Single Ribonucleotide Research Articles

Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme.

RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors. We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a 'first generation' of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates approximately 10(7)-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded 'second generation' DNAzymes that self-cleaved at rates of approximately 0.01 min-1 (a 10(8)-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes. Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.

Characterization and divalent metal-ion dependence of in vitro selected deoxyribozymes which cleave DNA/RNA chimeric oligonucleotides

By in vitro selection, a variety of catalytic DNA oligonucleotides were obtained which cleave chimeric oligonucleotides at a single ribonucleotide position embedded within a deoxyribonucleotide context in the presence or absence of divalent metal ions. After several cycles of selection/amplification in the absence and in the presence of low amounts of Mg 2+ two different types of catalysts emerged: one type depended strongly on Mg 2+ or other divalent metal ions, the other type performed cleavage reactions independently of Mg 2+ in the presence of spermine. Experimental analysis of the secondary structure of some of the selected deoxyribozymes was carried out by chemical probing. The ribonucleotide in the selected catalysts is unpaired and presents the cleavage site to the attacking nucleophile. Our results suggest that the main selection criterion under metal-free conditions was a favourable arrangement of the attacking nucleophile and the phosphate leaving group. The cleavage rates of the selected divalent metal independent catalysts are within the same order of magnitude as the rate of metal independent substrate hydrolysis in the hammerhead ribozyme. One of the metal dependent catalysts showed an unexpected preference for Ca 2+ instead of Mg 2+. In this deoxyribozyme binding of Ca 2+ ocurred co-operatively whereas binding of Mg 2+ did not. Comparison of the secondary structure and reactivity of this catalyst with Mg 2+ and Ca 2+ suggests that here a special binding pocket for Ca 2+ was selected. This deoxyribozyme achieved a rate acceleration of substrate cleavage in the order of at least 10 4 compared to the uncatalysed reaction performing a cleavage mechanism similar to that of the hammerhead or hairpin ribozyme.

Substrate properties of fluorescent ribonucleotides in the terminal transferase-catalyzed labeling of DNA sequencing primers.

Terminal deoxynucleotidyltransferase (terminal transferase, E.C. 2.7.7.31) has been used to add a single fluorescent ribonucleotide to the 3' terminus of DNA sequencing primers, thereby creating primers suitable for automated DNA sequence analysis. The previously introduced procedure using fluorescein-UTP for the postsynthetic labeling of primers can, under appropriate reaction conditions, now be extended to commercially available fluorescein-ATP and fluorescein-CTP permitting greater flexibility in primer design. The products of these addition reactions have been shown to provide sequence data qualitatively and quantitatively identical to those obtained with conventional 5'-terminally labeled primers using cycle sequencing conditions in conjunction with an automated sequencer. Ribonucleotide derivatives of four other dyes (coumarin, tetramethylrhodamine, lissamine and Texas Red) were also examined for their potential in the terminal transferase-catalyzed reaction. Whereas coumarin-UTP was efficiently incorporated giving a monoaddition product, the conjugates of all other dyes with ATP, CTP and UTP were extremely poor substrates under all conditions tested.

Evidence for the Rate-Limiting Departure of the 5‘-Oxygen in Nonenzymatic and Hammerhead Ribozyme-Catalyzed Reactions

Kinetic analysis, using substrates that consisted entirely of deoxynucleotides with the exception of the single mandatory ribonucleotide at the cleavage site which contained either a 5‘-oxy- or 5‘-...

Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta. virus RNA sequence

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.

Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.

Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNALys3-primer excision

Two model substrates were prepared to examine the mechanism of tRNA-primer excision catalyzed by reverse transcriptase associated ribonuclease H (RT-RNase H). The first model substrate contained sequences from the HIV genome and was designed to be structurally similar to the DNA-extended tRNA created by initiation of minus-strand DNA synthesis during retroviral replication. The DNA-extended RNA was a template and was annealed to a DNA oligonucleotide that primed reverse transcription of the RNA in the template. The second model substrate was structurally similar the first substrate but contained sequences unrelated to the HIV viral genome. The RT-RNase H catalyzed excision of the RNA from the template of the two model substrates was examined. Human immunodeficiency virus (HIV) and Moloney murine leukemia virus RT-RNase H hydrolyzed the substrates to leave a single ribonucleotide 5'-phosphate at the 5'-terminus of the model DNA genome. In contrast, avian myeloblastosis virus RT-RNase H hydrolyzed the phosphodiester bond at the DNA-RNA junction. These hydrolytic specificities were not highly dependent on substrate sequence. The importance of these specificities to retroviral integration is discussed. Additional data indicated that the HIV polymerase and RNase H active sites are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex.

Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity.

The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.

Observations concerning the discontinuous DNAs of cauliflower mosaic virus.

The double-stranded circular DNA encapsidated within cauliflower mosaic virus (CaMV) particles contains three single-stranded discontinuities, two in one strand and one in the other, so that, upon denaturation, three linear single-stranded DNAs are produced. Here we show that a fourth much smaller single-stranded DNA, termed alpha1, is also present in denatured CaMV DNA preparations. The 5' extremity of alpha1 is identical to that of the alpha-strand, the strand of DNA possessing only one interruption, while its 3' extremity lies just two nucleotides downstream from a major transcription initiation site. We also show that the interrupted strand at each discontinuity sometimes has a single ribonucleotide in place of a deoxyribonucleotide at its 5' extremity. Oligoribonucleotide chains of eight and 10 residues in length have also been detected at the 5' end of one of the discontinuities. These structures are thought to be the vestiges of primers which have not been completely excised prior to encapsidation of the DNA. The possibility that synthesis of the alpha-strand occurs by reverse transcription of viral RNA using initiator tRNA(met) as primer is discussed.

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.

Covalent Attachment of Ribonucleotides at 3′-Hydroxyl Ends of Deoxyribonucleic Acid Catalyzed by Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase of Escherichia coli

Abstract DNA-dependent RNA polymerase of Escherichia coli catalyzes covalent attachment of ribonucleotides at 3'-hydroxyl termini of synthetic as well as naturally occurring DNAs. Incorporation of a single ribonucleotide occurs at 3'-hydroxyl termini of poly[d(A-T)] in the presence of ATP or UTP, at termini of poly[(dG)·(dC)] in the presence of CTP or GTP and at termini of nicked colicin E1 DNA (termini introduced by the action of pancreatic DNase) in the presence of each of the four ribonucleotides. This end addition reaction occurs optimally over a broad pH range (7 to 10) and differs from general transcription in a number of properties. It is more than 20 times less sensitive to the action of streptolydigin than RNA synthesis. Furthermore, it is inhibited only 80 % by 60 µg per ml of rifampicin while RNA synthesis is inhibited more than 95 % by 5 µ per ml of this drug. Ribonucleotide(s) can thus be attached at 3'-hydroxyl termini of DNA under conditions where synthesis of free RNA is inhibited. While the initiation reaction leading to RNA synthesis is specific for purines (ATP and GTP), the end addition reaction shows no preferences for purines. The salt sensitivity and the rate of incorporation of AMP on poly[d(A-T)] parallels that of CMP on poly[(dG)·(dC)] but not GMP. In the absence of the σ subunit of RNA polymerase AMP and UMP end addition on poly [d(A-T)] show identical kinetics. In the presence of the σ subunit, however, AMP incorporation ceases within 10 min and no further incorporation occurs for at least 30 min. On a molar basis, the amount of AMP incorporated is equal to approximately 30 to 50 % of the added RNA polymerase which, as previously shown, corresponds to the proportion of active enzyme molecules (Wickner, S., Hurwitz, J., Nath, K., and Yarbrough, L. (1972) Biochem. Biophys. Res. Commun. 48, 619–627). Estimation of active RNA polymerase molecules can thus be obtained by end addition measurement. RNA chains are formed at 3'-hydroxyl termini of poly[d(A-T)] in the presence of ATP and UTP, and at the termini of nicked colicin E1 DNA in the presence of all four ribonucleotides. This covalently attached RNA, when isolated after heating in formaldehyde, Cs2SO4 banding, and removal of DNA by treatment with pancreatic DNase, shows a size distribution of 4 S for poly(A-U) and 4 to 10 S for RNA synthesized from nicked colicin E1 DNA. Formation of RNA covalently attached to nicked colicin E1 DNA is favored by the absence of the σ subunit of RNA polymerase, relatively low concentration of a single ribonucleoside triphosphate (20 to 50 µm), short incubation periods at 37° (less than 5 min), and a high ratio of DNA to RNA polymerase. While RNA polymerase holoenzyme catalyzes the synthesis predominantly of free RNA, the core enzyme preparations, depleted of σ subunit, synthesize predominantly covalently attached RNA at 3'-hydroxyl termini of DNA. Core enzyme preparations are also capable of initiating free RNA synthesis to a limited extent.

Turnover of nuclear RNA in HeLa cells: Evidence for a single ribonucleotide pool

The role of the ribonucleotide pool in measuring turn-over of RNA has been studied. Evidence presented previously has assumed a single pool. The nucleotide base ratios of two different species of nuclear RNA (heterogeneously sedimenting nuclear RNA and ribosomal precursor RNA) are known. We have studied the rate of approach to the equilibrium base ratio established by labeling, either via the salvage pathway or the endogenous route. In either case, normalization of the measured U:C ratios is accomplished by dividing the equilibrium U:C ratio in an isolated species by the measured base ratio (specific U:C ratio). These studies are consistent with the existence of a single ribonucleotide pool.

On the heterogeneity of the deoxyribonucleic acid associated with crystalline yeast cytochrome b2

1. 1. The buoyant density of cytochrome b 2 DNA before and after heating and quick-cooling suggests considerable helicity, and observations on the action of Escherichia coli exonucleases I and III on the DNA support this interpretation. 2. Enzymic and chemical investigation of the 3′- and 5′-phosphoryl and 3′- and 5′-hydroxyl end groups of the DNA indicate that these ends are heterogeneous. An average molecular weight range of 20 000–80 000 is estimated from these studies. 3. Cytochrome b 2 DNA serves as a primer for E. coli DNA polymerase, thereby permitting a study of overall nearest-neighbor base frequencies. Also, by the use of Mn 2+, a single ribonucleotide is incorporated in place of the corresponding deoxyribonucleotide in the product; alkaline hydrolysis of this product releases fragment which can be analyzed for base sequence. Both approaches support the conclusion that the cytochrome b 2 DNA is a heterogeneous population of molecules of the size indicated above or else that to be homogeneous in nucleotide sequence its weight must exceed 100 000.

Studies on the incorporation of deoxyribonucleotides and ribonucleotides into deoxyribonucleic acid

The purification of an enzyme fraction from calf-thymus nuclei catalyzing a limited incorporation of deoxyribonucleotides into the terminal positions of DNA has been described. The enzyme studied is apparently distinct from DNA polymerase. The same enzyme will also catalyze the incorporation of single ribonucleotides into the terminal positions of DNA.

Studies on the incorporation of deoxyribonucleotides and ribonucleotides into deoxyribonucleic acid

The purification of an enzyme fraction from calf-thymus nuclei catalyzing a limited incorporation of deoxyribonucleotides into the terminal positions of DNA has been described. The enzyme studied is apparently distinct from DNA polymerase. The same enzyme will also catalyze the incorporation of single ribonucleotides into the terminal positions of DNA.

Incorporation of deoxyribonucleotides into terminal positions of DNA

An enzyme fraction from calf thymus nuclei which incorporates single ribonucleotides into terminal positions of DNA has been described (1, 2, 3). It has been found that this same enzyme fraction (referred to in this communication as ‘nuclear enzyme’) catalyses the incorporation of deoxyribonucleotides into terminal positions of DNA and that this activity appears to be distinct from that of DNA polymerase.