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S Litvak

Publications and source records attributed to S Litvak.

At least 73 records · Page 4Linked to original sources
PubMed1993

Nuclear and kinetoplast DNA synthesis in Trypanosoma cruzi, autoradiographical study with DNA polymerase inhibitors.

DNA synthesis in epimastigote forms of Trypanosoma cruzi was studied autoradiographically by incorporation of [3H]thymidine in the nuclear and kinetoplast DNA. Both DNA were heavily labelled. Three eukaryotic DNA polymerase inhibitors (aphidicolin, aracytidine, and dideoxythimidine) were chosen to study the nuclear and kinetoplast DNA synthesis in vivo. Inhibition was mainly observed with the nuclear DNA.

Animals↗
PubMed1993

The ribonuclease H activity of HIV-1 reverse transcriptase: further biochemical characterization and search of inhibitors.

A recombinant homodimer p66/p66 of the HIV-1 reverse transcriptase (RT) was expressed in and purified from a protease-deficient strain of the yeast Saccharomyces cerevisiae. The RNase H activity associated with the homodimer was biochemically characterized. The effect of cations and the hybrid substrate specificity were studied. Some compounds which have been found to inhibit retroviral replication were tested as potential inhibitors of the retroviral DNA polymerase and RNase H activities. Most of these compounds inhibited preferentially the DNA polymerase activity. On the other hand, only suramin was found to inhibit RNase H more efficiently than DNA polymerase. As in the case of the DNA polymerase activity, the thiol-reacting agent N-ethylmaleimide (NEM) did not affect the RNAse H activity of HIV RT. When the effect of NEM was tested against E coli RNase H, a weak inhibitory effect was detected. Surprisingly, NEM strongly inhibits the same bacterial RNase H in the presence of a recombinant form of HIV RT devoid of nuclease activity. These results strongly suggest an interaction between E coli RNase H and HIV-1 RT.

Benzodiazepines↗
PubMed1993

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.

Animals↗
PubMed1992

In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription.

The molecular events involved in antisense-mediated inhibition of retroviral transcription were studied by analyzing the in vitro effect of antisense oligodeoxynucleotides on reverse transcription by Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT). Oligonucleotides have been designed to be complementary to three targets located in the 5' region of the HIV-1 RNA genome: the transactivating response element (TAR), the U5 region and a sequence contiguous to the primer binding site (PrePBS). Antisense oligodeoxynucleotides were used with their 3'-OH end either free or blocked by a dideoxynucleotide in order to avoid cDNA synthesis. Experiments with two recombinant forms of HIV RT, carrying or not RNase H activity, showed that antisense oligonucleotides can arrest reverse transcription by an RNase H-independent mechanism. The AntiTAR oligonucleotide did not affect reverse transcription. In contrast, the AntiU5 and AntiPrePBS oligonucleotides led to an efficient inhibition of both forms of HIV RT. In the case of the AntiU5, the inhibition obtained in the absence of the RNase H activity indicates that this effect can be related to features of the RNA secondary structure. The AntiPrePBS oligonucleotide did bind to its target only in the presence of PBS primer. Use of shifted oligonucleotides showed that the AntiPrePBS inhibitory effect depends on a cooperative annealing with the AntiPBS primer on the template.

Base Sequence↗
PubMed1992

Interaction of tRNALys with the p66/p66 form of HIV-1 reverse transcriptase stimulates DNA polymerase and ribonuclease H activities.

The precursor homodimeric p66/p66 form of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) possesses the DNA polymerase and RNase H activities involved in the synthesis of the double-stranded provirus DNA. Reverse transcription is initiated from tRNALys in the case of HIV-1. The present study confirmed that interactions between HIV-1 RT and tRNALys induce protein conformational changes and demonstrated that these interactions stimulate the enzymatic activities associated with the p66 subunit. Thus, the p66/p66 form of the enzyme is strongly stimulated in both DNA polymerase and RNase H activities. Preincubation of the enzyme with tRNA is an obligatory step to obtain the stimulatory effect. The affinity of template, primer, or substrate for RT p66/p66 did not change when the enzyme was preincubated with tRNALys at stimulatory concentrations; the interaction of tRNA with p66/p66 has an effect only on the maximal rate of polymerization. It is further shown that the RNase H domain of RT is much more accessible to protease attack than the DNA polymerase active site.

Chymotrypsin↗
PubMed1992

Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides.

Primer tRNA regions involved in the interactions between human immunodeficiency virus reverse transcriptase (HIV RT) and tRNA(Lys) were studied by digestion of primer with pancreatic ribonuclease in the presence or absence of HIV RT. The acceptor stem of tRNA(Lys) is not noticeably protected against nuclease action in the presence of HIV RT, while this enzyme clearly protects part of the anticodon and dihydrouridine loops of tRNA(Lys). The acceptor stem of primer tRNA was digested by RNase A only in the presence of the retroviral enzyme, suggesting a partial destabilization of this region by the HIV RT. Synthetic oligoribonucleotides, corresponding to the anticodon and the dihydrouridine loops, inhibited strongly reverse transcription, confirming the strong interaction of these tRNA regions with the enzyme.

Base Sequence↗
PubMed1992

Functional analysis of primers and templates in the synthesis of DNA catalyzed by human immunodeficiency virus type 1 reverse transcriptase.

The kinetics of copying of poly(A).(dT)n, poly(A).(U)n, poly(dA).(dT)n and poly(A).(dT)9-U by reverse transcriptase of human immunodeficiency virus-1 (HIV-1) has been studied and the binding affinity of the enzyme, for template or primer, determined. Short oligonucleotides and dTTP served as primers in the HIV-1 reverse-transcriptase-dependent DNA synthesis. Km and Vmax were measured as functions of the primer chain length; the logarithm of the values of both Km and Vmax increased linearly up to 10. For longer primers (n = 11 to n = 24) the increase of those values changes very little. The enhanced affinity of the primers, (dT)n or (U)n due to the formation of one complementary pair, A.dT, dA.dT, A.U was estimated as a factor of 2. A specific property of HIV-1 reverse transcriptase compared with other DNA polymerases (procaryotes, eucaryotes, other retroviruses and archaebacteria) was its higher affinity to riboprimers as compared to deoxyriboprimers. Relative initial rates when copying poly(A) or poly(dA) templates using different primers and various conditions were compared; the optimal temperature for the reaction of polymerization with poly(A) or poly(dA) templates and (U)10, (dT)10 or (dT)9-U primers was determined. The maximal activity of the enzyme in the case of poly(A) and decanucleotide primers was found at temperatures between 27-31 degrees C. An increase in the primer length results in the stabilization of the template.primer duplex complexed to the enzyme, thus increasing to more than 40 degrees C the optimal temperature of polymerization. The activation energy (Ea) values of the polymerization reaction for different template.primer complexes were evaluated.

DNA Replication↗
PubMed1992

Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme.

The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms. The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture.

Affinity Labels↗
PubMed1992

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.

Amino Acid Sequence↗
PubMed1992

Two DNA polymerases from Trypanosoma cruzi: biochemical characterization and effects of inhibitors.

1. Two DNA polymerases have been partially purified from Trypanosoma cruzi epimastigotes by DEAE-cellulose, phosphocellulose, and DNA agarose chromatography. 2. Both enzyme activities were characterized by several biochemical criteria. 3. They showed different sensitivity to KCl and displayed characteristic Mg2+ and Mn2+ requirements, although they exhibited almost identical primer-template utilization. 4. The preferred substrates were poly dC-oligo dG, activated calf thymus DNA, and poly dT-oligo rA. 5. Both enzyme fractions are not inhibited by aphidicolin while N-ethylmaleimide and phosphonacetic acid inhibited them to different extents. 6. ButylphenyldGTP strongly inhibited T. cruzi enzyme fraction I while it had no effect on enzyme fraction III. 7. This dGTP analog also inhibited the poly dT-directed polymerization of dAMP as described for other mammalian DNA polymerases. Kinetic studies indicated that butylphenyldGTP inhibited enzyme fraction I in a non-competitive fashion.

Animals↗
PubMed1991

DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase.

Studies in eucaryotic cells (mainly animals and yeast) indicate that at least two DNA polymerases are involved in DNA replication at the level of the replication fork: DNA polymerase alpha, which is associated with DNA primase, is involved in the replication of the lagging strand; DNA polymerase delta, associated with an exonuclease activity, synthesizes the forward continuous DNA strand. Much less information exists concerning plant systems. Previous work from this laboratory provided preliminary evidence of an association between DNA polymerase B from wheat embryo and an exonucleolytic activity. In this paper, we present additional data on the biochemical properties of DNA polymerase B. An improved purification procedure described in this article has been developed. During all the purification steps the nuclease activity was associated with DNA polymerase activity. A biochemical study of this enzyme activity shows that it is an exonuclease which hydrolyses DNA in the 3' to 5' direction. Moreover, this exonuclease confers a proofreading function to DNA polymerase B. Comparison of DNA polymerase B properties (template specificity, sensitivity to DNA replication inhibitors like aphidicolin and butyl-phenyl dGTP, copurification of DNA polymerase and exonuclease activities) with those of animal DNA polymerase delta indicates that these enzymes share many common features. To our knowledge, this is the first report of DNA polymerase delta in higher plants.

Adenosine Monophosphate↗
PubMed1991

Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis.

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.

Cell Fractionation↗
PubMed1990

Interactions with tRNA(Lys) induce important structural changes in human immunodeficiency virus reverse transcriptase.

Retroviral RNA-dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion-encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNA(Lys3) is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNA(Lys.) By fluorescence studies and alpha-chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNA(Lys) although the retroviral polymerase is able to interact with other tRNAs.

HIV↗
PubMed1990

Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.

DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.

Chromatography, DEAE-Cellulose↗
PubMed1990

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Amino Acid Sequence↗
PubMed1990

Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase.

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.

Chromatography, Affinity↗