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1.
Sci Rep ; 11(1): 8214, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859263

RESUMO

Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development, homeostasis, and regeneration. Here, we present a method to conditionally induce cre recombination in the mouse calvarial bone while avoiding systemic recombination in distal bones. To test our method, we utilized Prx1creER-egfp;td-Tomato mice and delivered 4-hydroxytamoxifen (4-OHT) to the mouse calvaria, subperiosteally. First, we showed that two calvaria subperiosteal injections of 10 µg of 4-OHT (3.3 mg of 4-OHT/kg of body weight) can induce local recombination as efficiently as two intraperitoneal systemic injections of 200 µg of tamoxifen (70 mg of tamoxifen/kg of body weight). Then, we studied the recombination efficiency of various subperiosteal calvaria dosages and found that two subperiosteal injections of 5 µg 4-OHT (1.65 mg of 4-OHT/kg of body weight) uphold the same recombination efficiency observed with higher dosages. Importantly, the result indicated that the low dosage does not induce significant systemic recombination in remote skeletal tissues. With the proposed local low dosage protocol, the recombination efficiency at the injection site (calvarial bone) reached 94%, while the recombination efficiency at the mandible and the digits was as low as the efficiency measured in control animals.


Assuntos
Integrases/metabolismo , Receptores de Estrogênio/genética , Recombinação Genética/fisiologia , Crânio/metabolismo , Animais , Osso e Ossos/metabolismo , Ativação Enzimática/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Marcação de Genes/métodos , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia
2.
Neurosci Lett ; 742: 135456, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33290837

RESUMO

A bitter substance induces specific orofacial and somatic behavioral reactions such as gapes in mice as well as monkeys and humans. These reactions have been proposed to represent affective disgust, and therefore, understanding the neuronal basis of the reactions would pave the way to understand affective disgust. It is crucial to identify and access the specific neuronal ensembles that are activated by bitter substances, such as quinine, the intake of which induces disgust reactions. However, the method to access the quinine-activated neurons has not been fully established yet. Here, we show evidence that a targeted recombination in active populations (TRAP) method, induces genetic recombination in the quinine-activated neurons in the central nucleus of the amygdala (CeA). CeA is one of the well-known emotional centers of the brain. We found that the intraoral quinine infusion, that resulted in disgust reactions, increased both cFos-positive cells and Arc-positive cells in the CeA. By using Arc-CreER;Ai3 TRAP mice, we induced genetic recombination in the quinine-activated neurons and labelled them with fluorescent protein. We confirmed that the quinine-TRAPed fluorescently-labelled cells preferentially coexpressed Arc after quinine infusion. Our results suggest that the TRAP method can be used to access specific functional neurons in the CeA.


Assuntos
Núcleo Central da Amígdala/metabolismo , Asco , Neurônios/metabolismo , Recombinação Genética/fisiologia , Percepção Gustatória/fisiologia , Paladar/fisiologia , Animais , Núcleo Central da Amígdala/química , Núcleo Central da Amígdala/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/química , Neurônios/efeitos dos fármacos , Quinina/administração & dosagem , Recombinação Genética/efeitos dos fármacos , Sacarina/administração & dosagem , Paladar/efeitos dos fármacos , Percepção Gustatória/efeitos dos fármacos
3.
J Neuroimmunol ; 344: 577245, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32335319

RESUMO

BACKGROUND: The Cre-lox system is a non-dynamic method of gene modification and characterization. Promoters thought to be relatively cell-specific are utilized for generation of cell-lineage-specific gene modifications. METHODS: CD11c.Cre+ITGA4fl/fl mice were generated to abolish the expression of ITGA (α4-integrin) in CD11c+ cells. Ex vivo flow cytometry studies were used to assess the expression of cellular surface markers in different lymphoid compartments and leukocytes subsets after Cre-mediated recombination. RESULTS: A significant reduction of α4-integrin expression among CD11c+- cells was achieved in CD11c.Cre+ITGA4fl/fl mice in primary and secondary lymphoid tissues. A similar reduction in the expression of α4-integrin was also observed in CD11c- cells. CONCLUSION: Cre-lox-mediated cell lineage-specific gene deletion is limited by the transient expression of recombination regulating sequences in hematopoietic cell lines. These methodological issues indicate the need to consider when to employ non-dynamic DNA recombination models in animal models of CNS autoimmunity. An experimental algorithm to address the biological complexities of non-dynamic gene recombination is provided.


Assuntos
Antígeno CD11c/biossíntese , Antígeno CD11c/genética , Linhagem da Célula/fisiologia , Integrinas/biossíntese , Integrinas/genética , Recombinação Genética/fisiologia , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
4.
PLoS Biol ; 17(6): e3000275, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170137

RESUMO

The origin and subsequent maintenance of sex and recombination are among the most elusive and controversial problems in evolutionary biology. Here, we propose a novel hypothesis, suggesting that sexual reproduction not only evolved to reduce the negative effects of the accumulation of deleterious mutations and processes associated with pathogen and/or parasite resistance but also to prevent invasion by transmissible selfish neoplastic cheater cells, henceforth referred to as transmissible cancer cells. Sexual reproduction permits systematic change of the multicellular organism's genotype and hence an enhanced detection of transmissible cancer cells by immune system. Given the omnipresence of oncogenic processes in multicellular organisms, together with the fact that transmissible cancer cells can have dramatic effects on their host fitness, our scenario suggests that the benefits of sex and concomitant recombination will be large and permanent, explaining why sexual reproduction is, despite its costs, the dominant mode of reproduction among eukaryotes.


Assuntos
Recombinação Genética/fisiologia , Reprodução/genética , Reprodução/fisiologia , Animais , Evolução Biológica , Transformação Celular Neoplásica/genética , Eucariotos , Genótipo , Humanos , Recombinação Genética/genética , Seleção Genética/genética , Comportamento Sexual/fisiologia
5.
Sci Rep ; 9(1): 4298, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862832

RESUMO

Human adenovirus (HAdV) group C are the common etiologic in infants with severe acute respiratory infections (SARI). In the study, we report that a novel recombinant HAdV-C group strain (SH2016) was isolated from an infant with SARI in Shanghai in Feb. 4, 2016. The whole-genome sequence of SH2016 strain was generated and compared to other HAdV genomes publicly available. The strain SH2016 genome contains 35,946 nucleotides and coded 40 putative proteins, which was divided into 11 regions. RDP and phylogenetic analyses of the complete genome showed that the SH2016 strain was arranged into a novel subtype and might be recombined with HAdV-1 and HAdV-2. Our finding indicated that the frequent recombination among the HAdV-C group played an important role in driving force for polymorphism of human HAdV-C group prevalent in Shanghai, China. Further epidemiological surveillance of HAdV-C group is necessary to explore whether the novel HAdV-C group will maintain long-term stability. And the pathogenicity and clinical characteristics of the novel HAdV-C group member should be done more.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Infecções por Adenoviridae/virologia , Adenovírus Humanos/classificação , Algoritmos , China , DNA Viral/genética , Genoma Viral/genética , Humanos , Filogenia , Recombinação Genética/genética , Recombinação Genética/fisiologia , Infecções Respiratórias/virologia , Sequenciamento Completo do Genoma
6.
Braz J Med Biol Res ; 49(5): e4738, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27074165

RESUMO

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.


Assuntos
Colite/fisiopatologia , Linfangiogênese/fisiologia , Neovascularização Patológica/fisiopatologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Doença Aguda , Adenoviridae/genética , Animais , Colite/etiologia , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética/fisiologia , Fator C de Crescimento do Endotélio Vascular/fisiologia
7.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;49(5): e4738, 2016. graf
Artigo em Inglês | LILACS | ID: lil-778341

RESUMO

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.


Assuntos
Animais , Feminino , Camundongos , Colite/fisiopatologia , Linfangiogênese/fisiologia , Neovascularização Patológica/fisiopatologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Doença Aguda , Adenoviridae/genética , Colite/etiologia , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Recombinação Genética/fisiologia , Fator C de Crescimento do Endotélio Vascular/fisiologia
8.
Proc Natl Acad Sci U S A ; 112(48): 14864-9, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26578802

RESUMO

The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes.


Assuntos
Cromossomos Humanos/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Homeostase do Telômero/fisiologia , Telômero/metabolismo , Animais , Cromossomos Humanos/genética , Dano ao DNA/fisiologia , DNA Helicases/genética , Células HeLa , Humanos , Camundongos , Recombinação Genética/fisiologia , Telômero/genética
9.
PLoS One ; 9(10): e109910, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25356735

RESUMO

Polyploidy plays a crucial role in plant evolution. Brassica napus (2n = 38, AACC), the most important oil crop in the Brassica genus, is an allotetraploid that originated through natural doubling of chromosomes after the hybridization of its progenitor species, B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC). A better understanding of the evolutionary relationship between B. napus and B. rapa, B. oleracea, as well as Arabidopsis, which has a common ancestor with these three species, will provide valuable information about the generation and evolution of allopolyploidy. Based on a high-density genetic map with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, we performed a comparative genomic analysis of B. napus with Arabidopsis and its progenitor species B. rapa and B. oleracea. Based on the collinear relationship of B. rapa and B. oleracea in the B. napus genetic map, the B. napus genome was found to consist of 70.1% of the skeleton components of the chromosomes of B. rapa and B. oleracea, with 17.7% of sequences derived from reciprocal translocation between homoeologous chromosomes between the A- and C-genome and 3.6% of sequences derived from reciprocal translocation between non-homologous chromosomes at both intra- and inter-genomic levels. The current study thus provides insights into the formation and evolution of the allotetraploid B. napus genome, which will allow for more accurate transfer of genomic information from B. rapa, B. oleracea and Arabidopsis to B. napus.


Assuntos
Brassica napus/genética , Cromossomos de Plantas/genética , Genoma de Planta/fisiologia , Polimorfismo de Nucleotídeo Único , Recombinação Genética/fisiologia , Tetraploidia
10.
Biochim Biophys Acta ; 1843(5): 1002-12, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24418621

RESUMO

RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA (ssDNA) filaments in vitro. However, the precise roles of RecQL5 in the cell remain elusive. Here, we present evidence suggesting that RecQL5 is involved in DNA interstrand crosslink (ICL) repair. Chicken DT40 RECQL5 gene knockout (KO) cells showed sensitivity to ICL-inducing agents such as cisplatin (CDDP) and mitomycin C (MMC) and a higher number of chromosome aberrations in the presence of MMC than wild-type cells. The phenotypes of RECQL5 KO cells resembled those of Fanconi anemia gene KO cells. Genetic analysis using corresponding gene knockout cells showed that RecQL5 is involved in the FANCD1 (BRCA2)-dependent ICL repair pathway in which Rad51-ssDNA filament formation is promoted by BRCA2. The disappearance but not appearance of Rad51-foci was delayed in RECQL5 KO cells after MMC treatment. Deletion of Rad54, which processes the Rad51-ssDNA filament in HR, in RECQL5 KO cells increased sensitivity to CDDP and further delayed the disappearance of Rad51-foci, suggesting that RecQL5 and Rad54 have different effects on the Rad51-ssDNA filament. Furthermore, the frequency and variation of CDDP-induced gene conversion at the immunoglobulin locus were increased in RECQL5 KO cells. These results suggest that RecQL5 plays a role in regulating the incidence and quality of ICL-induced recombination.


Assuntos
DNA/química , Genes Supressores de Tumor , RecQ Helicases/fisiologia , Recombinação Genética/fisiologia , Animais , Linhagem Celular , Galinhas , Técnicas de Silenciamento de Genes
11.
PLoS One ; 8(12): e84379, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349572

RESUMO

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G(2)). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes-two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit-granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.


Assuntos
Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Recombinação Genética/fisiologia , Animais , Cromossomos de Mamíferos/genética , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologia
12.
PLoS One ; 8(12): e85075, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24376867

RESUMO

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.


Assuntos
Aneuploidia , Meiose/fisiologia , Recombinação Genética/fisiologia , Caracteres Sexuais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Técnicas Citológicas , Quebras de DNA de Cadeia Dupla , Feminino , Marcadores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo Sinaptonêmico/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 110(52): 20906-11, 2013 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-24218579

RESUMO

In Escherichia coli, complete unlinking of newly replicated sister chromosomes is required to ensure their proper segregation at cell division. Whereas replication links are removed primarily by topoisomerase IV, XerC/XerD-dif site-specific recombination can mediate sister chromosome unlinking in Topoisomerase IV-deficient cells. This reaction is activated at the division septum by the DNA translocase FtsK, which coordinates the last stages of chromosome segregation with cell division. It has been proposed that, after being activated by FtsK, XerC/XerD-dif recombination removes DNA links in a stepwise manner. Here, we provide a mathematically rigorous characterization of this topological mechanism of DNA unlinking. We show that stepwise unlinking is the only possible pathway that strictly reduces the complexity of the substrates at each step. Finally, we propose a topological mechanism for this unlinking reaction.


Assuntos
Segregação de Cromossomos/fisiologia , Cromossomos Bacterianos/genética , DNA Catenado/química , Escherichia coli/genética , Modelos Biológicos , Recombinação Genética/fisiologia , Segregação de Cromossomos/genética , Proteínas de Escherichia coli/metabolismo , Integrases/metabolismo , Proteínas de Membrana/metabolismo
14.
J Virol ; 87(7): 3699-709, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23325691

RESUMO

Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Instabilidade Genômica/fisiologia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Plasmídeos/fisiologia , Recombinação Genética/fisiologia , Antígenos Virais/metabolismo , Bromodesoxiuridina , Proteínas de Transporte/metabolismo , Imunoprecipitação da Cromatina , Primers do DNA/genética , Proteínas de Ligação a DNA , Eletroforese em Gel de Campo Pulsado , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Instabilidade Genômica/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Sequências Repetidas Terminais/genética , Sequências Repetidas Terminais/fisiologia
15.
Mol Cell ; 47(3): 396-409, 2012 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-22704558

RESUMO

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Instabilidade Genômica/fisiologia , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estresse Fisiológico/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , DNA Helicases/genética , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Osteossarcoma , Ligação Proteica/fisiologia , Recombinação Genética/fisiologia , Troca de Cromátide Irmã/fisiologia , Ubiquitinação/fisiologia
16.
J Biol Chem ; 287(25): 21520-9, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22556412

RESUMO

Immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM) are critical for the maturation of the antibody response. Activation-induced cytidine deaminase (AID) initiates CSR and SHM by deaminating deoxycytidines (dCs) in switch (S) and V(D)J region DNA, respectively, to generate deoxyuracils (dUs). Processing of dUs by uracil DNA glycosylase (UNG) yields abasic sites, which are excised by apurinic/apyrimidinic endonucleases, eventually generating double strand DNA breaks, the obligatory intermediates of CSR. Here, we found that the bivalent iron ion (Fe(2+), ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iµ-C(H) transcripts, and reduced titers of secreted class-switched IgG1, IgG3, and IgA antibodies, without alterations in critical CSR factors, such as AID, 14-3-3γ, or PTIP, or in general germline I(H)-S-C(H) transcription. Fe(2+) did not affect B cell proliferation or plasmacytoid differentiation. Rather, it inhibited AID-mediated dC deamination in a dose-dependent fashion. The inhibition of intrinsic AID enzymatic activity by Fe(2+) was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn(2+), Mn(2+), Mg(2+), or Ni(2+), and the inability of Fe(2+) to inhibit UNG-mediated dU excision. Overall, our findings have outlined a novel role of iron in modulating a B cell differentiation process that is critical to the generation of effective antibody responses to microbial pathogens and tumoral cells. They also suggest a possible role of iron in dampening AID-dependent autoimmunity and neoplastic transformation.


Assuntos
Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Switching de Imunoglobulina/fisiologia , Ferro/metabolismo , Plasmócitos/metabolismo , Recombinação Genética/fisiologia , Animais , Diferenciação Celular/fisiologia , Citidina Desaminase/genética , Imunoglobulina A/genética , Imunoglobulina A/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Camundongos , Plasmócitos/citologia
17.
PLoS One ; 7(4): e33310, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22511919

RESUMO

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.


Assuntos
Proteínas de Bactérias/fisiologia , Helicobacter pylori/enzimologia , Recombinases/fisiologia , Recombinação Genética/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Segregação de Cromossomos/genética , Ciprofloxacina/farmacologia , DNA Topoisomerase IV/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação Puntual , Recombinases/química , Recombinases/genética , Recombinação Genética/genética
18.
J Biol Chem ; 287(4): 2328-41, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22102415

RESUMO

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Pyrococcus furiosus/enzimologia , Recombinação Genética/fisiologia , Trifosfato de Adenosina/genética , Proteínas Arqueais/genética , DNA Arqueal/genética , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Hidrólise , Complexos Multienzimáticos/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Pyrococcus furiosus/genética
19.
J Immunol ; 187(4): 1826-34, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21742970

RESUMO

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.


Assuntos
Quebra Cromossômica , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Fenômenos Imunogenéticos/fisiologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Deleção de Sequência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo
20.
J Cell Biochem ; 112(10): 2672-82, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21647941

RESUMO

Across the evolutionary spectrum, living organisms depend on high-fidelity DNA replication and recombination mechanisms to maintain genome stability and thus to avoid mutation and disease. The repair of severe lesions in the DNA such as double-strand breaks or stalled replication forks requires the coordinated activities of both the homologous recombination (HR) and DNA replication machineries. Growing evidence indicates that so-called "accessory proteins" in both systems are essential for the effective coupling of recombination to replication which is necessary to restore genome integrity following severe DNA damage. In this article we review the major processes of homology-directed DNA repair (HDR), including the double Holliday Junction (dHJ), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and error-free lesion bypass pathways. Each of these pathways involves the coupling of a HR event to DNA synthesis. We highlight two major classes of accessory proteins in recombination and replication that facilitate HDR: Recombination mediator proteins exemplified by T4 UvsY, Saccharomyces cerevisiae Rad52, and human BRCA2; and DNA helicases/translocases exemplified by T4 Gp41/Gp59, E. coli DnaB and PriA, and eukaryotic Mcm2-7, Rad54, and Mph1. We illustrate how these factors help to direct the flow of DNA and protein-DNA intermediates on the pathway from a double-strand break or stalled replication fork to a high-fidelity recombination-dependent replication apparatus that can accurately repair the damage.


Assuntos
Replicação do DNA/genética , Instabilidade Genômica/genética , Recombinação Genética/fisiologia , Animais , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Humanos , Recombinação Genética/genética
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