RESUMO
DNA double-strand breaks (DSBs) which occurs in cells after ionizing radiation (IR) or chemical agents are the most dangerous lesions in eukariotic cells, which leads to cell death or chromosome abberations and cancer. One of the earliest response of cells to DSBs formation is phosphorylation by 139 serine of core variant histone H2AX in megabase chromatin domains around DSB (gamma-H2AX), which amplify signal and makes it possible to identify even one DSB in genome. Effective formation of gamma-H2AX is very important for maintenance of genome stability. Here, using immunofluorescent and Western blotting techniques, we studied dynamics of gamma-H2AX formation in human lymphocytes of various individuals irradiated ex vivo. We have found that dynamics of gamma-H2AX formation in lymphocytes differ between individuals but have similar kinetics and statistically is independent on people age.
Assuntos
Cromossomos Humanos/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Histonas/metabolismo , Linfócitos/metabolismo , Raios X/efeitos adversos , Adulto , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos da radiaçãoRESUMO
Covalent modifications of histones in chromatin play an important role in regulation of eukaryotic gene expression and DNA repair. Formation of double-strand breaks (DSBs) in DNA is followed by the rapid local phosphorylation of the C-terminal serine in the replacement histone H2AX in megabase chromatin domains around DSBs and formation of discrete nuclear foci called gammaH2AX foci. This epigenetic modification of chromatin represents the "histone code" for DNA damage signaling and repair and has been extensively studied during last decade. It is known that after DSB rejoining gammaH2AX foci are eliminated from the nucleus, but molecular mechanism of this elimination remains to be established. However, gammaH2AX elimination can serve as a useful marker of DSB repair in normal cells and tissues. In this paper the available data on kinetics and possible mechanisms of gammaH2AX elimination are reviewed.
Assuntos
Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Humanos , Cinética , Modelos Genéticos , FosforilaçãoRESUMO
Forskolin is a natural compound found in the coleus herb that activates the enzyme adenylate cyclase and increases the concentration of intracellular cyclic AMP (cAMP). This chemical is widely used as a stimulating food additive. It is unknown whether forskolin can effect cellular responses to ionizing radiation, such as induction of phosphorylation of histone H2AX (gamma-H2AX) in megabase chromatin domains near DNA double-strand breaks (DSBs). Here we report that treatment with forskolin decreases H2AX phosphorylation after irradiation detected by immunoblotting or by analysis of the overall gamma-H2AX-associated fluorescence in the nuclei. However, this chemical does not affect the number of gamma-H2AX foci, the frequency of radiation-induced chromosome aberrations, or cell survival after X irradiation, which is consistent with the view that it does not change the induction of repair of DSBs. We suggest that the overall decrease of H2AX phosphorylation after treatment with forskolin in irradiated cells reflects a lesser extent of apparent H2AX modification at individual DSBs that may be caused by inhibition of the initial spread of gamma-H2AX and/or by stimulation of elimination of gamma-H2AX from chromatin after DSB rejoining.
Assuntos
Colforsina/farmacologia , Histonas/química , Histonas/fisiologia , Animais , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos da radiação , Cromatina/química , Aberrações Cromossômicas/efeitos da radiação , Cricetinae , Cricetulus , Humanos , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estrutura Terciária de Proteína , Radiação Ionizante , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Published data are reviewed on the mechanism of inheritance of repressive epigenetic modifications of chromatin, histone and DNA, which suggest that they depend on RNA. RNA is transcribed from most of genome compartments and when it forms duplex structures because of overlapping transcription or because of resynthesis by RNA-dependent RNA polymerase, heterochromatin is generated. Mitotic phosphorylation of Ser-10 in histone H3 stimulates transcription of heterochromatin and its recovery in the next cell cycle by RNA interference.
Assuntos
Cromatina/metabolismo , Epigênese Genética , RNA/fisiologia , Animais , Metilação de DNA , Heterocromatina/metabolismo , Histonas/metabolismo , RNA/genética , Interferência de RNA , Transcrição GênicaRESUMO
Different fragments of promoters of deoxyribonuclease II (DNAse II) and calcium-modulating cyclophilin ligand (CAML) associated with Alu family repeats have been inserted into a luciferase reporter vector. These constructions were introduced into A549 and HEK293 cell lines and after transient transfection we lysed cells and analysed luciferase activities in these lysates. It has been shown that Alu repeats localized in constructions influence expression of luciferase. Therefore, Alu copies which are associated with cis-regulatory modules of protein-coding genes have biological activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Elementos Alu/genética , Retroelementos/genética , Linhagem Celular , Endodesoxirribonucleases/genética , Regulação da Expressão Gênica , Humanos , Transcrição GênicaRESUMO
Using computer-based methods we determined the global distribution of short interspersed nuclear elements (SINEs) in the human and mouse X chromosomes. It has been shown that this distributions is similar to the distributions of CpG islands and genes but is different from the distribution of LINE1 elements. Since SINEs (human Alu and mouse B2) may have binding sites for Polycomb protein YY1, we suggest that these repeats can serve as additional signals ("boosters") in Polycomb-dependent silencing of gene rich segments during X inactivation.
Assuntos
Elementos Nucleotídeos Curtos e Dispersos/genética , Cromossomo X/genética , Elementos Alu/genética , Animais , Processamento Eletrônico de Dados , Feminino , Heterocromatina/metabolismo , Humanos , Mamíferos/genética , Camundongos , Elementos Nucleotídeos Curtos e Dispersos/fisiologia , Cromossomo X/metabolismo , Inativação do Cromossomo X , Fator de Transcrição YY1/metabolismoRESUMO
We have used FISH with microdissected chromosome arm-specific DNA probes that allow direct visualization of chromosome 1 folding in human sperm nuclei. We described the overall topology and the mode of packaging of sperm chromosome 1 and thus established new elements of ordered genome architecture in these specialized cells.
Assuntos
Núcleo Celular/genética , Cromossomos Humanos Par 1/metabolismo , Espermatozoides/citologia , Cromossomos Humanos Par 1/ultraestrutura , Genoma , Humanos , Masculino , Microscopia de FluorescênciaRESUMO
The synaptic vesicle cycle sustains neurotransmission and keeps pace between exo- and endocytosis in synapses. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in fission of clathrin-coated vesicles from the presynaptic membrane during endocytosis. The present study addresses the effect of the non-hydrolysable GTP analog, GTPgammaS, on the assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPgammaS induced distinct ultrastructural changes in synapses: the number of synaptic vesicles at active zones was reduces, and the number of docked vesicles was increased; at the same time the number of clathrin-coated intermediates at the synaptic endocytic zone was increased, indicating that synaptic vesicle recycling was inhibited. Clathrin-coated intermediates with unusual shape were found. At low concentrations of GTPgammaS they were represented by long tubules decorated by spirals containing dynamin and clathrin-coated vesicles on the top. At high concentrations of GTPgammaS the tubulular structures were shorted and branched. The pitch of the spiral and tubule's diameter were significantly reduced (23.1 +/- 0.4 and 19.0 +/- 0.5 nm, respectively, as compared to those at low concentration of GTPgammaS, 26.6 +/- 0.4 and 23.3 +/- 0.4 nm; P < 0.001). We suggest that these structural changes correspond to distinct steps in the fission reaction. A model is proposed. It implies that the fast GTP hydrolysis leads to an increase in length of the spiral due to the straightening of the dynamin dimmers, composing the spiral. This leads to a fast increase both in the pitch and the diameter of the helix. The shift in diameter breaks the local hydrophobic interactions between the inner and the outer leaflets of the lipid membrane at the sites of dynamin binding. Stretching of the spiral leads to an expansion of the neck in the longitudinal direction and promotes severing of the membrane that subsequently results in the release of the clathrin-coated vesicle.
Assuntos
Axônios/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Membranas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Animais , Axônios/ultraestrutura , Células Cultivadas , Dinaminas/metabolismo , Endocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/administração & dosagem , Lampreias , Microinjeções , Medula Espinal/citologia , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Postreplication DNA repair was studies in three strains of Micrococcus luteus having different sensitivity to ultraviolet light: a wild type ATCC 5698, a ultraviolet-sensitive mutant G7, deficient in the incision step of repair and in ultraviolet-resistant transformant obtained from G7 by treatment with DNA of wild type cells, Trf(G7). It is shown that the G7 mutant has a low capacity for repair of postreplication DNA gaps compared with the wild type or Trf(G7). It seems to be that postreplication repari capacity contributes significantly to the ultraviolet resistance of M. luteus in addition to the excision repair. In contrast with G7 the size of the DNA fragments synthesized immediately after ultraviolet irradiation in the wild type (and Trf(G7)) seems to be much higher than that expected if each dimer produces one DNA gap in the daughter strand. Since this cannot only be explained by the excision of dimers from parental DNA we have suggested that a rapid repair of postreplication DNA gap occurs in M. luteus.
Assuntos
Reparo do DNA/efeitos da radiação , Micrococcus/efeitos da radiação , Centrifugação com Gradiente de Concentração , Cloranfenicol/farmacologia , Replicação do DNA , Dinitrofenóis/farmacologia , Genes , Micrococcus/metabolismo , Peso Molecular , Mutação , Raios UltravioletaRESUMO
The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination which ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one.
Assuntos
Reparo do DNA , DNA Bacteriano/efeitos da radiação , Endonucleases/farmacologia , Micrococcus/efeitos da radiação , Matemática , Dímeros de Pirimidina , Raios UltravioletaRESUMO
In ultraviolet-irradiated Micrococcus luteus wild type the replication of DNA was not interrupted at every pyrimidine dimer, in contrast to that in ultraviolet-sensitive G7 and some other mutants. The contribution of uninterrupted replication to the ultraviolet resistance of M. luteus proved to be equal to the contributions of excision repair and inducible postreplication repair. It was found that some postreplication gaps could be filled by constitutive pathways of postreplication repair when inducible pathways were suppressed by chloramphenicol. Prolonged treatment with chloramphenicol was shown to block not only inducible repair but also other processes essential for ultraviolet irradiation survival.
Assuntos
Reparo do DNA , Micrococcus/efeitos da radiação , Tolerância a Radiação , Cloranfenicol/farmacologia , Replicação do DNA , Raios UltravioletaRESUMO
Available data on possible genetic impacts of mammalian retroposons are reviewed. Most important is the growing number of established examples showing the involvement of retroposons in modulation of expression of protein-coding genes transcribed by RNA polymerase II (Pol II). Retroposons contain conserved blocks of nucleotide sequence for binding of some important Pol II transcription factors as well as sequences involved in regulation of stability of mRNA. Moreover, these mobile genes provide short regions of sequence homology for illegitimate recombinations, leading to diverse genome rearrangements during evolution. Therefore, mammalian retroposons representing a significant fraction of noncoding DNA cannot be considered at present as junk DNA but as important genetic symbionts driving the evolution of regulatory networks controlling gene expression.
Assuntos
Regulação da Expressão Gênica , Mamíferos/genética , Retroelementos , Elementos Alu , Animais , Sequência de Bases , DNA/genética , Evolução Molecular , Expressão Gênica , Rearranjo Gênico , Genoma Humano , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , RNA Polimerase II/genética , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
In some vertebrates tandem repeats (TTAGGG)n are located not only in telomeres, but also in intrachromosomal sites. In Chinese hamster cells such interstitial repeats which may be called "telomeric" heterochromatin (THC), representing up to 5% of genome. Earlier we have shown, that blocks of THC dynamically bind telomeric protein TRF1 in Chinese hamster cells. In this work question has been studied whether this interaction depends on a transcription. In cells with the normal transcription around 85% of initial fluorescence intensity of GFP-TRF1 is restored in 60 sec after the photobleaching. Treatment of the cells with transcription inhibitor actinomycin D (ActD) in the concentration completely inhibiting activity of DNA-dependent RNA polymerases I and II (RPI and RPII) leads to fast and practically full suppression of exchange GFP-TRF1 (10% of initial fluorescence is restored only) whereas an inhibitor of protein synthesis cycloheximide (CHD) has not effect. At the low ActD concentration, suppressing only RPI, efficiency of recovery of fluorescence was not changed. Since some fractions of heterochromatin in mammalian cells are actively transcribed, exchange of GFP-TRF 1 can be connected to transcription of THC which may be necessary for synthesis of small interfering RNA and self-maintenance of the heterochromatin, or with inhibition of expression of other genes effecting TRF1 stability.
Assuntos
Cromossomos/metabolismo , Heterocromatina/metabolismo , Repetições Minissatélites/fisiologia , RNA Polimerase I/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Cricetinae , Cricetulus , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , RNA Polimerase I/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacosRESUMO
The deletion (D) allele of the human ACE gene is associated with higher ACE activity than the insertion (I) allele. There is controversy as to whether the ACE genotype may be associated with elite athletic status; recent studies have identified no significant associations amongst those drawn from mixed sporting disciplines. However, such lack of association may reflect the mixed nature of such cohorts, given that an excess frequency of the I allele has been reported amongst elite endurance athletes, and an excess of the D allele amongst those engaged in more power-orientated sports. We examined this hypothesis by determining ACE I/D allele frequency amongst 217 Russian athletes (swimmers, skiers, triathletes and track-and-field participants) prospectively stratified by performance ('outstanding' or 'average'), and the duration of their event (SDA (<1 min), MDA (1 to 20 min), and LDA (>20 min): short, middle and long distance athletes respectively). ACE genotype and allele frequencies were compared to 449 controls. ACE genotype frequency amongst the whole cohort, or the outstanding athletes alone, was no different to that amongst sedentary controls. However, there was an excess of the D allele (frequency 0.72, P=0.001) amongst the outstanding SDA group, and an excess of the I allele (frequency 0.63, P=0.032) amongst the outstanding MDA group. These findings were replicated in the outstanding swimmers, with track and field SDA similarly demonstrating an excess of the D allele (P=0.01). There was no association found between the outstanding LDA and ACE genotype (P=0.27). These data not only confirm an excess of the D allele in elite SDA, and I allele in elite MDA, but also offer an explanation as to why any such association may be hard to detect amongst a heterogeneous cohort of mixed athletic ability and discipline.
Assuntos
Peptidil Dipeptidase A/genética , Aptidão Física , Polimorfismo Genético/genética , Adulto , Alelos , Feminino , Frequência do Gene , Humanos , Masculino , Federação Russa , EsportesRESUMO
Escherichia coli recA+ and recA- cells were co-transformed with a mixture of pMB9 (Tcr) and pST8-26 (Apr, pBR325 derivative) plasmid DNAs followed by selection on plates containing both tetracycline and ampicillin. A set of stable Tcr Apr derivatives was isolated from these transformants. Many of the stable Tcr Apr segregants contained fused pMB9::pST8-26 plasmids with lengths that were about 0.8 kb longer than the sum of the lengths of the parental plasmids; one plasmid (pTF8) was about 1.5 kb shorter. The fusion was not stimulated by UV irradiation of co-transformants and occurred both in recA+ and recA- genetic backgrounds. Restriction analysis of the fused plasmids showed the two replicons were in the same relative orientation, and also indicated unique points of fusion in most cases (in 9 out of 10) which are localised within the 1.6-kb regions around the replication origins (RO). Because the fusion of plasmids of the type used in this study was not described before we have tentatively named it RO-fusion (Replication-Origin-fusion).
Assuntos
Escherichia coli/genética , Plasmídeos , Recombinação Genética , Transformação Bacteriana , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , DNA BacterianoRESUMO
Human retrotransposons, Alu-family DNA repeats (AFRs), have variable nucleotide sequence but conservative short elements, which may have important functions, are also present. In our previous reports we have described human nuclear DNA-binding protein interacting with AFRs and evidence was presented that the protein recognizes sequence motif 5'-GGAGGC-3' which is conserved in the spacer of RNA polymerase III promoter of AFRs and in the SV40 T-antigen-dependent replication origin of AFRs. In this study it was found that double-stranded synthetic oligonucleotides containing indicated conservative sequences of AFRs actually have high-affinity binding site for HeLa nuclear protein. The data suggest that non-infected human cells contain nuclear DNA-binding protein which recognizes the conservative sequence motif of AFRs - GGAGGC.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase III/genética , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Escherichia coli/genética , Células HeLa/análise , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Sequências Repetitivas de Ácido NucleicoRESUMO
Internal promoters of some genes transcribed by RNA polymerase III (e.g. tRNA genes, adenovirus VA1 RNA gene, human retroposons of the Alu family) contain a conserved sequence element, B-box, interacting with basal transcription factor TFIIIC2 which initiates assembly of the full transcription complex on the genes, and which represents the major determinant of the efficiency of their expression. In this study we have identified in human nuclear extracts a protein which interacts with VA1 B-box DNA and forms a high-affinity complex which is very stable after the addition of a large excess of competitor DNA. Unlike TFIIIC2, the B-box-binding activity of the B-box-binding protein is found to be decreased in adenovirus 5-transformed human cells. In these cells (line 293) increased transcription of VA1 and tRNA genes in vivo and in vitro was previously detected by other workers. Our results suggest that besides TFIIIC2, an additional B-box-binding protein factor may be involved in the regulation of expression of the RNA polymerase III-transcribed genes.
Assuntos
Adenovírus Humanos/genética , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Fatores de Transcrição TFIII , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Ligação Competitiva , Linhagem Celular Transformada , Núcleo Celular/química , Transformação Celular Viral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese/métodos , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Human cells contain a nuclear protein interacting with Alu repeats, and this protein seems to recognize a conserved sequence motif, GGAGGC, present within the RNA polymerase III promoter and within the SV40 T-antigen-dependent ARS-like element. To study the potential functional role of this element, we have inserted the sequence into a chloramphenicolacetyltransferase (CAT) expression vector with a SV40 promoter and enhancer element from the up-stream region of the human c-myc gene, and transfected HeLa cells with the resulting plasmid. Analysis of expression by the CAT assay indicates that the Alu-derived sequence supresses transcription of the CAT gene driven by the c-myc enhancer/SV40 promoter. The Alu-derived sequence also inhibits ARS activity of the c-myc enhancer. The data allow the explanation of the transcriptional inactivity of Alu repeats in HeLa cells, and suggest the existence of a negative control of Alu transcription.
Assuntos
Replicação do DNA , Elementos de DNA Transponíveis , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proto-Oncogenes , Sequências Repetitivas de Ácido Nucleico , Transcrição Gênica , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc , RNA Polimerase III/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Nuclear protein which selectively binds to the Alu-family DNA repeat (AFR, Blur8) is partially purified from human HeLa cells using a gel retention assay. At low protein concentrations only a single complex of the protein with AFR is formed (CII). Increasing protein concentrations lead to the gradual disappearance of CII, being replaced by complexes with higher (CI) and lower (CIII, CIV) electrophoretic mobilities. Differential binding of AFR restriction subfragments indicates that multiple protein-binding sites are present within AFR. We discuss two models explaining the anomalous electrophoretic mobility of CII by DNA bending or looping upon cooperative multi-site binding of the protein to AFR.
Assuntos
DNA , Sequências Repetitivas de Ácido Nucleico , Antígenos Transformantes de Poliomavirus/genética , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/metabolismo , Enzimas de Restrição do DNA , Eletroforese em Gel de Poliacrilamida , Células HeLa/análise , Humanos , Proteínas Nucleares/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
DNA repair is an important factor of stability of pro- and eukaryotic genomes which plays a central role in mutagenesis and carcinogenesis. Genetic control of nucleotide excision repair (NER) in mammalian cells is well studied, but little is known about molecular mechanisms of postreplication repair (PRR) which allows bypass of base lesions in template strands after DNA replication. In Saccharomyces cerevisiae PRR is controlled by the RAD61RAD18 pathway which involves POL30 gene encoding proliferating cell nuclear antigen (PCNA), and in human cells PCNA is known to be closely associated with the newly replicated chromatin where PRR probably takes place. In UV-irradiated human cells distinct PCNA foci may be detected in some cells which accumulate phosphorylated breast cancer susceptibility protein BRCA1 and another protein BARD1. Human PCNA is also known to be phosphorylated after UV-irradiation. In this study we found that the known inhibitor of protein kinases staurosporine supresses PRR in NER-deficient cells which is consistent with the view that BRCA1 and PCNA are required for PRR. We also have shown that the distinct PCNA foci in UV-irradiated NER-deficient cells are actually associated with the newly replicated chromatin. Since RAD18 protein is not essential for normal DNA replication and directly controls PRR in yeast, we analysed whether this protein as well as its human homologs (HR18A and HR18B) have common domains with BRCA1 and BARD1. It is found that HR18A has a subregion of homology to BARD1 and HR18A-to BRCA1. Taken together the results indicate that BRCA1 and BARD1 may be involved in PRR in human cells.