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1.
Cell Stem Cell ; 30(9): 1262-1281.e8, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37582363

RESUMO

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.


Assuntos
Síndromes Mielodisplásicas , Sítios de Splice de RNA , Humanos , Multiômica , Splicing de RNA/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Mutação/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo
2.
DNA Repair (Amst) ; 84: 102614, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975634

RESUMO

The RNase H2 complex is a conserved heterotrimeric enzyme that degrades RNA:DNA hybrids and promotes excision of rNMPs misincorporated during DNA replication. Failure to remove ribonucleotides from DNA leads to genomic instability in yeast and humans. The monogenic Aicardi-Goutières syndrome (AGS) results from mutation in one of several genes, among which are those encoding the RNase H2 subunits. The complete cellular and genomic consequences of RNASEH2 mutations and the precise connection to disease remain unclear. To learn more about the effect of RNASEH2 mutations on the cell, we used yeast as a model of AGS disease. We have generated yeast strains bearing AGS-associated mutations in RNASEH2 genes. There is a range of disease presentation in patients bearing these RNASEH2 variants. Here we report on in vivo phenotypes of genomic instability, including mutation and recombination rates, and synthetic gene interactions. These phenotypes provide insight into molecular consequences of RNASEH2 mutations, and lay the groundwork for further study of genomic instability as a contributing factor to AGS disease.


Assuntos
Alelos , Doenças Autoimunes do Sistema Nervoso/genética , Instabilidade Genômica , Malformações do Sistema Nervoso/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Domínio Catalítico , Humanos , Mutação com Perda de Função , Fenótipo , Ribonucleases/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química
3.
Cell Cycle ; 15(3): 331-6, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26716562

RESUMO

The replicative DNA polymerases insert ribonucleotides into DNA at a frequency of approximately 1/6500 nucleotides replicated. The rNMP residues make the DNA backbone more susceptible to hydrolysis and can also distort the helix, impeding the transcription and replication machineries. rNMPs in DNA are efficiently removed by RNaseH2 by a process called ribonucleotides excision repair (RER). In the absence of functional RNaseH2, rNMPs are subject to cleavage by Topoisomerase I, followed by further processing to result in deletion mutations due to slippage in simple DNA repeats. The topoisomerase I-mediated cleavage at rNMPs results in DNA ends that cannot be ligated by DNA ligase I, a 5'OH end and a 2'-3' cyclic phosphate end. In the budding yeast, the mutation level in RNaseH2 deficient cells is kept low via the action of the Srs2 helicase and the Exo1 nuclease, which collaborate to process the Top1-induced nick with subsequent non-mutagenic gap filling. We have surveyed other helicases and nucleases for a possible role in reducing mutagenesis at Top1 nicks at rNMPs and have uncovered a novel role for the RecQ family helicase Sgs1 in this process.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Ribonucleotídeos/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , DNA Helicases/genética , Reparo do DNA , Enzimas Reparadoras do DNA/genética , DNA Topoisomerases Tipo I/genética , Exodesoxirribonucleases/genética , Humanos , Mutagênese
4.
Microb Cell ; 3(6): 248-254, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28203566

RESUMO

Ribonucleotides can become embedded in DNA from insertion by DNA polymerases, failure to remove Okazaki fragment primers, R-loops that can prime replication, and RNA/cDNA-mediated recombination. RNA:DNA hybrids are removed by RNase H enzymes. Single rNMPs in DNA are removed by RNase H2 and if they remain on the leading strand, can lead to mutagenesis in a Top1-dependent pathway. rNMPs in DNA can also stimulate genome instability, among which are homologous recombination gene conversion events. We previously found that, similar to the rNMP-stimulated mutagenesis, rNMP-stimulated recombination was also Top1-dependent. However, in contrast to mutagenesis, we report here that recombination is not stimulated by rNMPs incorporated by the replicative polymerase epsilon. Instead, recombination seems to be stimulated by multiple contiguous rNMPs, which may arise from R-loops or replication priming events.

5.
BMC Ecol ; 15: 22, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26219534

RESUMO

For the third time, BMC Ecology is delighted to announce the winners of our Image Competition. This year featured entries from all over the world and showcased not only the creativity and talent of the participants, but also the exquisite beauty and diversity of our planet. We are pleased to present the winning selections of the editorial board of the journal and guest judge Dr. Ana Luz Porzecanski, as well as some highly commended images that are sure to impress.


Assuntos
Distinções e Prêmios , Ecologia , Fotografação
6.
Nucleic Acids Res ; 42(16): 10226-34, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25159610

RESUMO

Ribonucleotides are misincorporated into replicating DNA due to the similarity of deoxyribonucleotides and ribonucleotides, the high concentration of ribonucleotides in the nucleus and the imperfect accuracy of replicative DNA polymerases in choosing the base with the correct sugar. Embedded ribonucleotides change certain properties of the DNA and can interfere with normal DNA transactions. Therefore, misincorporated ribonucleotides are targeted by the cell for removal. Failure to remove ribonucleotides from DNA results in an increase in genome instability, a phenomenon that has been characterized in various systems using multiple assays. Recently, however, another side to ribonucleotide misincorporation has emerged, where there is evidence for a functional role of misinserted ribonucleotides in DNA, leading to beneficial consequences for the cell. This review examines examples of both positive and negative effects of genomic ribonucleotide misincorporation in various organisms, aiming to highlight the diversity and the utility of this common replication variation.


Assuntos
Replicação do DNA , DNA/metabolismo , Ribonucleotídeos/metabolismo , DNA/química , Genoma , Instabilidade Genômica , Humanos , Ribonuclease H/metabolismo , Ribonucleotídeos/química
7.
Nature ; 511(7508): 251-4, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-24896181

RESUMO

Srs2 helicase is known to dismantle nucleofilaments of Rad51 recombinase to prevent spurious recombination events and unwind trinucleotide sequences that are prone to hairpin formation. Here we document a new, unexpected genome maintenance role of Srs2 in the suppression of mutations arising from mis-insertion of ribonucleoside monophosphates during DNA replication. In cells lacking RNase H2, Srs2 unwinds DNA from the 5' side of a nick generated by DNA topoisomerase I at a ribonucleoside monophosphate residue. In addition, Srs2 interacts with and enhances the activity of the nuclease Exo1, to generate a DNA gap in preparation for repair. Srs2-Exo1 thus functions in a new pathway of nick processing-gap filling that mediates tolerance of ribonucleoside monophosphates in the genome. Our results have implications for understanding the basis of Aicardi-Goutières syndrome, which stems from inactivation of the human RNase H2 complex.


Assuntos
DNA Helicases/metabolismo , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Mutação/genética , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Animais , Linhagem Celular , Dano ao DNA/genética , DNA Helicases/genética , Escherichia coli/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
Mol Cell ; 44(6): 848-50, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22195960

RESUMO

RNA:DNA hybrids in the genome are constantly being generated as a by-product of transcription; in this issue, two papers, from Helmrich et al. (2011) and Wahba et al. (2011), provide insight into how RNA:DNA hybrids lead to genetic instability.

10.
BMC Microbiol ; 11: 214, 2011 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-21951709

RESUMO

BACKGROUND: The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. RESULTS: The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. CONCLUSIONS: Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.


Assuntos
Candida albicans/enzimologia , Candida albicans/genética , Dano ao DNA , DNA Helicases/metabolismo , Proteínas Fúngicas/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , Reparo do DNA/efeitos dos fármacos , Proteínas Fúngicas/genética , Recombinação Homóloga/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia
12.
FEMS Microbiol Lett ; 220(2): 181-6, 2003 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-12670679

RESUMO

Mutants of Salmonella Enteritidis selected following exposure to the sanitizer chlorine or to the preservatives sodium nitrite, sodium benzoate or acetic acid show resistance to multiple antibiotics (tetracycline, chloramphenicol, nalidixic acid, and ciprofloxacin). Complementation experiments with a functional marR restored antibiotic susceptibility of selected mutants to levels similar to wild-type strains, suggesting that mar mutation was responsible for resistance. The multiple antibiotic resistance (mar) operon is a global regulator controlling intrinsic resistance towards structurally and functionally unrelated antibiotics and other noxious agents. Mutants selected after exposure to an inducing agent maintained elevated antibiotic resistance after serial subculture in media void of the inducing agent. Results highlight the importance of monitoring the use of antimicrobial agents to ensure that concentrations capable of inactivating target pathogens are used.


Assuntos
Cloro/farmacologia , Conservantes de Alimentos/farmacologia , Salmonella enteritidis/efeitos dos fármacos , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Genes Bacterianos/genética , Teste de Complementação Genética , Mutação , Salmonella enteritidis/genética , Tetraciclina
13.
J Food Prot ; 65(4): 673-6, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11952218

RESUMO

In this study, the transmission of Escherichia coli O157:H7 to lettuce plants through spray and surface irrigation was demonstrated. For all treatments combined, the number of plants testing positive following a single exposure to E. coli O157: H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following surface irrigation (6 of 32 plants). E. coli O157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E. coli O157:H7 cells. The results of this study suggest that regardless of the irrigation method used, crops can become contaminated; therefore, the irrigation of food crops with water of unknown microbial quality should be avoided.


Assuntos
Cloro/farmacologia , Escherichia coli O157/fisiologia , Lactuca/microbiologia , Agricultura/métodos , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Microbiologia da Água
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