in ALT cells.


Assuntos
Replicação do DNA/genética , Recombinação Genética/genética , Telômero/genética , Linhagem Celular , Estruturas Cromossômicas/genética , DNA/genética , Reparo do DNA/genética , Humanos , Telomerase/genética , Homeostase do Telômero/genética
20.
PLoS Genet ; 15(2): e1007685, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30779731

RESUMO

Cohesin acetyltransferases ESCO1 and ESCO2 play a vital role in establishing sister chromatid cohesion. How ESCO1 and ESCO2 are controlled in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show a critical role of CUL4-RING ligases (CRL4s) in cohesion establishment via regulating ESCO2 in human cells. Depletion of CUL4A, CUL4B or DDB1 subunits substantially reduces the normal cohesion efficiency. We also show that MMS22L, a vertebrate ortholog of yeast Mms22, is one of DDB1 and CUL4-associated factors (DCAFs) involved in cohesion. Several lines of evidence show selective interaction of CRL4s with ESCO2 through LxG motif, which is lost in ESCO1. Depletion of either CRL4s or ESCO2 causes a defect in SMC3 acetylation, which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing ESCO2 on chromatin and catalyzing SMC3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA promote ESCO2-dependent establishment of sister chromatid cohesion.


Assuntos
Acetiltransferases/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Culina/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Ubiquitina/genética , Acetilação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética
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