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1.
Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages
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Synthetic oligodeoxyribonucleotides containing CpG-dinucleotides (CpG DNA) in specific sequence contexts activate the vertebrate immune system. We have examined the effect of 3′-deoxy-2′–5′-ribonucleoside (3′-deoxynucleoside) incorporation into CpG DNA on the immunostimulatory activity. Incorporation of 3′-deoxynucleosides results in the formation of 2′–5′-internucleotide linkages in an otherwise 3′–5′-linked CpG DNA. In studies, both in vitro and in vivo, CpG DNA containing unnatural 3′-deoxynucleoside either within the CpG-dinucleotide or adjacent to the CpG-dinucleotide failed to induce immunostimulatory activity, suggesting that the modification was not recognized by the receptors. Incorporation of the same modification distal to the CpG-dinucleotide in the 5′-flanking sequence potentiated the immunostimulatory activity of the CpG DNA. The same modification when incorporated in the 3′-flanking sequence had an insignificant effect on immunostimulatory activity of CpG DNA. Interestingly, substitution of a 3′-deoxynucleoside in the 5′-flanking sequence distal to the CpG-dinucleotide resulted in increased IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. The incorporation of the same modification in the 3′-flanking sequence resulted in lower IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. These results suggest that site-specific incorporation of 3′-deoxynucleotides in CpG DNA modulates immunostimulatory properties. 相似文献
2.
Characterization of intracellular DNA strand breaks induced by neocarzinostatin in Escherichia coli cells. 总被引:1,自引:1,他引:0
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DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed. 相似文献
3.
Repair of DNA strand gaps and nicks containing 3'-phosphate and 5'-hydroxyl termini by purified mammalian enzymes.
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A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-phosphate and 3'-hydroxyl termini. To further assess this possibility, we compared the activity of the 3'-phosphatase of purified calf thymus polynucleotide kinase towards a variety of substrates. The rate of removal of 3'-phosphate groups from nicked or short (1 nt) gapped sites in double-stranded DNA was observed to be similar to that of 3'-phosphate groups from single-stranded substrates. Thus this activity of polynucleotide kinase does not appear to be influenced by steric accessibility of the phosphate group. We subsequently demonstrated that the concerted reactions of polynucleotide kinase and purified human DNA ligase I could efficiently repair DNA nicks possessing 3'-phosphate and 5'-hydroxyl termini, and similarly the combination of these two enzymes together with purified rat DNA polymerase beta could seal a strand break with a 1 nt gap. With a substrate containing a nick bounded by 3'- and 5'-OH termini, the rate of gap filling by polymerase beta was significantly enhanced in the presence of polynucleotide kinase and ATP, indicating the positive influence of 5'-phosphorylation. The reaction was further enhanced by addition of DNA ligase I to the reaction mixture. This is due, at least in part, to an enhancement by DNA ligase I of the rate of 5'-phosphorylation catalyzed by polynucleotide kinase. 相似文献
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DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.
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A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function. 相似文献
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7.
Y Uesawa J Kuwahara Y Sugiura 《Biochemical and biophysical research communications》1989,164(2):903-911
Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min. 相似文献
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The binding of the wedge-shaped isostructural analogue of the biradical species of the chromphore of antitumor antibiotic neocarzinostatin to sequence-specific bulged DNAs results in alterations in ellipticity of the DNAs. Circular dichroism (CD) spectroscopic results suggest that the drug specifically recognizes bulges of DNA via a combination of conformational selection and induced fit, not by binding to a preorganized site. Analysis of circular dichroism spectra indicates that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the bulge cavity. The effective recognition of the bulge site on duplex DNA appears to depend to a significant extent on the bent groove space being flexible enough to be able to adopt the geometrically optimal conformation compatible with the wedge-shaped drug molecule, rather than involving 'lock and key' recognition. The spectroscopic results indicate a change of DNA conformation, consistent with an allosteric binding model. Spectroscopic studies with various bulged DNAs also reveal that the binding strength directly correlates with the stability of the bulge structures. 相似文献
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11.
DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini.
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In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress. 相似文献
12.
Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common. 相似文献
13.
J M Neumann T Huynh-Dinh S K Kan B Genissel J Igolen S Tran-Dinh 《European journal of biochemistry》1982,121(2):317-323
14.
degradation of DNA to acid soluble products was induced by the combined action of neocarzinostatin and sulfhydryl agent as 2-mercaptoethanol, dithiothreitol, or reduced glutathione, but not other reducting agent as ascorbic acid or NaBH4. From the analysis by Sephadex G-10 gel filtration, acid soluble products were found to be thymine and oligonucleotide, but not thymidylic acid and thymidine. Release of adenine or guanine from DNA was not detected.From these results, it is suggested that DNA chain breakage by the combined action of neocarzinostatin and 2-mercaptoethanol may be due to an indirect phosphodiester bond breakage with release of thymine. 相似文献
15.
Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. 总被引:147,自引:0,他引:147
P R Shank S H Hughes H J Kung J E Majors N Quintrell R V Guntaka J M Bishop H E Varmus 《Cell》1978,15(4):1383-1395
Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV. 相似文献
16.
The sequences of the first 194 base pairs at both termini of adenovirus type 5 (Ad5) DNA have been determined, using the chemical degradation technique developed by Maxam and Gilbert (Proc. Nat. Acad. Sci. USA 74 (1977), pp. 560-564). The nucleotide sequences 1-75 were confirmed by analysis of labeled RNA transcribed from the terminal HhaI fragments in vitro. The sequence data show that Ad5 DNA has a perfect inverted terminal repetition of 103 base pairs long. 相似文献
17.
Two enzymatically modified derivatives of tobramycin have been prepared by gentamicin nucleotidyl transferase-catalyzed adenylylation of tobramycin, using ATP and (Sp)-ATP alpha S as adenylylation substrates. (EC 2.7.7.46). The 1H, 13C, and 31P NMR spectra have been assigned for tobramycin, 2'-(adenosine-5'-phosphoryl)-tobramycin (TbAMP) and 2'-(adenosine-5'-thiophosphoryl)-tobramycin (TbAMPS). Several one- and two-dimensional NMR techniques have been utilized, notably, 1H-1H homonuclear correlation spectroscopy at 470 or 500 MHz and 13C-1H heteronuclear correlation spectroscopy at 50.3 MHz. The 1H assignments for tobramycin are similar to those previously reported for rings I and III of kanamycin A. The 13C assignments for tobramycin were similar to those previously reported, except for reversal of the assignments for anomeric carbons in the glycosyl rings. The 1H and 13C assignments for tobramycin were used to guide the assignments of the spectra for TbAMP and TbAMPS. Nearly complete assignments were obtained for these two derivatives of tobramycin. From the measured proton coupling constants, only small conformational changes were observed upon modification of tobramycin by adenylylation. From the proton and carbon spectra of the adenylylated derivatives the 2' position is shown to be the site of adenylation. Large downfield shifts of the 2'proton and carbon resonances are easily observed and are more pronounced for TbAMPS than for TbAMP. 相似文献
18.
Conformational properties of trimeric and tetrameric 2′,5′-linked oligonucleotides, 3′-MOE-A32′,5′ (1) and 3′-MOE-A42′,5′ (2), and their 3′,5′-linked analogs, 2′-MOE-A33′,5′ (3) and 2′-MOE-A43′,5′ (4), were examined with the use of heteronuclear NMR spectroscopy. The temperature-dependent 3JHH, 3JHP and 3JCP coupling constants, acquired in the range of 273–343 K, gave insight into the conformation of sugar rings in terms of a two-state North ↔ South (N ↔ S) pseudorotational equilibrium and into the conformation of the sugar–phosphate backbone in the model antisense oligonucleotides 1–4. 2′,5′-linked oligomers 3′-MOE-A32′,5′ (1) and 3′-MOE-A42′,5′ (2) show preference for N-type conformers and indication of A-type conformational features, which is prerequisite for antisense hybridization. The drive of N ↔ S equilibrium in 1–4 has been rationalized with the competing gauche effects of 2′/3′-phosphodiester and 3′/2′-MOE groups, anomeric and steric effects. Furthermore, the pairwise comparisons of 3′-MOE with 3′-OH and 3′-deoxy 2′,5′-linked adenine trimers emphasized the fine tuning of N ↔ S equilibrium in 3′-MOE-A32′,5′ (1) and 3′-MOE-A42′,5′ (2) by the steric effects of 3′-MOE group and the possibility of water-mediated H-bonds with vicinal phosphodiester functionality. In full correspondence, the drive of N ↔ S equilibrium towards N by 2′-MOE in 3′,5′-linked analogs 2′-MOE-A33′,5′ (3) and 2′-MOE-A43′,5′ (4) is weaker in comparison with 3′-OH group in the corresponding ribo analogs. βt, γ+ and ε– rotamers are preferred in both 2′,5′- and in 3′,5′-linked oligonucleotides 1–4. 相似文献
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20.
Regulation of mRNA translation by 5'- and 3'-UTR-binding factors 总被引:12,自引:0,他引:12
The translational regulation of specific mRNAs is important for controlling gene expression. The past few years have seen a rapid expansion in the identification and characterization of mRNA regulatory elements and their binding proteins. For the majority of these examples, the mechanism by which translational regulation is achieved is not well understood. Nevertheless, detailed analyses of a few examples show that almost every event in the initiation pathway, from binding of the cap complex to the joining of the 60S ribosomal subunit, is subject to regulation. 相似文献