Phosphate End Research Articles

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Characterization of Telomere-Binding Activity of Replication Factor C Large Subunit p140

The large subunit of RFC (RFC p140) has been suggested to be associated with the 3′-end of elongating DNA primer and to recruit proliferating cell nuclear antigen (PCNA) onto DNA polymerase δ. Previously, we isolated a cDNA clone encoding a DNA-binding domain of RFC p140 as a telomeric repeat (TTAGGG)n binding protein. This domain was shown to have a specific affinity for the 5′-phosphate ends of a telomere repeat sequence. In order to investigate the structure and function of RFC p140, we constructed the full-length recombinant RFC p140 as well as N- and/or C-terminal deleted mutants and analyzed their telomere-binding activities. South-Western blot and gel mobility shift analyses revealed that deletion of the N- but not the C-terminal region enhances recognition of the telomeric repeat sequence and 5′-phosphate ends, suggesting the negative effect of the N-terminal region of the RFC p140 binding to the telomeric repeat. On the other hand, the C-terminal truncated RFC inhibits the telomerase activity more than the N-terminal-deleted and full-length RFC p140. The inhibitory effect of RFC p140 on telomerase activity is completely diminished by both terminal deletions. Thus, a certain interaction of the N- and C-terminal regions is considered to be required for RFC p140 to suppress telomerase activity. Taken together, these results suggest that both telomeric repeat-binding and telomerase inhibitory activities of RFC p140 are finely regulated by the intrinsic N- and C-terminal regions.

RNA-binding and capping activities of proteins in rotavirus open cores.

Guanylyltransferases are members of the nucleotidyltransferase family and function in mRNA capping by transferring GMP to the phosphate end of nascent RNAs. Although numerous guanylyltransferases have been identified, studies which define the nature of the interaction between the capping enzymes of any origin and their RNA substrates have been limited. Here, we have characterized the RNA-binding activity of VP3, a minor protein component of the core of rotavirions that has been proposed to function as the viral guanylyltransferase and to direct the capping of the 11 transcripts synthesized from the segmented double-stranded RNA (dsRNA) genome of these viruses. Gel shift analysis performed with disrupted (open) virion-derived cores and virus-specific RNA probes showed that VP3 has affinity for single-stranded RNA (ssRNA) but not for dsRNA. While the ssRNA-binding activity of VP3 was found to be sequence independent, the protein does exhibit preferential affinity for uncapped over capped RNA. Like the RNA-binding activity, RNA capping assays performed with open cores indicates that the guanylyltransferase activity of VP3 is nonspecific and is able to cap RNAs initiating with a G or an A residue. These data establish that all three rotavirus core proteins, VP1, the RNA polymerase; VP2, the core capsid protein; and VP3, the guanylyltransferase, have affinity for RNA but that only in the case of the RNA polymerase is the affinity sequence specific.

Cyclases of the 3'-terminal phosphate in RNA: a new family of RNA processing enzymes conserved in eucarya, bacteria and archaea.

The 2',3'-cyclic phosphate termini are produced, as either intermediates or final products, during RNA cleavage by many different endoribonucleases. Likewise, ribozymes such as hammerheads, hairpins, or the hepatitis delta ribozyme, generate 2',3'-cyclic phosphate ends. Discovery of the RNA 3'-terminal phosphate cyclase has indicated that cyclic phosphate termini in RNA can also be produced by an entirely different mechanism. The RNA 3'-phosphate cyclase converts the 3'-terminal phosphate in RNA into the 2',3'-cyclic phosphodiester in the ATP-dependent reaction which involves formation of the covalent cyclase-AMP and the RNA-N3' pp5' A intermediates. The findings that several eukaryotic and prokaryotic RNA ligases require the 2',3'-cyclic phosphate for the ligation of RNA molecules raised a possibility that the RNA 3'-phosphate cyclase may have an anabolic function in RNA metabolism by generating terminal cyclic groups required for ligation. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity. This article reviews properties of the human and bacterial cyclases, their mechanism of action and substrate specificity. Possible biological functions of the enzymes are also discussed.

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.

Acousto-optic surface-plasmon resonance measurements of thin films on gold

In this work a highly sensitive optical technique was used to study the adsorption of thin organic layers onto a gold film from water, in situ and in real-time. Optical excitation of a surface-plasmon resonance (SPR) was used to probe the gold/water interface. The use of an acousto-optic tunable filter then provides a differential technique for monitoring the SPR position in optical wavelength. This allows optical changes at the metal/liquid interface to be measured as the adsorption of thin organic layers occurs. Adsorption of a poly(ethylene glycol) monododecyl ether surfactant (C12E8) and a 30-mer DNA oligonucleotide with a mercaptohexyl group at the 5′-phosphate end (DNA-SH) onto gold from water were examined. Conventional, angle-dependent reflectivity measurements taken on the same system provided complementary SPR data, allowing the sensitivity of the two techniques to be compared.

Quartz Crystal Microbalance Study of DNA Immobilization and Hybridization for Nucleic Acid Sensor Development

The immobilization of two 30-mer oligonucleotides, one biotinylated (biotin-DNA) and the other having a mercaptohexyl group at the 5'-phosphate end (BS1-SH), onto modified gold surfaces has been examined using a quartz crystal microbalance (QCM). Both single-layer and multilayer DNA films were prepared. The single-layer films of biotin-DNA were constructed by binding to a precursor layer of avidin, which had been attached to the QCM either covalently using a water-soluble carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or via electrostatic interaction with poly(allylamine hydrochloride) (PAH). Single-layer films of BS1-SH were also formed on PAH via the electrostatic attraction between the amine groups on PAH and the negatively charged phosphate backbone of DNA. Multilayer films of DNA were fabricated by the successive deposition of avidin and poly(styrenesulfonate) (PSS), up to a total of nine avidin/PSS layers, followed by DNA adsorption. DNA immobilization and hybridization of the immobilized DNAs was monitored in situ from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. Equal frequency changes were observed for the DNA immobilization and hybridization steps for the single-layer films, indicating a DNA probe-to-hybridized DNA target ratio of 1:1. The multilayer DNA films also exhibited DNA hybridization, with a greater quantity of DNA hybridized compared with the single-layer films. The multilayer films provide a novel means for the fabrication of DNA-based thin films with increased capacity for nucleic acid detection.

DNA binding and hybridization on gold and derivatized surfaces

The binding of various single- and double-stranded DNAs onto gold and derivatized gold surfaces, and their hybridization with complementary DNA species, have been investigated using a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). The DNA species employed were a 21-mer oligonucleotide (Mbo21), several double-stranded plasmid DNAs (7.2 kilobases) modified by incorporation of α-phosphothio-nucleotides into the ends of the linearized plasmid DNA (pPS-S x , where x represents the number of α-phosphothio-nucleotides), and a 30-mer oligonucleotide having a mercaptohexyl group at the 5′-phosphate end (BS1-SH). Both QCM and SPR data reveal that unmodified DNA does not spontaneously adsorb onto underivatized gold surfaces from aqueous solutions. Modification of the gold surface through the attachment of an ionizable thiol compound, 2-dimethylaminoethanethiol hydrochloride (DMAET), allows DNA to adsorb through electrostatic interactions. SPR measurements confirm the presence of Mbo21 DNA on the DMAET-modified gold surface. Immobilized Mbo21, however, does not undergo hybridization. QCM and SPR data suggest that pPS-S 4, pPS-S 50 and BS1-SH DNA all assume a flat orientation on gold. No hybridization of single-stranded DNA to gold-immobilized pPS-S 4 and pPS-S 50 could be detected. In contrast 30-mer DNA binding from solution to the complement BS1-SH immobilized on gold reveals hybridization of the DNA strands.

The yeast site-specific recombinase Flp mediates alcoholysis and hydrolysis of the strand cleavage product: mimicking the strand-joining reaction with non-DNA nucleophiles

The yeast site-specific recombinase Flp is covalently linked to DNA via a 3′-phosphotyrosyl bond during the strand-breakage step of recombination. We show that this phosphotyrosyl diester bond formed between Flp and DNA can serve as the target for alcoholysis or hydrolysis in an Flp-assisted reaction. Flp does not mediate alcoholysis of the labile phosphodiester bond within the DNA chain under our assay conditions. The body of available evidence supports the notion that the alcoholysis/hydrolysis reaction is mechanistically analogous to the strand-joining step of the recombination pathway. The only difference is that the DNA 5′-hydroxyl group that acts as the nucleophile during recombination is substituted by a non-DNA nucleophile. We find that the alcoholysis reaction occurs only within the normal cleavage complex produced by the “shared active site” assembled at the interface of two Flp monomers. Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate linked at its 3′-phosphate end to a short tyrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer. However, even in this substrate that mimics the strand-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol.

An apoptotic endonuclease activated either by decreasing pH or by increasing calcium.

DNA fragmentation in isolated nuclei from the murine IL3-dependent bone marrow cell line BAF3 could be stimulated either by decreasing pH below 6.5 or by adding microM calcium at neutral pH. An endonuclease which could also be stimulated either by a decrease in pH, to 6.5, or by the presence of microM calcium at neutral pH, was purified 10(4)-fold from nuclei of BAF3 cells. Digestion of DNA with the purified enzyme resulted in 5'-terminal hydroxyl and 3'-terminal phosphate ends. These characteristics are distinct from those described for other mammalian endonucleases. The possible role of this enzyme in genome digestion during apoptosis is discussed.

The environment of two metal ions surrounding the splice site of a group I intron.

Several divalent metal ions (Ca2+, Sr2+ and Pb2+) do not promote splicing, but instead induce cleavage at a single site in the conserved group I intron core in the absence of the guanosine cofactor at elevated pH, generating products with 5'-OH and 3'-phosphate ends. The reaction is competed by Mg2+, which does not cleave at this position, but hydrolyses the splice sites producing 3'-OH and 5'-phosphate ends. Mn2+ promotes both core cleavage and splice site hydrolysis under identical conditions, suggesting that two different metal atoms are involved, each responsible for one type of cleavage, and with different chemical and geometric requirements. Based on the core cleavage position and on the previously proposed coordination sites for Mg2+, we propose a structural location for two metal ions surrounding the splice site in the Michel-Westhof three-dimensional model of the group I intron core. The proposed location was strengthened by a first mutational analysis which supported the suggested interaction between one of the metal ions and the bulged residue in P7.

3'- and 5'-strand cleavage reactions catalyzed by the Fpg protein from Escherichia coli occur via successive beta- and delta-elimination mechanisms, respectively.

The Fpg protein from Escherichia coli is a multifunctional protein that excises damaged purine bases from DNA to generate aldehydic abasic sites and then catalyzes the successive cleavage of the phosphodiester bonds first on the 3'-side and then on the 5'-side of the abasic site to generate 5'- and 3'-phosphate ends, respectively, thereby excising the deoxyribose residue. The mechanisms of the 3'- and 5'-strand cleavage reactions have been studied by nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). The 3'-strand cleavage reaction is a beta-elimination reaction in which the 2'-hydrogen is abstracted and the 3'-phosphate is eliminated. The 5'-strand cleavage reaction is a delta-elimination reaction in which the 4'-hydrogen is abstracted and the 5'-phosphate is eliminated. Two types of experiments were performed to establish the occurrence of the sequential elimination reactions. First, when the reaction was performed in H2(18)O, 31P NMR demonstrated that neither phosphate group contained 18O. Second, the five-carbon product derived from the deoxyribose residue was stabilized by reduction with NaBH4 and characterized by GC-MS. The mass spectrum of the reduced product was identical to that of authentic 4-oxo-2-pentenal, the tautomerized product of successive beta- and delta-elimination reactions.

Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye

A synthesized 20-mer DNA probe complementary to a part of an oncogene v-myc region having a mercaptohexyl group at the 5'-phosphate end was immobilized on a gold electrode by chemisorption. The immobilized DNA was detected voltammetrically using Hoechst 33258 with a DNA minor groove binder and an electrochemically active dye. The modified electrode was immersed into a 100 mumol/L Hoechst 33258 solution and washed with a phosphate buffer (pH 7.0). The anodic peak current (ipa) of Hoechst 33258 on the modified electrode was higher than that on a bare gold electrode (128 and 75 nA, respectively). It was considered that Hoechst 33258 was concentrated on the electrode surface due to its association with DNA. When the modified electrode was hybridized in a solution of a model targeted gene (10(-7) g/mL), single-stranded pVM623 containing the PstI fragment of a 1.5-kilobase pair of oncogene v-myc, the ipa was 192 nA. On the other hand, the ipa was 128 nA when the modified electrode was reacted in a solution of single-stranded pUC119 without a region complementary to v-myc in pVM623. The ipa was related to the concentration of the targeted DNA in the hybridization reaction. The use of Hoechst 33258 resulted in a sequence-specific detection of the targeted DNA quantitatively ranging from 10(-7) to 10(-13) g/mL in a buffer solution.

Purification and characterization of human ribonuclease HII.

A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.

Sites of strand breakage in DNA irradiated by fast neutrons

Therapeutic fast neutrons are densely ionizing particles, with a high relative biological effectiveness relative to 60Co γ rays (RBE) and a low oxygen enhancement ratio (OER). The molecular basis of their properties is not yet entirely understood. In a previous work, we have shown that neutrons induce a different number of DNA frank strand breaks as compared to γ photons, and we have revealed the presence of breaks due to the direct effects of neutrons. In the present work, we searched for eventual differences in the chemical nature of the attacked sites in DNA irradiated in oxygenated diluted solution. We compare our results with neutrons to those previously reported by other authors using γ- or X-rays. Using sequencing gel electrophoresis of short natural DNA restriction fragments, or synthetic oligonucleotides, we have shown that, in the case of neutrons, the attack occurs with almost the same probability, at each nucleotide, as reported for γ- and X-rays. The doubling of bands in the bottom of gels shows the presence of two types of termini, the 3′-phosphate and the 3′-phosphoglycolate. Upon neutron irradiation, the 3′-phosphate end appears with a higher yield than the 3′-phosphoglycolate, whereas equal amounts were obtained with γ- or X-rays.

Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site-specific DNA-cleaving/DNA-joining activity.

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGC-CCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.

Nonenzymic cleavage and ligation of DNA at a three A.cntdot.T base pair site. A two-step pseudohydrolysis of DNA

The chemical nuclease Mn-TMPyP/KHSO 5 is able to hydroxylate 5' carbon-hydrogen bonds of deoxyribose units at three contiguous A-T base pairs sites and initiate DNA rupture. Double-strand cleavage sites consist of trinucleotide sequences with a four nucleotide 3' stagger of the cleaved residues. Both strand nicks are identical and consist of 3'-phosphate termini facing a 5'-aldehyde residue which is easily reduced to a 5'-alcohol by NaBH 4 . These two successive treatments (oxidation plus reduction) are equivalent to a hydrolysis of the phosphodiester bond. Such mechanism is reminiscent of DNA restriction enzymes but with 5'-OH and 3'-phosphate ends at the site of cleavage

Influence of nitrogen, oxygen, and nitroimidazole radiosensitizers on DNA damage induced by ionizing radiation.

Oxygen and nitroaromatic compounds are known to enhance the sensitivity of cells to ionizing radiation. Employing calf thymus DNA and oligo(dA)12/poly(dT), we have examined the differences to DNA damage, in particular thymine glycols and the 3'-DNA termini at strand breaks, arising from irradiation under anoxic and oxic conditions and the presence and absence of misonidazole [1-(2-nitro-1-imidazoyl)-3-methoxy-2-propanol]. We show that (i) irradiation under nitrogen generates strand breaks almost exclusively with 3'-phosphate termini; (ii) irradiation under oxic conditions increases the yield of strand breaks 3-fold, and the 3' termini consist of 3'-phosphoglycolate and 3'-phosphate end groups in a ratio of approximately 1.6; (iii) the patterns of base and sugar damage detectable by a postlabeling assay [Weinfield, M., & Soderlind, K.-J. (1991) Biochemistry 30, 1091-1097] differ completely between DNA irradiated under oxic vs anoxic conditions; (iv) the presence of misonidazole under anoxic conditions does not increase the level of strand breakage but, like oxygen, significantly enhances the formation of 3'-phosphoglycolate end groups; (v) the presence of misonidazole during anoxic irradiation does not increase the yield of any other type of 'oxic' damage detectable by the postlabeling assay, such as thymine glycols; and (vi) misonidazole at concentrations greater than 50 microM affords significant protection to naked DNA, probably by OH radical scavenging, and both the nitroaromatic ring and methoxyisopropanol side chain contribute to this protective action.

Construction of nucleosome cores from defined DNA sequences of prokaryotic origin

A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described. Efficient de novo reconstitution without added carrier DNA is demonstrated. DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA. As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident. The possibility that a phosphate end group positions DNA fragments on the histone is considered. The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.

11] Using hydroxyl radical to probe DNA structure

Publisher Summary This chapter describes the application of the hydroxyl radical as a reagent for elucidating the structural details of DNA. The hydroxyl radical cleaves DNA by abstracting a hydrogen atom from a deoxyribose residue in the DNA backbone. Subsequently, the sugar breaks down, leaving a single-nucleoside gap with predominantly 5´- and 3´-phosphate ends. A small amount of 3´-phosphoglycolate is formed as well. This product migrates faster than the corresponding 3´-phosphate and can be seen when high percentage polyacrylamide gels are used to separate the products of hydroxyl radical cleavage of 5´-radiolabeled DNA. The examples presented in the chapter show that the hydroxyl radical cleavage experiment yields high resolution, reproducible structural data for DNA in solution. Adenine tracts in both a bent restriction fragment and a short oligonucleotide duplex have a characteristic pattern of hydroxyl radical cleavage that is related to the unusual conformation adopted by these tracts. The four strands of an immobile Holliday junction show distinctive hydroxyl radical protections that report on the crossover configuration of the molecule. Not only does this technique provide structural data at the nucleotide level, it can easily be performed by any laboratory that is experienced in the methods of molecular biology.