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1.
The past year has seen a coming-of-age in DNA enzyme research. Far from being laboratory curiosities, the activities of new DNA enzymes have broadened the known catalytic repertoire of nucleic acid enzymes, provided valuable insights into different mechanistic possiblities open to nucleic acid catalysts, and explored the importance for catalysis of native functionalities within DNA and RNA, as well as of a diversity of extrinsic cofactors. Thus, the first amino acid cofactor-utilizing DNA enzyme has been described, as well as DNA enzymes that cleave RNA without the assistance of any external cofactor. On the practical side, the most efficient RNA-cleaving nucleic acid enzyme described to date is a DNA enzyme. 相似文献
2.
DNA unwinding enzyme II of Escherichia coli. 2. Characterization of the DNA unwinding activity. 总被引:2,自引:0,他引:2
The DNA-stimulated 75000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme - DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells. 相似文献
3.
Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II 总被引:2,自引:0,他引:2
T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme. 相似文献
4.
Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreover, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme. 相似文献
5.
以Lineweave-Burk plot双倒数作图法测得该酶对底物S-腺苷酰甲硫氨酸(SAM)的K_m=7.69×10~(-6)mol/L,在1mmol/LS-腺苷酰高半胱氨酸(SAH)存在下,Ki=7.33×10~(-4)mol/L,两条直线相交于纵轴,证明SAH是该酶的竞争性抑制剂。该酶最适pH为7.8,对热不稳定。同时还测定了该酶对不同DNA底物的专一性及盐浓度、代谢相关物’两价阳离子、某些酸根等对该酶调节性质的影响。以碘代乙酰胺修饰该酶的SH基’及用二硫苏糖醇(DTT)和巯基乙醇(MSH)保护该酶SH基所作的实验表明SH基是该酶活性中心所必需的,用高效液相色谱(HPLC)法证明该酶所甲基化的碱基为刘氏小球菌(M·L、DNA)分子中的胞嘧啶,且求得甲基化30min后所得甲基化水平为2.39%。同时也证明当用该酶将λDNA甲基化后,可使BamHI限制性核酸内切酶对甲基化后的λDNA丧失切割作用。 相似文献
6.
Eukaryotic DNA polymerases in DNA replication and DNA repair 总被引:16,自引:0,他引:16
Peter M. J. Burgers 《Chromosoma》1998,107(4):218-227
DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair.
Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures
to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the
cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to
some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA
polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication
and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint
control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both
leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA
polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment
maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase
β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that
function.
Received: 20 April 1998 / Accepted: 8 May 1998 相似文献
7.
A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG. 相似文献
8.
Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication 总被引:23,自引:0,他引:23
L Blanco A Bernad J M Lázaro G Martín C Garmendia M Salas 《The Journal of biological chemistry》1989,264(15):8935-8940
The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication. 相似文献
9.
Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA 总被引:1,自引:0,他引:1
F Palitti D Carotti S Grünwald M Rispoli E P Whitehead C Salerno R Strom D Drahovsky 《Biochimica et biophysica acta》1987,910(3):292-296
The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme. 相似文献
10.
Dillingham MS Webb MR Kowalczykowski SC 《The Journal of biological chemistry》2005,280(44):37069-37077
We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed. 相似文献
11.
A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation. 相似文献
12.
The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA 总被引:1,自引:0,他引:1
Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme. 相似文献
13.
DNA gyrase: structure and function. 总被引:27,自引:0,他引:27
DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme. 相似文献
14.
《Critical reviews in biochemistry and molecular biology》2013,48(3-4):335-375
AbstractDNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents and as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme. 相似文献
15.
Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase 总被引:14,自引:0,他引:14
The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA. 相似文献
16.
The polA6 mutation is an allele of the polA gene of Escherichia coli which produces a DNA polymerase I species readily distinguishable from that produced by the wild type allele. Experiments described here show that this enzyme has an altered pH optimum for polymerization and a lower binding affinity for DNA. The defect clearly lies within the carboxyl-terminal large fragment of the enzyme produced by in vivo or in vitro proteolysis since the fragment has the same pH optimum for polymerization as the intact enzyme. The polA6 enzyme and its fragment are more sensitive to phosphate ions than the wild type polymerase, and the large fragment is less efficient at binding poly d(AT) in in vitro binding assays. Although the specific nucleolytic activity of the polA6 enzyme is higher than that of the wild type, there is no apparent alteration in pH optimum for the hydrolysis of eigher double or single stranded DNA. 相似文献
17.
During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA. In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA. How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme. We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands. The type I restriction enzyme is able to destroy the ‘naked’ DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA. 相似文献
18.
Chemiluminescent detection of DNA: application for DNA sequencing and hybridization. 总被引:7,自引:4,他引:3
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A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range. 相似文献
19.
The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA. 相似文献
20.
Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage. 总被引:4,自引:3,他引:1
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Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage. 相似文献