2--3--dideoxyguanosine-5--triphosphate and 2--3--dideoxyadenosine-triphosphate

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Topics: Chloroplasts; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; Fluorescent Dyes; Gene Components; NADH Dehydrogenase; Nicotiana; Plant Proteins; Regulatory Sequences, Ribonucleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA Editing; RNA, Messenger; RNA, Plant; Sequence Analysis, RNA

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.

Topics: Binding Sites; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA; DNA-Directed DNA Polymerase; Kinetics; Models, Molecular; Nucleotides; Protein Binding; Protein Structure, Secondary; Taq Polymerase; Thymine Nucleotides

The mechanism and dynamics of translesion DNA synthesis were evaluated using primer/templates containing a tetrahydrofuran moiety designed to mimic an abasic site. Steady-state kinetic analysis reveals that the T4 DNA polymerase preferentially incorporates dATP across from the abasic site with 100-fold higher efficiency than the other nucleoside triphosphates. Under steady-state conditions, the catalytic efficiency of dATP incorporation across from an abasic site is only 220-fold lower than that across from T. Surprisingly, misincorporation across from T is favored 4-6-fold versus replication across an abasic site, suggesting that the dynamics of the polymerization cycle are differentially affected by formation of aberrant base pairs as opposed to the lack of base-pairing capabilities afforded by the abasic site. Linear pre-steady-state time courses were obtained for the incorporation of any dNTP across from an abasic site, indicating that chemistry or a step prior to chemistry is rate-limiting for the polymerization cycle. Low elemental effects (<3) measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP indicate that chemistry is not solely rate-limiting. Single-turnover experiments yield kpol/Kd values that are essentially identical to kcat/Km values and provide further evidence that the conformational change preceding chemistry is rate-limiting. Extension beyond an A:abasic mispair is approximately 20-fold and 100-fold faster than extension beyond a G:abasic mispair or C:abasic mispair, respectively. Extension from the G:abasic or A:abasic site mispair generates significant elemental effects (between 5 and 20) and suggests that chemistry is at least partially rate-limiting for extension beyond either mispair.

Topics: Apurinic Acid; Bacteriophage T4; Base Pair Mismatch; Catalysis; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; DNA Damage; DNA-Directed DNA Polymerase; DNA, Viral; Exonucleases; Kinetics; Nucleic Acid Synthesis Inhibitors; Substrate Specificity

Although techniques such as biopanning rely heavily upon the screening of randomized gene libraries, there is surprisingly little information available on the construction of those libraries. In general, it is based on the cloning of 'randomized' synthetic oligonucleotides, in which given position(s) contain an equal mixture of all four bases. Yet, many supposedly 'randomized' libraries contain significant elements of bias and/or omission. Here, we report the development and validation of a new, PCR-based assay that enables rapid examination of library composition both prior to and after cloning. By using our assay to analyse model libraries, we demonstrate that the cloning of a given distribution of sequences does not necessarily result in a similarly composed library of clones. Thus, while bias in randomized synthetic oligonucleotide mixtures can be virtually eliminated by using unequal ratios of the four phosphoramidites, the use of such mixtures does not ensure retrieval of a truly randomized library. We propose that in the absence of a technique to control cloning frequencies, the ability to analyse the composition of libraries after cloning will enhance significantly the quality of information derived from those libraries.

Topics: Cloning, Molecular; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA; DNA-Directed DNA Polymerase; Gene Library; Oligonucleotides; Polymerase Chain Reaction; Templates, Genetic; Thymine Nucleotides

The Taq DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of Taq polymerase are deficient in two respects: (i) these enzymes incorporate each of the four dideoxynucleoside 5' triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (ii) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the Taq DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP. When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate. As a result, the G track generated during DNA sequencing by these Taq polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected. Another property of these Taq polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing.

Topics: Crystallography, X-Ray; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Secondary; Taq Polymerase; Thymine Nucleotides

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Cloning, Molecular; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Dideoxynucleotides; Escherichia coli; Humans; Nucleoside-Diphosphate Kinase; Phosphorylation; Recombinant Proteins

A set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), which confers on the virus a reduced sensitivity to multiple therapeutic dideoxynucleosides (ddNs), has been identified. In this study, we defined the biochemical properties of RT with such mutations by using site-directed mutagenesis, overproduction of recombinant RTs, and steady-state kinetic analyses. A single mutation, Q151M, which developed first among the five mutations in patients receiving therapy, most profoundly reduced the sensitivity of RT to multiple ddN 5'-triphosphate (ddNTPs). Addition of other mutations to Q151M further reduced the sensitivity of RT to ddNTPs. RT with the five mutations proved to be resistant by 65-fold to 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP), 12-fold to ddCTP, 8.8-fold to ddATP, and 3.3-fold to 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP), compared with wild-type RT (RTwt). Steady-state kinetic studies revealed comparable catalytic efficiency (kcat/Km) of RTs carrying combined mutations as compared with that of RTwt (< 3-fold), although a marked difference was noted in inhibition constants (Ki) (e.g. Ki of a mutant RT carrying the five mutations was 62-fold higher for AZTTP than that of RTwt). Thus, we conclude that the alteration of RT's substrate recognition, caused by these mutations, accounts for the observed multi-ddN resistance of HIV-1. The features of multi-ddNTP-resistant RTs should provide insights into the molecular mechanism of RT discriminating ddNTPs from natural substrates.

Topics: Antiviral Agents; Base Sequence; Binding Sites; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; DNA Primers; DNA, Viral; Drug Resistance, Multiple; HIV Infections; HIV Reverse Transcriptase; HIV-1; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Thymine Nucleotides; Zidovudine

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.

Topics: Autoanalysis; Base Sequence; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA; Fluorescent Dyes; Nucleotides; Plasmids; Polymerase Chain Reaction; Thymine Nucleotides

Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.

Topics: Animals; Autoanalysis; Base Sequence; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; DNA; DNA-Directed DNA Polymerase; Drosophila; Genetic Carrier Screening; Magnesium; Manganese; Molecular Sequence Data; Mutation; Phosphates; Polymerase Chain Reaction; Pyrophosphatases; T-Phages; Thymine Nucleotides