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1.
PLoS Genet ; 15(11): e1008486, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738749

RESUMO

To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in Caenorhabditis elegans. Mutants of the CRL4 complex (cul-4, ddb-1, and gad-1) show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Endodesoxirribonucleases/genética , Recombinação Homóloga/genética , Ligases/genética , Complexo Sinaptonêmico/genética , Animais , Caenorhabditis elegans/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Células Germinativas/crescimento & desenvolvimento , Meiose/genética , Prófase Meiótica I/genética
2.
Nucleic Acids Res ; 47(19): 10202-10211, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31504832

RESUMO

The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/genética , Genoma Bacteriano/genética , Plasmídeos/genética , RNA/genética , DNA Polimerase Dirigida por RNA , Vibrio vulnificus/genética
3.
Nat Commun ; 10(1): 4388, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558727

RESUMO

Meiosis is a conserved tenet of sexual reproduction in eukaryotes, yet this program is seemingly absent from many extant species. In the human fungal pathogen Candida albicans, mating of diploid cells generates tetraploid products that return to the diploid state via a non-meiotic process of depolyploidization known as concerted chromosome loss (CCL). Here, we report that recombination rates are more than three orders of magnitude higher during CCL than during normal mitotic growth. Furthermore, two conserved 'meiosis-specific' factors play central roles in CCL as SPO11 mediates DNA double-strand break formation while both SPO11 and REC8 regulate chromosome stability and promote inter-homolog recombination. Unexpectedly, SPO11 also promotes DNA repair and recombination during normal mitotic divisions. These results indicate that C. albicans CCL represents a 'parameiosis' that blurs the conventional boundaries between mitosis and meiosis. They also reveal parallels with depolyploidization in mammalian cells and provide potential insights into the evolution of meiosis.


Assuntos
Candida albicans/genética , Diploide , Recombinação Homóloga/genética , Meiose/genética , Tetraploidia , Candidíase/microbiologia , Cromossomos Fúngicos/genética , Dano ao DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Mitose/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação
4.
BioDrugs ; 33(5): 503-513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385197

RESUMO

The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , RNA Guia
5.
Nucleic Acids Res ; 47(17): 8988-9004, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31299084

RESUMO

Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Genoma Fúngico , Humanos , Complexo Mediador/genética , Modelos Moleculares , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
6.
Oncol Rep ; 42(3): 1133-1140, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322271

RESUMO

A 42­year­old woman presented with ~30 adenomatous polyps of the left sided­colon with early rectosigmoid cancer. The patient had no previous medical history and no familial history of inherited colorectal disease. No germline gene mutations associated with colorectal adenomatous polyposis, including APC regulator of WNT signaling pathway, mutY DNA glycosylase, DNA polymerase­Îµ, catalytic subunit, DNA polymerase δ1, catalytic subunit, and mismatch repair genes, were detected via germline genetic testing. A heterozygous germline mutation in methyl­CpG binding domain 4, DNA glycosylase (MBD4), c.217C>T/p.Gln73*, which resulted in the generation of a stop codon, was identified by genetic analyses including whole­exome sequencing. Immunohistochemical staining analysis revealed that the expression of MBD4 protein was absent in the cancer tissue, while it was expressed in the normal epithelium. Sequencing and copy­number analyses demonstrated the loss of the remaining allele of MBD4 in the cancer tissue. Furthermore, somatic mutation signature analysis showed preferential transition of cytosine to thymine residues at CpG dinucleotides in cancer tissues. Although it has been previously reported that germline missense mutations and somatic mutations of MBD4 are associated with the development of colorectal cancer, this is the first report, to the best of our knowledge, in which a germline nonsense mutation of the MBD4 gene has been identified in an early­onset colorectal cancer patient with oligopolyposis.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Endodesoxirribonucleases/genética , Mutação em Linhagem Germinativa , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/patologia , Adulto , Idade de Início , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Feminino , Testes Genéticos , Humanos , Masculino , Linhagem , Prognóstico
7.
Mol Cell ; 75(5): 1020-1030.e4, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31350119

RESUMO

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.


Assuntos
Bacteriófagos/fisiologia , DNA Viral/metabolismo , Escherichia coli/virologia , Ilhas Genômicas , Montagem de Vírus/fisiologia , DNA Viral/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Genetics ; 212(4): 1259-1278, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31248887

RESUMO

Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammatory responses that can lead to tissue degeneration. Simpler metazoan models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathologies. Here, we show that in a DNase II-defective Caenorhabditis elegans strain, persistent cytoplasmic DNA leads to systemic tissue degeneration and loss of tissue functionality due to impaired proteostasis. These pathological outcomes can be therapeutically alleviated by restoring protein homeostasis, either via ectopic induction of the ER unfolded protein response or N-acetylglucosamine treatment. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.


Assuntos
DNA/imunologia , Retículo Endoplasmático/metabolismo , Imunidade Inata , Proteostase , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo
9.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
10.
J Biol Chem ; 294(26): 10120-10130, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31073030

RESUMO

In Saccharomyces cerevisiae, Tel1 protein kinase, the ortholog of human ataxia telangiectasia-mutated (ATM), is activated in response to DNA double-strand breaks. Biochemical studies with human ATM and genetic studies in yeast suggest that recruitment and activation of Tel1ATM depends on the heterotrimeric MRXMRN complex, composed of Mre11, Rad50, and Xrs2 (human Nbs1). However, the mechanism of activation of Tel1 by MRX remains unclear, as does the role of effector DNA. Here we demonstrate that dsDNA and MRX activate Tel1 synergistically. Although minimal activation was observed with 80-mer duplex DNA, the optimal effector for Tel1 activation is long, nucleosome-free DNA. However, there is no requirement for DNA double-stranded termini. The ATPase activity of Rad50 is critical for activation. In addition to DNA and Rad50, either Mre11 or Xrs2, but not both, is also required. Each of the three MRX subunits shows a physical association with Tel1. Our study provides a model of how the individual subunits of MRX and DNA regulate Tel1 kinase activity.


Assuntos
DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nucleossomos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
11.
PLoS One ; 14(4): e0215479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022206

RESUMO

Deoxyribonucleases (DNases) might play a role in prevention of autoimmune conditions such as systemic lupus erythematosus through clearance of cell debris resulting from apoptosis and/or necrosis. Previous studies have suggested that variations in the in vivo activities of DNases I-like 3(1L3) and II have an impact on autoimmune-related conditions. The genes for these DNases are known to show copy number variations (CNVs) whereby copy loss leads to a reduction of the in vivo activities of the enzymes, thereby possibly affecting the pathophysiological background of autoimmune diseases. Using a simple newly developed quantitative real-time PCR method, we investigated the distributions of the CNVs for DNASE1L3 and DNASE2 in Japanese and German populations. It was found that only 2 diploid copy numbers for all of these DNASE CNVs was distributed in both of the study populations; no copy loss or gain was evident for any of the autoimmune-related DNase genes. Therefore, it was demonstrated that these human autoimmune-related DNase genes show low genetic diversity of CNVs resulting in alterations of the in vivo levels of DNase activity.


Assuntos
Autoimunidade/genética , Variações do Número de Cópias de DNA , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , Heterogeneidade Genética , Doenças Autoimunes , Alemanha , Humanos , Japão
12.
Genetics ; 212(2): 431-443, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31015193

RESUMO

During meiosis, formation of double-strand breaks (DSBs) and repair by homologous recombination between homologs creates crossovers (COs) that facilitate chromosome segregation. CO formation is tightly regulated to ensure the integrity of this process. The DNA damage response kinases, Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) have emerged as key regulators of CO formation in yeast, flies, and mice, influencing DSB formation, repair pathway choice, and cell cycle progression. The molecular networks that ATM and ATR influence during meiosis are still being resolved in other organisms. Here, we show that Caenorhabditis elegans ATM and ATR homologs, ATM-1 and ATL-1 respectively, act at multiple steps in CO formation to ultimately ensure that COs are formed on all chromosomes. We show a role for ATM-1 in regulating the choice of repair template, biasing use of the homologous chromosome instead of the sister chromatid. Our data suggest a model in which ATM-1 and ATL-1 have antagonistic roles in very early repair processing, but are redundantly required for accumulation of the RAD-51 recombinase at DSB sites. We propose that these features of ATM-1 and ATL-1 ensure both CO formation on all chromosomes and accurate repair of additional DSBs.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Segregação de Cromossomos/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Reparo de DNA por Recombinação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Caenorhabditis elegans/metabolismo , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Rad51 Recombinase/metabolismo
13.
FEBS Open Bio ; 9(6): 1137-1143, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31001930

RESUMO

Abdominal arterial aneurysm (AAA) shares many features with autoimmune diseases and appears to be a T-cell-mediated process. In addition, certain epigenetic changes, including DNA methylation, are associated with AAA. In this study, we investigated epigenetic modifications in regulatory T cells (Tregs) from AAA patients. We used flow cytometry to sort FOXP3+ CD4+ CD25+ Tregs from the peripheral blood of AAA patients and from healthy controls (HC), and then detected DNA methylation and histone modifications by ELISA. The DNA methylation rate of Tregs was significantly higher in AAA patients than in the HC group (0.159 ± 0.08% vs 0.098 ± 0.03%, P < 0.05), while the acetylation rates of H3 and H3K9 histones were lower in the AAA than in the HC group. We also examined the expression of mRNA encoding enzymes that catalyze making and removing epigenetic modifications by real-time PCR: we found that mRNA levels of DNA methyltransferase (DNMT) 1 and DNMT3A were higher in the AAA than in the HC group, mRNA levels of methyl-CpG-binding domain protein (MBD) 2 and MBD4 were higher in the AAA than in the HC group (MBD2: 6.21 ± 2.57 vs 3.04 ± 1.45; MBD4: 7.76 ± 3.48 vs 4.97 ± 3.10; both P < 0.05), and mRNA levels of histone deacetylase (HDAC) 1 and HDAC5 were significantly up-regulated in the AAA compared with the HC group (HDAC1: 2.17 ± 1.18 vs 1.51 ± 0.99; HDAC5: 1.35 ± 0.49 vs 0.94 ± 0.76; both P < 0.05). Together, our results reveal that rates of DNA methylation and histone modifications of Tregs are significantly altered in AAA patients.


Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/genética , Epigênese Genética/genética , Linfócitos T Reguladores/fisiologia , Acetilação , Idoso , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Feminino , Histona Desacetilase 1/genética , Histona Desacetilases/genética , Histonas/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Regulação para Cima/genética
14.
Nucleic Acids Res ; 47(8): 4136-4152, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30892613

RESUMO

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , Reparo do DNA , DNA Bacteriano/química , Endodesoxirribonucleases/química , Proteínas de Escherichia coli/química , Geobacillus stearothermophilus/enzimologia , ortoaminobenzoatos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Cinética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Thermotoga maritima/química , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , ortoaminobenzoatos/metabolismo
15.
Genes (Basel) ; 10(3)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893921

RESUMO

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.


Assuntos
Cromossomos Humanos/genética , Replicação do DNA , Endonucleases/genética , Neoplasias/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Resolvases de Junção Holliday/genética , Humanos , Recombinases/genética , Estresse Fisiológico
16.
J Biol Chem ; 294(19): 7931-7941, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30894417

RESUMO

A recently identified and widely prevalent prokaryal gene cluster encodes a suite of enzymes with imputed roles in nucleic acid repair. The enzymes are as follows: MPE, a DNA endonuclease; Lhr-Core, a 3'-5' DNA helicase; LIG, an ATP-dependent DNA ligase; and Exo, a metallo-ß-lactamase-family nuclease. Bacterial and archaeal MPE proteins belong to the binuclear metallophosphoesterase superfamily that includes the well-studied DNA repair nucleases Mre11 and SbcD. Here, we report that the Pseudomonas putida MPE protein is a manganese-dependent DNA endonuclease that incises either linear single strands or the single-strand loops of stem-loop DNA structures. MPE has feeble activity on duplex DNA. A crystal structure of MPE at 2.2 Å resolution revealed that the active site includes two octahedrally coordinated manganese ions. Seven signature amino acids of the binuclear metallophosphoesterase superfamily serve as the enzymic metal ligands in MPE: Asp33, His35, Asp78, Asn112, His124, His146, and His158 A swath of positive surface potential on either side of the active site pocket suggests a binding site for the single-strand DNA substrate. The structure of MPE differs from Mre11 and SbcD in several key respects: (i) MPE is a monomer, whereas Mre11 and SbcD are homodimers; (ii) MPE lacks the capping domain present in Mre11 and SbcD; and (iii) the topology of the ß sandwich that comprises the core of the metallophosphoesterase fold differs in MPE vis-à-vis Mre11 and SbcD. We surmise that MPE exemplifies a novel clade of DNA endonuclease within the binuclear metallophosphoesterase superfamily.


Assuntos
Proteínas de Bactérias , Endodesoxirribonucleases , Manganês , Família Multigênica , Pseudomonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Manganês/química , Manganês/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Pseudomonas/genética
17.
J Biol Chem ; 294(19): 7632-7643, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30885940

RESUMO

dsDNA breaks (DSBs) are resected in a 5'→3' direction, generating single-stranded DNA (ssDNA). This promotes DNA repair by homologous recombination and also assembly of signaling complexes that activate the DNA damage checkpoint effector kinase Chk1. In fission yeast (Schizosaccharomyces pombe), genetic screens have previously uncovered a family of three xeroderma pigmentosum G (XPG)-related nucleases (XRNs), known as Ast1, Exo1, and Rad2. Collectively, these XRNs are recruited to a euchromatic DSB and are required for ssDNA production and end resection across the genome. Here, we studied why there are three related but distinct XRN enzymes that are all conserved across a range of species, including humans, whereas all other DSB response proteins are present as single species. Using S. pombe as a model, ChIP and DSB resection analysis assays, and highly efficient I-PpoI-induced DSBs in the 28S rDNA gene, we observed a hierarchy of recruitment for each XRN, with a progressive compensatory recruitment of the other XRNs as the responding enzymes are deleted. Importantly, we found that this hierarchy reflects the requirement for different XRNs to effect efficient DSB resection in the rDNA, demonstrating that the presence of three XRN enzymes is not a simple division of labor. Furthermore, we uncovered a specificity of XRN function with regard to the direction of transcription. We conclude that the DSB-resection machinery is complex, is nonuniform across the genome, and has built-in fail-safe mechanisms, features that are in keeping with the highly pathological nature of DSB lesions.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , DNA Fúngico/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , Humanos , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
18.
Viruses ; 11(3)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841485

RESUMO

Herpesvirus infection is an orderly, regulated process. Among these viruses, the encapsidation of viral DNA is a noteworthy link; the entire process requires a powered motor that binds to viral DNA and carries it into the preformed capsid. Studies have shown that this power motor is a complex composed of a large subunit, a small subunit, and a third subunit, which are collectively known as terminase. The terminase large subunit is highly conserved in herpesvirus. It mainly includes two domains: the C-terminal nuclease domain, which cuts the viral concatemeric DNA into a monomeric genome, and the N-terminal ATPase domain, which hydrolyzes ATP to provide energy for the genome cutting and transfer activities. Because this process is not present in eukaryotic cells, it provides a reliable theoretical basis for the development of safe and effective anti-herpesvirus drugs. This article reviews the genetic characteristics, protein structure, and function of the herpesvirus terminase large subunit, as well as the antiviral drugs that target the terminase large subunit. We hope to provide a theoretical basis for the prevention and treatment of herpesvirus.


Assuntos
Endodesoxirribonucleases/metabolismo , Infecções por Herpesviridae/tratamento farmacológico , Herpesviridae/efeitos dos fármacos , Herpesviridae/enzimologia , Subunidades Proteicas/metabolismo , Proteínas Virais/metabolismo , Empacotamento do DNA , DNA Viral , Desenvolvimento de Medicamentos , Endodesoxirribonucleases/genética , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/genética , Proteínas Virais/genética , Montagem de Vírus
19.
Immunogenetics ; 71(5-6): 433-436, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30714079

RESUMO

There is currently no effective treatment for metastasised uveal melanoma (UM). Recently, it was reported that a UM patient was responsive to checkpoint inhibitor (CI) treatment, due to a high tumour mutation burden correlated with a germline loss-of-function MBD4 mutation. Here, we report on another UM patient who carried an MBD4 germline nonsense variant (p.Leu563Ter) and her tumour showed a fivefold higher than average mutation burden. We confirmed the association between germline loss-of-function variant in MBD4 and CI response. The patient experienced stable disease (10 months) and survived 2 years with metastatic disease, which is twice as long as median survival. Additionally, the frequency of MBD4 loss-of-function variants in reported UM cohorts was > 20 times higher than in an aggregated population genome database (P < 5 × 10-5), implying a potential role as UM predisposition gene. These findings provide a strong basis for the inclusion of MBD4 in the screening of potential UM-prone families as well as stratification of immunotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Códon sem Sentido , Endodesoxirribonucleases/genética , Mutação em Linhagem Germinativa , Melanoma/tratamento farmacológico , Melanoma/genética , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/genética , Alelos , Substituição de Aminoácidos , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Ipilimumab/administração & dosagem , Melanoma/diagnóstico , Resultado do Tratamento , Neoplasias Uveais/diagnóstico
20.
Mol Cell ; 73(4): 727-737.e3, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30709710

RESUMO

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Edição de Genes/métodos , Integrases/genética , Pareamento de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Integrases/metabolismo , Motivos de Nucleotídeos , Especificidade por Substrato
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