Inhibition of the initiation of HIV-1 reverse transcription by 3'-azido-3'-deoxythymidine. Comparison with elongation.
(41/803)
Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA(3)(Lys), and reverse transcriptase. Initiation, corresponding to addition of the first six nucleotides to tRNA(3)(Lys), is distinguished from elongation by its high specificity and low efficiency (processivity). Here, we compared the inhibition of initiation and elongation of reverse transcription by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deoxythymidine. We report the first detailed study of nucleotide binding, discrimination, and pyrophosphorolysis by the authentic initiation complex. We showed that the initiation and elongation complexes bound AZTTP and dTTP with the same affinity, while the polymerization rates were reduced by 148-160-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced by the same extent, indicating that the polymerization equilibrium is the same in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis significantly relieved inhibition of DNA synthesis during elongation in the presence of physiological pyrophosphate concentrations. Remarkably, although pyrophosphorolysis of dTMP and AZTMP were equally efficient during elongation, reverse transcriptase was almost totally unable to unblock the AZTMP-terminated primer during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription, despite a reduced selectivity of incorporation. (+info)
Nucleotide pools and regulation of ribonucleic acid synthesis in yeast.
(42/803)
Nucleotide pools were measured in growing and amino acid-starved Saccharomyces cerevisiae. During amino acid starvation there are neither significant changes in the endogeneous nucleoside triphosphate pool levels nor measurable synthesis of guanosine 5'-diphosphate, 3'-diphosphate. Stable ribonucleic acid synthesis does not appear to be regulated by changes in the triphosphate pools or by the unusual nucleotide guanosine 5'-diphosphate, 3'-diphosphate. (+info)
Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus.
(43/803)
The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus. (+info)
Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.
(44/803)
During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved. (+info)
Universal bases for hybridization, replication and chain termination.
(45/803)
Several unnatural, predominantly hydrophobic nucleobases that pack efficiently in duplex DNA without hydrogen bonding functionalities are reported to circumvent the hydrogen bonding-based specificity, both during oligonucleotide hybridization and enzymatic DNA synthesis. The reported nucleoside analogs are efficient 'universal bases' for hybridization, template directed DNA synthesis and chain termination. Moreover, several of the universal bases function in their biological role, hybridization or replication, with an efficiency not significantly reduced relative to their natural counterparts. (+info)
Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro.
(46/803)
DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments. (+info)
Binding of lactose repressor to poly d(A-T) : OD AND CD melting of the complex.
(47/803)
The binding of lactose repressor to poly d(A-T) at low ionic strength has been investigated by heat denaturation. The poly d(A-T) melting is monitored by optical density and the protein melting by circular dichroism. From the modification of the poly d(A-T) melting curve we estimate a maximum binding ratio of about one tetrameric repressor to about 20 basic pairs. The repressor melting can be interpreted as a global shift from a to b structure of about 25 residues per subunit. The melting curves of poly d(A-T) and repressor have not a shape easy to interpret; nevertheless both show a cooperative transition in the same temperature range where we can evaluate that about 3.8 aminoacid residues shift from a to b structure when 1 bases pair melt. (+info)
(dA-dT) dependent inactivation of the DNA template properties by interaction with netropsin and distamycin A.
(48/803)
The inhibitory effect of the polypeptide antibiotics netropsin and distamycin A on DNA dependent nucleic acid synthesis has been shown to be related to the base composition of the template DNA. A number of natural DNA's of quite different dA-dT content as well as poly (dI-dC)-poly (dI-dC), poly (dA-dT)-poly (dA-dT), poly (dA) - poly (dT) and poly (dG) - poly (dC) has been studied as templates in DNA and in part in RNA polymerase reaction. The highest binding efficiency of netropsin existing for (dA-dT) - containing DNA polymers and the less pronounced interaction with the (dI-dC)-containing polymer shown by the melting and CD spectrral behaviour of the complexes are entirely reflected in the template inactivation. The same is evident for distamycin A. However, in contrast to netropsin the antibiotic distamycin A exhibits some binding tendency to poly (dG) - poly (dC). Binding effects of a netropsin derivative to DNA and (dA-dT) -containing polymers suggest the importance of hydrogen bonds of the peptide groups in the complex formation. (+info)