RESUMEN
BACKGROUND: A wide variety of DNA lesions interfere with replication and transcription, leading to mutations and cell death. DNA repair mechanisms act upon these DNA lesions present in the genomic DNA. To investigate a DNA repair mechanism elaborately, an in vitro DNA repair substrate containing DNA lesions at a specific site is required. Previously, to prepare the substrate, phagemid ssDNA and DNA lesion-harboring oligonucleotides were employed with considerable amounts of DNA polymerase and DNA ligase. However, preparing in vitro DNA repair substrate in general is difficult and labor intensive. RESULTS: Here, we modified the construction method of in vitro mismatch repair substrate using a nicking-endonuclease, which produces gap corresponding to the ssDNA in the plasmid DNA, and swaps DNA lesion-containing oligonucleotide upon addition of restriction enzyme and T5 exonuclease. This modified method is able to produce in vitro DNA repair substrates containing adenine:cytosine mismatch basepair, 8-oxoG, and uracil. The DNA repair enzyme, each Fpg, hOGG1 could cleave an 8-oxoG-containing DNA substrate, the mixture of UDG and APE1 could cleave a uracil-containing DNA substrate. Omitting a column purification step, DNA repair substrates were prepared by one-pot synthesis. CONCLUSIONS: We were able to prepare in vitro DNA repair substrates using this simple method involving restriction enzymes and T5 exonuclease. It is anticipated that this method, termed as "Oligo Swapping Method", will be valuable for understanding the DNA repair machinery.
RESUMEN
Novel proteins were isolated from the sera of Chinese Mamushi (Gloydius blomhoffi brevicaudus) and Habu (Trimeresurus flavoviridis). The primary structures of these proteins were determined by protein sequencing, and the nucleotide sequences were established by cDNA cloning from the liver mRNAs. They belonged to the fetuin family having a double-headed cystatin-like domain and a His-rich domain, akin to HSF, an antihemorrhagic factor isolated from Habu serum. They showed no antihemorrhagic activity and were designated HSF-like proteins (HLPs). Mamushi serum contained two different HLPs termed HLP-A and HLP-B. Both HLP-A and Habu HLP had a unique 17-residue deletion in their His-rich domains. HLP-B comprised two glycosylated polypeptide chains and inhibited the precipitation of calcium phosphate as potently as does bovine fetuin. HLP-B was hence identified as a snake fetuin. The phylogenetic analysis of the fetuin family of proteins showed that antihemorrhagins and HLPs have evolved from this snake fetuin.
Asunto(s)
Venenos de Crotálidos/química , Proteínas de Reptiles/química , alfa-Fetoproteínas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Fosfatos de Calcio/química , Bovinos , Clonación Molecular , ADN Complementario/química , Hígado/metabolismo , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas de Reptiles/genética , Proteínas de Reptiles/aislamiento & purificación , Alineación de Secuencia , Viperidae , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.
Asunto(s)
Proteínas Sanguíneas/genética , Evolución Molecular , Proteínas de Secreción Prostática/genética , Trimeresurus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/química , Secuencia Conservada , ADN Complementario/genética , Disulfuros/química , Japón , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Polimorfismo de Nucleótido Simple , Señales de Clasificación de Proteína , Selección Genética , Homología de Secuencia de AminoácidoRESUMEN
Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Venenos de Crotálidos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Cromatografía en Gel , Venenos de Crotálidos/química , Venenos de Crotálidos/genética , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Protones , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Trimeresurus , UltrafiltraciónRESUMEN
An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.
Asunto(s)
Proteínas Sanguíneas/farmacología , Hemorragia/prevención & control , Viperidae , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , China , Clonación de Organismos , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Cartilla de ADN , ADN Complementario/química , Hemorragia/inducido químicamente , Japón , Ratones , Datos de Secuencia MolecularRESUMEN
Three small serum proteins (SSP-1, -2, and -3), with molecular masses of 6.5-10kDa, were isolated from Habu (Trimeresurus flavoviridis) serum, and the amino acid sequences were determined by protein and cDNA analysis. Despite only limited sequence identity to any mammalian prostatic secretory protein of 94 amino acids (PSP94), all of the Cys residues in these SSPs were well conserved. SSPs are the first PSP94 family proteins to be identified in reptiles. SSP-1 and -3 weakly inhibited the proteolytic activity of a snake venom metalloproteinase. On the other hand, SSP-2 formed a tight complex with triflin, a snake venom-derived Ca(2+) channel blocker that suppresses the smooth muscle contraction. This suggests a role for SSP-2 in the self defense system of venomous snakes.
Asunto(s)
Proteínas de Secreción Prostática/metabolismo , Trimeresurus/sangre , Secuencia de Aminoácidos , Animales , Canales de Calcio/metabolismo , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , ADN Complementario/metabolismo , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Conejos , Factores de TiempoRESUMEN
Certain snakes have antihemorrhagic proteins in their sera. Habu serum factor (HSF), an antihemorrhagic protein isolated from the serum of the Japanese habu snake (Trimeresurus flavoviridis) is composed of two cystatin-like domains (D1 and D2) and a His-rich domain, and it inhibits several snake venom metalloproteinases (SVMPs). The activity of HSF can be abolished by trinitrophenylation of Lys residues with 2,4,6-trinitrobenzene sulphonic acid. Upon complex formation of HSF with SVMP, however, the loss of its inhibitory activity by the chemical modification was suppressed, and Lys(15), Lys(41), and Lys(103) residues in HSF were not trinitrophenylated. In order to identify the domain that is critical to the inhibitory activity on SVMPs, native HSF was digested with papain followed by cleavage with cyanogen bromide, yielding a low-molecular mass fragment that was composed of two peptide chains (residues 5-89 and 312-317) linked by a disulfide bond. This fragment inhibited several SVMPs and showed significant antihemorrhagic activity. This indicates that the N-terminal half of D1 is indispensable for the antihemorrhagic activity of HSF. Furthermore, a three-dimensional model of two cystatin-like domains constructed by the homology modeling has indicated that three Lys residues (15, 41, and 103) are exposed to the same surface of HSF molecule.
Asunto(s)
Proteínas Sanguíneas/análisis , Modelos Moleculares , Fragmentos de Péptidos/farmacología , Trimeresurus/sangre , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacología , Biología Computacional , Bromuro de Cianógeno , Cartilla de ADN , Espectrometría de Masas , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Datos de Secuencia Molecular , Papaína , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Análisis de Secuencia de ADNRESUMEN
Habu serum factor (HSF) is a metalloproteinase inhibitor that is isolated from the serum of habu snake (Trimeresurus flavoviridis), and it can suppress snake venom-induced hemorrhage. In the present study, the inhibitory property and fundamental structure of HSF were analyzed in detail. HSF inhibited all the hemorrhagic and most of the non-hemorrhagic metalloproteinases tested from the venoms of T. flavoviridis and Gloydius halys brevicaudus. HSF was extremely stable in a broad range of temperature and pH, and the treatments with a temperature of 100 degrees C or pH ranging from 1 to 13 barely affects its reactivity against G. halys brevicaudus H6 protease. Gel filtration chromatography revealed that HSF binds to the H6 protease with a 1:1 molar ratio. A secondary structure profile of HSF that was monitored by circular dichroism spectrum remained unvaried up to 2 M urea. The activity of HSF was stoichiometrically abolished by chemical modification with 2,4,6-trinitrobenzene sulfonic acid and N-bromosuccinimide; this indicates that Lys and Trp residues in its sequence play a role in the inhibitory mechanism. In this study, the amino acid sequence of HSF that was obtained by cDNA cloning was identical to that reported previously, except for five substitutions. We concluded that these discrepancies reflect a difference in the places of capture of the snake specimens.