Lyubov S. Victorova

Gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases.

Several 2′-deoxythymidine 5′-triphosphate and 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate analogs containing a hydrophobic phosphonate group instead of the γ-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases α and β, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some γ-ester and γ-amide derivatives of dTTP and 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the γ-position. Modification of the γ-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase α. γ-Methylphosphonate and γ-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the γ-phosphate in AZTTP with γ-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.

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

The 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.

Nevirapine-selected mutations Y181I/C of HIV-1 reverse transcriptase confer cross-resistance to stavudine.

Stavudine administration did not increase the frequency of Y181I/C reverse transcriptase (RT) mutations in non-nucleoside reverse transcriptase inhibitor (NNRTI)-treated patients. However, recombinant Y181C HIV-1 showed reduced stavudine susceptibility with respect to both recombinant wild-type and K103N HIV-1 strains. In addition, recombinant Y181I RT enzyme showed reduced susceptibility to stavudine with respect to both wild-type and K103N RT. A previously unnoticed role of Y181I/C RT changes selected by nevirapine or other NNRTI in determining stavudine resistance is documented.

Effect of P-chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase

Phosphorothioate analogues of oligonucleotides (PS‐oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [RP] configuration, while the presence of the linkage(s) of the [SP] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3′‐end. The presence of [SP] linkage at this position strongly reduces the enzyme activity while the [RP] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.

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

Some 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.

The donor site of the peptidyltransferase center of ribosomes: Equilibrium association constants of model substrates and inhibitors

1. Introduction There are several pubhcatlons concernmg deter- mmatlon of the bmdmg constants (K,,) of model acceptor substrates of the peptldyltransferase center of Eksckcrrcha co11 rlbosomes such as CACCA-Phe [ 1,2] and puromycm [3] However, no quantitative data on bmdmg of model substrates to the donor site have been reported so far Here we report determination of the equlhbnum bmdmg constants of the pentanucleotlde fragment of peptldyl-tRNA CACCA-Leu+Ac and several other model peptlde donors [4,5] and donor site mhtbltors of the peptldyltransferase center of E colz rlbosomes It has been found that K,, changes mslgmficantly from the pentanucleotlde to the trmucleotlde model substrates and mlubltors of the donor site and decreases 2--3 orders of magnitude, upon transition to their dl- and mononucleotlde analogs 2. Materials and methods 2 1. E co11 MRE-600 nbosotnes These were isolated by two methods (A) by treat-

Trivaline ‘catalyzes’ 5′‐pdGTT oligomerization in solution

We have found that the 5′‐pdGTT molecules at a concentration of 10−4 M are oligomerized in solution in the presence of 10−4 M tripeptide ‐ (L‐Val)3‐NH‐NH‐DNS‐CF3COOH and the condensation reagents (carbodiimide and imidazole). Oligonucleotides not less than 12 bases long were formed in the yield which was over 15%. It is known that in the absence of peptide 10−2 M mono‐ or dinucleotides are required. Thus trivaline can be considered as one of the simplest enzymes. This oligomerization seems to be an essential way for the synthesis of long enough oligonucleotides of the random GC‐sequence, which could be used at the earliest steps of evolution.

Furano- and pyrrolo [2,3-d] pyrimidine nucleosides and their 5'-O-triphospates: synthesis and enzymatic activity.

A series of bicyclic [2,3-d]furano- and pyrrolopyrimidine ribonucleosides were synthesized and converted chemically into corresponding 5′-O-triphosphates. Substrate properties of the triphosphates toward some RNA and DNA polymerases are reported

Human DNA polymerases and retroviral reverse transcriptases: selectivity in respect to dNTPs modified at triphosphate residues.

Abstract A series of dTTP and ddTTP(3′N3) γ-phosphonates and β,γ-diphosphonates are studied as substrates or terminating substrates towards different human DNA polymerases and retroviral reverse transcriptases.

Synthesis and Substrate Properties of Thymidine 5′-Triphosphate Analogs with Large Hydrophobic Substituent Groups At α-P Atom

Abstract The new thymidine 5′-O-triphosphate analogs—thymidine 5′-O-α-phenylphosphonyl-β, γ-diphosphate 4, 3′-fluoro-3′-deoxythymidine 5′-O-α-phenylphosphonyl-β, γ-diphosphate 5 and thymidine 5′-O-α-decylphosphonyl-β, γ-diphosphate 6 were synthesized. Their substrate properties towards avian myeloblastosis virus and human immunodeficience virus reverse transcriptases, DNA polymerase β from rat liver and terminal deoxynucleotidyltransferase from calf thymus were demonstrated. The mixture of Rp and Sp diastereomers of 4 was separated by reversed phase HPLC, diastereomeric selectivity of the reaction of DNA chain elongation was noted.