RESUMO
The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Polimorfismo Genético , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/metabolismo , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Polimerase I/química , DNA Polimerase I/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Hidrólise , Cinética , Modelos Químicos , Sondas de Oligonucleotídeos/química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Especificidade por Substrato , TemperaturaRESUMO
DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/metabolismo , RNA/metabolismo , Thermus thermophilus/enzimologia , Thermus/enzimologia , Sequência de Aminoácidos , Archaea/enzimologia , Sequência de Bases , Clonagem Molecular , Cristalografia por Raios X , Interleucina-6/genética , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Quaternária de Proteína , RNA/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Moldes GenéticosRESUMO
The 5'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Dados de Sequência Molecular , Fosfodiesterase I , Diester Fosfórico Hidrolases/genética , Análise de Sequência , Especificidade por SubstratoRESUMO
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
Assuntos
DNA/metabolismo , Sondas de Oligonucleotídeos , Polimorfismo de Fragmento de Restrição , Archaeoglobus fulgidus/genética , Bacteriófago M13/genética , DNA/isolamento & purificação , Endonucleases/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Leucócitos/metabolismo , Modelos Biológicos , Mutagênese Insercional , Pyrococcus furiosus/genética , Espectrometria de FluorescênciaRESUMO
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element.
Assuntos
Genes Fúngicos , Genes Letais , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Transcrição Gênica , Alelos , Composição de Bases , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA Fúngico/genética , DNA Fúngico/metabolismo , Expressão Gênica , Genes Recessivos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Homologia de Sequência do Ácido NucleicoRESUMO
The promoters of vertebrate and yeast U6 small nuclear RNA genes are structurally dissimilar, although both are recognized by RNA polymerase III. Vertebrate U6 RNA genes have exclusively upstream promoters, while the U6 RNA gene from the yeast Saccharomyces cerevisiae (SNR6) has internal and downstream promoter elements that match the tRNA gene intragenic A- and B-block elements, respectively. Substitution of the SNR6 A or B block greatly diminished U6 RNA accumulation in vivo, and a subcellular extract competent for RNA polymerase III transcription generated nearly identical DNase I protection patterns over the SNR6 downstream B block and a tRNA gene intragenic B block. We conclude that the SNR6 promoter is functionally similar to tRNA gene promoters, although the effects of extragenic deletion mutations suggest that the downstream location of the SNR6 B block imposes unique positional constraints on its function. Both vertebrate and yeast U6 RNA genes have an upstream TATA box element not normally found in tRNA genes. Substitution of the SNR6 TATA box altered the site of transcription initiation in vivo, while substitution of sequences further upstream had no effect on SNR6 transcription. We present a model for the SNR6 transcription complex that explains these results in terms of their effects on the binding of transcription initiation factor TFIIIB.
Assuntos
Genes Fúngicos , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Alelos , Animais , Sequência de Bases , Sondas de DNA , Desoxirribonuclease I , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , RNA Fúngico/genética , Deleção de Sequência , TATA Box , Fator de Transcrição TFIIIB , Fatores de Transcrição/metabolismo , VertebradosRESUMO
Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.