Inhibition of reverse transcription in rat liver intracisternal A-particles by thymidine derivatives

AbstractThe thymidine derivatives araAzT, dTTP(3′N3), TTP(3′NH2), and araTTP(3′N3), were studied as inhibitors of the reverse transcription taking place within endogenous retroviral A-type particles, where retroviral RNAs served as templates and primers. dTTP(3′N3) was shown to be the most efficient inhibitor of retroviral particle reverse transcription. Termination of DNA chain elongation is the basic mechanism of the inhibitory action of dTTP(3′N3). The compound has a very low inhibitory effect on mammalian DNA-dependent DNA polymerases α, β and γ

Effect of triphosphate modifications in 2′-deoxynucleoside 5′-triphosphates on their specificity towards various DNA polymerases

AbstractSome natural and glycon-modified dNTPs with β,γ-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases α and β, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of β,γ-methylenediphosphonate substituted dTTP and 3′-azido-3′-deoxy-dTTP decreased in the following order: CF2=CHF>CBr2>CFMe≫CH2. This order is individual for each DNA polymerase. It is interesting to mention that β,γ-CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase β. This specificity distinguishes DNA polymerase β from other DNA polymerases studied

Selectivity of DNA polymerases toward α and β nucleotide substrates of d and l series

AbstractThe substrate properties of four carbocyclic d and l nucleoside 5′-triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the d-β and l-β series were found to be terminating substrates for these enzymes, while the derivatives of the d-α and l-α series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template-independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed

The Puromycin Route to Assess Stereo- and Regiochemical Constraints on Peptide Bond Formation in Eukaryotic Ribosomes

We synthesized a series of puromycin analogues to probe the chemical specificity of the ribosome in an intact eukaryotic translation system. These studies reveal that both d-enantiomers and β-amino acid analogues can be incorporated into protein, and provide a quantitative means to rank natural and unnatural residues. Modeling of a d-amino acid analogue into the 50S ribosomal subunit indicates that steric clash may provide part of the chiral discrimination. The data presented provide one metric of the chiral and regiospecificity of mammalian ribosomes

Terminal Deoxynucleotidyl Transferase: The Story of A Misguided DNA Polymerase

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis