Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 53
Filtrar
Filtros adicionais











País/Região como assunto
Intervalo de ano
1.
Nat Rev Genet ; 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455890

RESUMO

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.

2.
Genome Res ; 28(11): 1720-1732, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30341161

RESUMO

Despite the rapid development of sequencing technologies, the assembly of mammalian-scale genomes into complete chromosomes remains one of the most challenging problems in bioinformatics. To help address this difficulty, we developed Ragout 2, a reference-assisted assembly tool that works for large and complex genomes. By taking one or more target assemblies (generated from an NGS assembler) and one or multiple related reference genomes, Ragout 2 infers the evolutionary relationships between the genomes and builds the final assemblies using a genome rearrangement approach. By using Ragout 2, we transformed NGS assemblies of 16 laboratory mouse strains into sets of complete chromosomes, leaving <5% of sequence unlocalized per set. Various benchmarks, including PCR testing and realigning of long Pacific Biosciences (PacBio) reads, suggest only a small number of structural errors in the final assemblies, comparable with direct assembly approaches. We applied Ragout 2 to the Mus caroli and Mus pahari genomes, which exhibit karyotype-scale variations compared with other genomes from the Muridae family. Chromosome painting maps confirmed most large-scale rearrangements that Ragout 2 detected. We applied Ragout 2 to improve draft sequences of three ape genomes that have recently been published. Ragout 2 transformed three sets of contigs (generated using PacBio reads only) into chromosome-scale assemblies with accuracy comparable to chromosome assemblies generated in the original study using BioNano maps, Hi-C, BAC clones, and FISH.

3.
Sci Rep ; 8(1): 10439, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29992973

RESUMO

ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.

4.
Nat Neurosci ; 21(8): 1139, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29875394

RESUMO

In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."

5.
Bioinformatics ; 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29931085

RESUMO

Summary: Standardised interfaces for efficiently accessing high-throughput sequencing data are a fundamental requirement for large-scale genomic data sharing. We have developed htsget, a protocol for secure, efficient and reliable access to sequencing read and variation data. We demonstrate four independent client and server implementations, and the results of a comprehensive interoperability demonstration. Availability and implementation: http://samtools.github.io/hts-specs/htsget.html. Supplementary information: Supplementary data are available at Bioinformatics online.

6.
Genome Res ; 28(4): 448-459, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29563166

RESUMO

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.


Assuntos
Evolução Molecular , Genoma/genética , Muridae/genética , Filogenia , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromossomos/genética , Cariotipagem/métodos , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Retroelementos/genética , Especificidade da Espécie
7.
Nat Neurosci ; 21(2): 207-217, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29311744

RESUMO

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.

8.
Microorganisms ; 5(3)2017 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-28926970

RESUMO

Armillaria mellea is a major plant pathogen. Yet, the strategies the organism uses to infect susceptible species, degrade lignocellulose and other plant material and protect itself against plant defences and its own glycodegradative arsenal are largely unknown. Here, we use a combination of gel and MS-based proteomics to profile A. mellea under conditions of oxidative stress and changes in growth matrix. 2-DE and LC-MS/MS were used to investigate the response of A. mellea to H2O2 and menadione/FeCl3 exposure, respectively. Several proteins were detected with altered abundance in response to H2O2, but not menadione/FeCl3 (i.e., valosin-containing protein), indicating distinct responses to these different forms of oxidative stress. One protein, cobalamin-independent methionine synthase, demonstrated a common response in both conditions, which may be a marker for a more general stress response mechanism. Further changes to the A. mellea proteome were investigated using MS-based proteomics, which identified changes to putative secondary metabolism (SM) enzymes upon growth in agar compared to liquid cultures. Metabolomic analyses revealed distinct profiles, highlighting the effect of growth matrix on SM production. This establishes robust methods by which to utilize comparative proteomics to characterize this important phytopathogen.

9.
Sci Rep ; 7(1): 3935, 2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28638050

RESUMO

Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.

10.
Genetics ; 206(2): 603-619, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28592499

RESUMO

Meiotic recombination is an essential feature of sexual reproduction that ensures faithful segregation of chromosomes and redistributes genetic variants in populations. Multiparent populations such as the Diversity Outbred (DO) mouse stock accumulate large numbers of crossover (CO) events between founder haplotypes, and thus present a unique opportunity to study the role of genetic variation in shaping the recombination landscape. We obtained high-density genotype data from [Formula: see text] DO mice, and localized 2.2 million CO events to intervals with a median size of 28 kb. The resulting sex-averaged genetic map of the DO population is highly concordant with large-scale (order 10 Mb) features of previously reported genetic maps for mouse. To examine fine-scale (order 10 kb) patterns of recombination in the DO, we overlaid putative recombination hotspots onto our CO intervals. We found that CO intervals are enriched in hotspots compared to the genomic background. However, as many as [Formula: see text] of CO intervals do not overlap any putative hotspots, suggesting that our understanding of hotspots is incomplete. We also identified coldspots encompassing 329 Mb, or [Formula: see text] of observable genome, in which there is little or no recombination. In contrast to hotspots, which are a few kilobases in size, and widely scattered throughout the genome, coldspots have a median size of 2.1 Mb and are spatially clustered. Coldspots are strongly associated with copy-number variant (CNV) regions, especially multi-allelic clusters, identified from whole-genome sequencing of 228 DO mice. Genes in these regions have reduced expression, and epigenetic features of closed chromatin in male germ cells, which suggests that CNVs may repress recombination by altering chromatin structure in meiosis. Our findings demonstrate how multiparent populations, by bridging the gap between large-scale and fine-scale genetic mapping, can reveal new features of the recombination landscape.


Assuntos
Variações do Número de Cópias de DNA/genética , Recombinação Homóloga/genética , Meiose/genética , Recombinação Genética , Animais , Mapeamento Cromossômico , Cromossomos/genética , Troca Genética , Genoma , Genótipo , Haplótipos , Masculino , Camundongos
11.
Elife ; 62017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28438259

RESUMO

The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify cis-acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.


Assuntos
Variação Genética , Condutos Olfatórios/fisiologia , Neurônios Receptores Olfatórios/classificação , Receptores Odorantes/genética , Animais , Perfilação da Expressão Gênica , Camundongos , Neurônios Receptores Olfatórios/fisiologia , Análise de Sequência de RNA
12.
Nat Chem Biol ; 13(1): 12-14, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27820796

RESUMO

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.


Assuntos
Testes Genéticos , Genoma/efeitos dos fármacos , Genoma/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos
13.
Genome Res ; 27(2): 300-309, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27986821

RESUMO

We are rapidly approaching the point where we have sequenced millions of human genomes. There is a pressing need for new data structures to store raw sequencing data and efficient algorithms for population scale analysis. Current reference-based data formats do not fully exploit the redundancy in population sequencing nor take advantage of shared genetic variation. In recent years, the Burrows-Wheeler transform (BWT) and FM-index have been widely employed as a full-text searchable index for read alignment and de novo assembly. We introduce the concept of a population BWT and use it to store and index the sequencing reads of 2705 samples from the 1000 Genomes Project. A key feature is that, as more genomes are added, identical read sequences are increasingly observed, and compression becomes more efficient. We assess the support in the 1000 Genomes read data for every base position of two human reference assembly versions, identifying that 3.2 Mbp with population support was lost in the transition from GRCh37 with 13.7 Mbp added to GRCh38. We show that the vast majority of variant alleles can be uniquely described by overlapping 31-mers and show how rapid and accurate SNP and indel genotyping can be carried out across the genomes in the population BWT. We use the population BWT to carry out nonreference queries to search for the presence of all known viral genomes and discover human T-lymphotropic virus 1 integrations in six samples in a recognized epidemiological distribution.


Assuntos
Genoma Humano/genética , Genômica , Alinhamento de Sequência/métodos , Sequenciamento Completo do Genoma/métodos , Alelos , Compressão de Dados , Genótipo , Humanos , Mutação INDEL/genética , Análise de Sequência de DNA , Software
14.
G3 (Bethesda) ; 6(12): 4211-4216, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27765810

RESUMO

Wild-derived mouse inbred strains are becoming increasingly popular for complex traits analysis, evolutionary studies, and systems genetics. Here, we report the whole-genome sequencing of two wild-derived mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of Mus musculus domesticus origin. These two inbred strains were selected based on their geographic origin, karyotype, and use in ongoing research. We generated 14× and 18× coverage sequence, respectively, and discovered over 1.1 million novel variants, most of which are private to one of these strains. This report expands the number of wild-derived inbred genomes in the Mus genus from six to eight. The sequence variation can be accessed via an online query tool; variant calls (VCF format) and alignments (BAM format) are available for download from a dedicated ftp site. Finally, the sequencing data have also been stored in a lossless, compressed, and indexed format using the multi-string Burrows-Wheeler transform. All data can be used without restriction.


Assuntos
Animais Selvagens/genética , Diploide , Genoma , Camundongos Endogâmicos/genética , Animais , Animais Selvagens/classificação , Feminino , Variação Genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos/classificação , Filogenia
15.
Genome Biol ; 17(1): 167, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27480531

RESUMO

BACKGROUND: The Mouse Genomes Project is an ongoing collaborative effort to sequence the genomes of the common laboratory mouse strains. In 2011, the initial analysis of sequence variation across 17 strains found 56.7 M unique single nucleotide polymorphisms (SNPs) and 8.8 M indels. We carry out deep sequencing of 13 additional inbred strains (BUB/BnJ, C57BL/10J, C57BR/cdJ, C58/J, DBA/1J, I/LnJ, KK/HiJ, MOLF/EiJ, NZB/B1NJ, NZW/LacJ, RF/J, SEA/GnJ and ST/bJ), cataloguing molecular variation within and across the strains. These strains include important models for immune response, leukaemia, age-related hearing loss and rheumatoid arthritis. We now have several examples of fully sequenced closely related strains that are divergent for several disease phenotypes. RESULTS: Approximately 27.4 M unique SNPs and 5 M indels are identified across these strains compared to the C57BL/6 J reference genome (GRCm38). The amount of variation found in the inbred laboratory mouse genome has increased to 71 M SNPs and 12 M indels. We investigate the genetic basis of highly penetrant cancer susceptibility in RF/J finding private novel missense mutations in DNA damage repair and highly cancer associated genes. We use two highly related strains (DBA/1J and DBA/2J) to investigate the genetic basis of collagen-induced arthritis susceptibility. CONCLUSIONS: This paper significantly expands the catalogue of fully sequenced laboratory mouse strains and now contains several examples of highly genetically similar strains with divergent phenotypes. We show how studying private missense mutations can lead to insights into the genetic mechanism for a highly penetrant phenotype.


Assuntos
Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos Endogâmicos/genética , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Dano ao DNA/genética , Reparo do DNA/genética , Homozigoto , Mutação INDEL/genética , Camundongos , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único
16.
Eukaryot Cell ; 14(9): 941-57, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26150413

RESUMO

Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ΔgliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ΔgliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ΔgliK strain, but not the ΔgliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.


Assuntos
Aspergillus fumigatus/metabolismo , Gliotoxina/biossíntese , Metionina/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliotoxina/toxicidade , Metilação , S-Adenosil-Homocisteína/metabolismo
17.
Mamm Genome ; 26(9-10): 403-12, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26123534

RESUMO

The Mouse Genomes Project was initiated in 2009 with the goal of using next-generation sequencing technologies to catalogue molecular variation in the common laboratory mouse strains, and a selected set of wild-derived inbred strains. The initial sequencing and survey of sequence variation in 17 inbred strains was completed in 2011 and included comprehensive catalogue of single nucleotide polymorphisms, short insertion/deletions, larger structural variants including their fine scale architecture and landscape of transposable element variation, and genomic sites subject to post-transcriptional alteration of RNA. From this beginning, the resource has expanded significantly to include 36 fully sequenced inbred laboratory mouse strains, a refined and updated data processing pipeline, and new variation querying and data visualisation tools which are available on the project's website ( http://www.sanger.ac.uk/resources/mouse/genomes/ ). The focus of the project is now the completion of de novo assembled chromosome sequences and strain-specific gene structures for the core strains. We discuss how the assembled chromosomes will power comparative analysis, data access tools and future directions of mouse genetics.


Assuntos
Genoma , Camundongos Endogâmicos/genética , Polimorfismo de Nucleotídeo Único/genética , Processamento Pós-Transcricional do RNA/genética , Animais , Sequência de Bases , Elementos de DNA Transponíveis/genética , Genômica , Camundongos
18.
Cell ; 161(2): 319-32, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25843629

RESUMO

Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.


Assuntos
Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas B-raf/genética , Pseudogenes , RNA/metabolismo , Animais , Sequência de Bases , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas B-raf/metabolismo
19.
EMBO J ; 34(11): 1509-22, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-25899817

RESUMO

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.


Assuntos
Reparo do DNA/fisiologia , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Fúngico/genética , DNA de Cadeia Simples/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
20.
Nature ; 517(7535): 489-92, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25363767

RESUMO

Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.


Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Genes ras/genética , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Mutação/genética , Proteína Oncogênica p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Animais , Carcinógenos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/genética , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Feminino , Instabilidade Genômica/genética , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Metilnitrosoureia/toxicidade , Camundongos , Modelos Genéticos , Mutação Puntual/genética , Uretana/toxicidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA