共查询到20条相似文献,搜索用时 46 毫秒
1.
S Kimura M Kai H Kobayashi A Suzuki H Morioka E Otsuka K Sakaguchi 《Canadian Metallurgical Quarterly》1997,25(24):4970-4976
A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative. 相似文献
2.
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication. 相似文献
3.
DK Nagi WC Knowler V Mohamed-Ali PH Bennett JS Yudkin 《Canadian Metallurgical Quarterly》1998,21(1):127-133
Starting with crude yeast mitochondria, the intron homing endonuclease, I-SecIV, was purified to near homogeneity. This highly purified enzyme differs from some other well-characterized yeast mitochondrial intron-encoded endonucleases in terms of its structure and DNA cleavage specificity. The enzyme is a heterodimer with a native molecular mass of 92 kDa. A small catalytic subunit (32 kDa) is probably encoded largely or entirely by intron 5 alpha of the cytochrome oxidase subunit I gene. A larger polypeptide subunit (60 kDa) may be a nuclear factor necessary for intron mobility. I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates. Analysis of kinetic parameters shows that the purified enzyme has a very high affinity for DNA and exhibits low turnover which may have implications for subsequent steps in the intron homing process. 相似文献
4.
We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight. 相似文献
5.
TP Hilbert RJ Boorstein HC Kung PH Bolton D Xing RP Cunningham GW Teebor 《Canadian Metallurgical Quarterly》1996,35(8):2505-2511
We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny. 相似文献
6.
Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH. 相似文献
7.
PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Single-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a single-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer/template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions. 相似文献
8.
I Poggesi R Spinelli E Frigerio M Strolin Benedetti L Carra D Sassella R Rimoldi 《Canadian Metallurgical Quarterly》1997,40(2):291-294
Mitochondrial DNA (mtDNA) suffers extensive damage from the environment, however, the enzymes involved in the repair of mtDNA are still unknown. Here, we partially purified mitochondrial endonuclease G (Endo G) from the bovine heart, and examined the action of Endo G on damaged DNA. Treatment of DNA with L-ascorbic acid or peplomycin, that introduces single-strand breaks through active oxygen radicals, greatly enhanced the susceptibility to nucleolytic attacks from Endo G. The enzyme cleaved at or near sites where single-strand breaks were present in the opposite strand. Cisplatin-mediated DNA damage, which causes intrastrand crosslinks between adjacent guanine residues, also facilitated Endo G digestion, indicating that the enzyme can recognize local distortions in the duplex DNA introduced by adducts. These nucleolytic properties of Endo G in vitro suggest its possible involvement in the maintenance of mtDNA by eliminating defective genomes from the multicopy pool. 相似文献
9.
Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substrates, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRNA(Leu), exactly at the 5' end of the mature molecules. The activity was abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. The most abundant polypeptides in the purified enzyme fractions have molecular masses of about 100, 44 and 35 kDa. The enzyme requires divalent cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structural requirements for the purified enzyme were analyzed with different tRNA precursor models. The analysis of the derivatives of tRNA(Leu) precursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzyme. 相似文献
10.
The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed that CET1 is essential for yeast cell growth. The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe. 相似文献
11.
An NADPH-dependent succinic semialdehyde reductase has been purified from bovine brain by several chromatographic procedures. The preparation appeared homogeneous on SDS/PAGE. The enzyme is a monomeric protein with a molecular mass of 28 kDa. A number of properties of the bovine brain enzyme, such as substrate specificity, specific activity, molecular mass, optimum pH, amino acid composition, and kinetic parameters, have been determined and compared with those reported for preparations from other sources. The results indicate that the enzyme isolated from bovine brain in the present study is different from those reported for preparations from other sources. The inhibition kinetic patterns obtained when the products of the reaction or substrate analogs are used as inhibitor of the reaction catalyzed by the enzyme are consistent with an ordered sequential mechanism involving the formation of an intermediate ternary complex and in which NADPH is the first substrate to bind the enzyme. 相似文献
12.
RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates. 相似文献
13.
We have previously shown that in developing chicken embryos and differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs through the replacement of 5-methylcytosine by cytosine (Jost, J. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4685-4688; Jost, J. P. & Jost, Y.C. (1994) J. Biol. Chem. 269, 10040-10043). We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from 12-day-old chicken embryos. The enzyme copurifies with a mismatch-specific thymine-DNA glycosylase and an apyrimidic-endonuclease. The reaction product of the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified apyrimidic-endonuclease activity cleaves 3' from the apyrimidic sugar. A 52.5-kDa peptide, isolated as a single band from preparative SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase activities. 5-Methylcytosine-DNA glycosylase has an apparent pI of 5.5-7.5 and maximal activity between pH 6.5 and 7.5. The Km for hemimethylated oligonucleotide substrate is 8 x 10(-8) M with a Vmax of 4 x 10(-11) mol/h/micrograms proteins. 5-Methylcytosine-DNA glycosylase binds equally well to methylated and non-methylated DNA. The enzyme reacts six times faster with the hemimethylated DNA than with the same bifilarly methylated DNA sequence, and single-stranded methylated DNA is not a substrate. The action of the enzyme is distributive. 相似文献
14.
Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B 总被引:2,自引:0,他引:2
The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S. Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207-212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An alpha-agarase, i.e. specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium. In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues. 相似文献
15.
Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II 总被引:4,自引:0,他引:4
A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone. 相似文献
16.
VS Mikhailov AL Mikhailova M Iwanaga S Gomi S Maeda 《Canadian Metallurgical Quarterly》1998,72(4):3107-3116
A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3'-->5' exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication. 相似文献
17.
MA Kerzhner SA Shiryaev LA Zheleznaya NI Matvienko 《Canadian Metallurgical Quarterly》1997,62(8):883-889
Screening of thermophilic bacterial strains revealed a strain containing site-specific endonuclease BspMKI. This endonuclease was purified to functional homogeneity during sequential chromatographic steps. The enzyme recognizes sequence 5'-G decreases TCGAC-3' on DNA molecule and is isoschizomer of endonuclease SalI. The molecular mass of BspMKI is about 45 kD. The enzyme is maximally active at 55 degrees C and MRB (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol) is the optimal buffer. The enzyme is highly stable and retains its activity during two weeks at room temperature. 相似文献
18.
19.
A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl. 相似文献
20.
A stable DNA/protein complex having an apparent molecular mass of approximately 150 kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions. 相似文献