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1.
Cell Rep ; 20(7): 1553-1562, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28813668

RESUMO

Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with it.


Assuntos
Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
2.
PLoS One ; 11(12): e0168873, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28033325

RESUMO

Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.


Assuntos
Proteínas de Plantas/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribose/metabolismo , Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sequência de Bases , Cromatografia de Fase Reversa , Metilação , Mutação Puntual , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
3.
PLoS One ; 11(12): e0168832, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28036383

RESUMO

The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.


Assuntos
Aspergillus oryzae/enzimologia , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação/fisiologia , Catálise , Domínio Catalítico/fisiologia , Cinética , Alinhamento de Sequência , Especificidade por Substrato
4.
Genes Dev ; 30(11): 1327-38, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27298336

RESUMO

R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.


Assuntos
Expressão Gênica , Genoma Fúngico/genética , Poli A/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mapeamento Cromossômico , DNA Fúngico/metabolismo , Genômica , Histonas/metabolismo , Poli A/química , Poli A/metabolismo , Conformação Proteica , RNA Fúngico/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
5.
Biosci Biotechnol Biochem ; 80(9): 1681-92, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27151561

RESUMO

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T1 (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S1 (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man10GlcNAc2 and Man11GlcNAc2 were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.


Assuntos
Aspergillus oryzae/enzimologia , Biotecnologia , Fermentação , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Japão , Ribonuclease T1/isolamento & purificação , Ribonuclease T1/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/isolamento & purificação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Tripsinogênio/metabolismo
6.
Biosens Bioelectron ; 83: 169-76, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27125839

RESUMO

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo.


Assuntos
DNA de Cadeia Simples/metabolismo , Fabaceae/enzimologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/análise , Endonucleases Específicas para DNA e RNA de Cadeia Simples/antagonistas & inibidores , Endonucleases Específicas para DNA e RNA de Cadeia Simples/análise , Espectrometria de Fluorescência/métodos , Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/metabolismo , Fabaceae/efeitos dos fármacos , Fabaceae/crescimento & desenvolvimento , Fluorescência , Grafite/química , Limite de Detecção , Óxidos/química , Proteínas de Plantas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
7.
Mol Cell Biochem ; 412(1-2): 131-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26699908

RESUMO

SAW1 is required for efficient removal by the Rad1-Rad10 nuclease of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break (DSB) repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing. Saw1 was shown in vitro to bind flaps with high affinity, but displayed diminished affinity when flaps were short (<30 deoxynucleotides [nt]), consistent with it not being required for short flap cleavage. Accordingly, this study, using in vivo fluorescence microscopy showed that SAW1 was not required for recruitment of Rad10-YFP to DNA DSBs during their repair by SSA when the flaps were ~10 nt. In contrast, recruitment of Rad10-YFP to DSBs when flaps were ~500 nt did require SAW1 in G1 phase of cell cycle. Interestingly, we observed a substantial increase in colocalization of Saw1-CFP and Rad10-YFP at DSBs when short flaps were formed during repair, especially in G1, indicating significant recruitment of Saw1 despite there being no requirement for Saw1 to recruit Rad10. Saw1-CFP was seldom observed at DSBs without Rad10-YFP. Together, these results support a model in which Saw1 and Rad1-Rad10 are recruited as a complex to short and long flaps in all phases of cell cycle, but that Saw1 is only required for recruitment of Rad1-Rad10 to DSBs when long flaps are formed in G1.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Quebras de DNA de Cadeia Dupla
8.
Anal Biochem ; 494: 76-81, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26548958

RESUMO

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.


Assuntos
Técnicas de Química Analítica/métodos , Ensaio de Imunoadsorção Enzimática , Poli Adenosina Difosfato Ribose/análise , Anticorpos/imunologia , Dano ao DNA , Desoxirribonuclease I/metabolismo , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribose/imunologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ensaio de Radioimunoprecipitação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Ácido Tricloroacético/química
9.
Cell Mol Biol Lett ; 20(4): 549-61, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26124052

RESUMO

Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-ß sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-ß DHFR sequence. We show that the topologically closed DHFR ori-ß replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação , Tetra-Hidrofolato Desidrogenase/genética , Sequência Rica em At , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cricetulus , Replicação do DNA , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plasmídeos/química , Plasmídeos/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
10.
J Vet Med Sci ; 77(9): 1163-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25843154

RESUMO

Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5-28.4- and 10.1-208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Cadeia Dupla/genética , Infecções por Rotavirus/veterinária , Rotavirus/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/virologia , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/virologia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia
11.
Anal Chim Acta ; 866: 84-9, 2015 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-25732696

RESUMO

As an important antitumor drug, bleomycin (BLM) is widely used in the treatment of a variety of cancers. In addition, nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancer development. Thus, the development of BLM and nuclease detection methods is of great significance in cancer therapy and related biological mechanism research. Here, a WS2 nanosheet-based turn-on fluorescent sensing platform for simple, fast and sensitive detection of BLM and nuclease was reported. WS2 nanosheet exhibits different affinity toward ssDNA with different length and excellent fluorescence quenching ability. A fluorescein (FAM)-labeled long ssDNA could be adsorbed on the surface of WS2 nanosheet and the fluorescence was therefore quenched. In the presence of BLM·Fe(II) or S1 nuclease (a ssDNA-specific nuclease which was used as a model enzyme), an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis. Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface were then produced, resulting in a weak fluorescence quenching after mixing WS2 nanosheets. Thus, the fluorescence signal was restored. The proposed sensor displays a wide linear range and a high sensitivity with a detection limit of 0.3 nM for BLM and 0.01 U mL(-1) for S1 nuclease. It also exhibits a good performance in complex biological samples. This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.


Assuntos
Bleomicina/análise , Nanoestruturas/química , Endonucleases Específicas para DNA e RNA de Cadeia Simples/análise , Espectrometria de Fluorescência , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Hidrólise , Limite de Detecção , Oxirredução , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
12.
Anal Biochem ; 468: 34-8, 2015 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-25263815

RESUMO

S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uµl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uµl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis.


Assuntos
Proteínas Fúngicas/análise , Nanopartículas Metálicas , Endonucleases Específicas para DNA e RNA de Cadeia Simples/análise , DNA de Cadeia Simples/metabolismo , Difosfatos/farmacologia , Inibidores Enzimáticos/farmacologia , Estudos de Viabilidade , Corantes Fluorescentes , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Nanopartículas Metálicas/química , Oligonucleotídeos , Poli C , Prata , Endonucleases Específicas para DNA e RNA de Cadeia Simples/antagonistas & inibidores , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Espectrometria de Fluorescência/métodos
13.
Appl Microbiol Biotechnol ; 99(3): 1145-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25472432

RESUMO

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.


Assuntos
Proteínas Fúngicas/metabolismo , Penicillium/enzimologia , Penicillium/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Reatores Biológicos/microbiologia , Carboidratos/análise , Células Imobilizadas/enzimologia , Células Imobilizadas/metabolismo , Carvão Vegetal , Meios de Cultura/química , Fermentação , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Oxigênio/análise , Penicillium/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
14.
Nucleic Acids Res ; 42(22): 13456-68, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25398895

RESUMO

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.


Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Animais , Células Cultivadas , Proteínas Fúngicas/metabolismo , Camundongos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ribonuclease H/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Estereoisomerismo , Temperatura Ambiente
15.
PLoS One ; 9(10): e108401, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25285445

RESUMO

The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg · mL(-1) Lambda DNA, and 50 nM target ssDNA in 5 µg · mL(-1) Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes.


Assuntos
DNA de Cadeia Simples/análise , Corantes Fluorescentes , Proteínas Fúngicas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Coloração e Rotulagem/métodos , Pareamento Incorreto de Bases/genética , Sequência de Bases , DNA de Cadeia Simples/genética , DNA Viral/análise , DNA Viral/genética , Fluorescência
16.
PLoS One ; 9(7): e103491, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25058678

RESUMO

Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.


Assuntos
DNA de Cadeia Simples/metabolismo , Microfluídica/métodos , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase/métodos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Análise de Sequência de DNA
17.
J Am Soc Mass Spectrom ; 25(7): 1136-45, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24845355

RESUMO

Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Bases , HIV-1/química , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Espectrometria de Massas por Ionização por Electrospray
18.
Biochem Biophys Res Commun ; 445(3): 602-7, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24565838

RESUMO

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Deleção de Genes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/análise , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
19.
Mol Cell ; 52(1): 63-74, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24119400

RESUMO

Homology-dependent repair of double-strand breaks (DSBs) from nonsister templates has the potential to generate loss of heterozygosity or genome rearrangements. Here we show that the Saccharomyces cerevisiae Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage. A role for Yen1 is only apparent in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair of a single DSB, suggesting that the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules (JMs) accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the mus81Δ mutant contain a single Holliday junction (HJ) explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize an early strand exchange intermediate and direct repair to noncrossover or crossover outcomes, respectively.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , RNA Helicases DEAD-box/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Resolvases de Junção Holliday/metabolismo , Mutação , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Fatores de Tempo
20.
EMBO J ; 32(3): 461-72, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23299942

RESUMO

The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3' tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3'-flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3' tailed substrates in vivo and in vitro, and enhances 3' tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3' tail removal in recombination.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Imunoprecipitação da Cromatina , Primers do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Mutagênese , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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