Search

The potential relationship between helicase structural organization and unwinding mechanism is examined by performing in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. The results have outlined the differences as well as the similarities in the mechanism of unwinding between Rho and other hexameric helicases, which are discussed in relation with the biological function of the Rho helicase.

Site-specific fluorescent labeling of RNA molecules is achieved by specific transcription using an unnatural base pair system. This method is useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.

The steady-state kinetic measurements are extended to two replicative DNA polymerases, exonuclease-deficient T7 DNA polymerase T7(super -) and HIV reverse transcriptive (HIV-RT). The influences of G[8-5]C on the thermal stability and base pairing properties of DNA duplexes are examined.

An approach for inhibiting the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-I) reverse transcriptase (RT) is demonstrated by targeting RNase H RNA.DNA hybrid substrates. In this approach the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin and ribostamycin, to two different chimeric RNA-DNA duplexes inhibits specific RT-mediated RNase cleavage in competitive manner.

A helix-turn-helix motif in the crystal structure of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was proposed to be a conserved nucleic acid binding domain among several nucleotide polymerizing enzymes (Hermann, T.; Meier, T.; Gotte, M.; Heumann, H. Nucleic Acids Res. 1994, 22, 4625-4633). The sequence of this domain is homologous to (super 259)KLVGKL-(X)(sub 16)KLLR(super 284) of HIV-1 RT, which acts as a 'helix clamp' grasping the template-primer (T-P) complex. We characterized the helix clamp motif using MALDI-TOF MS and surface plasmon resonance (BIAcore). Our studies showed that the 'helix clamp' has a nucleic acid binding function that may not be sequence specific. This evidence suggests that ionic interactions between the helix clamp and oligonucleotide backbone are not solely responsible for binding. Secondary and tertiary structures of the protein may also play a significant role in nucleic acid binding. The association and dissociation constants, k(sub a) and k(sub d), for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 x 10(super 3) M(super -1) s(super -1) and 1.22 x 10(super 3) s(super -1), respectively.

Research has been conducted on the YXDD motif of the reverse transcriptase family. The evidence that reverse transcriptases catalyze DNA synthesis is presented.

The HIV-1 reverse transcriptase-RNA/RNA-dCTP ternary complex has a different conformation than complexes involving DNA/DNA and DNA/RNA. Because of this, dCTP incorporation is significantly less than that for DNA/DNA and DNA/RNA substrates.

Researchers developed a synthetic peptide that inhibits the conformational change that HIV reverse transcriptase undergoes during dimerization. The enzyme is a heterodimer and the thumb domain of p51 is essential for dimerization.

Research was conducted to identify the inhibitors of DNA strand transfer reactions. A chemical mass screening assay was created using a novel application of scintillation proximity technology that was specifically aimed at detecting inhibition of one or more steps of the DNA strand transfer process. The identification of a broad range of inhibitors against DNA strand transfer indicates that the complex reaction could be an important target for drug discovery and development.

Most of the pre-steady-state kinetic studies of HIV-1 reverse transcriptase (HIV-1 RT) have shown substoichiometric bursts of product formation in the initial reaction cycle. The extinction coefficient of the HIV-1 RT heterodimer was altered by applying quantitative amino acid analysis. It was observed that normal nucleotide incorporation occurs with quantitative bursts in the first cycle. Findings showed that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with altered systems are well-defined by the presence of nonproductive ternary complexes, which can isomerize to productive complexes.

Research was conducted to examine the kinetics for the incorporation of dCMP, ddCMP and (+)SddCMP and its unnatural beta-L-(-) enantiomer, (-)SdCMP, into DNA/DNA and DNA/RNA primer-templates catalyzed by HIV-1 RT. The objective was to gain an insight into the differences between RNA-dependent and DNA-dependent polymerization. Results suggest that perturbations on the ribose ring of cytidine analogues decrease the rate and efficiency of incorporation but improves the binding affinity.

The multifunctional HIV reverse transcriptase (RT) enzyme catalyzes RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and RNase H activities. Four crystallographic analyses of HIV-1 RT are reported in complexes with different carboxailides, with the aim of gaining an understanding of the differing inhibition of mutant RTs, and clarifying previous studies.

The topography and functional features of the complex produced in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNA(super Lys3) were investigated. Chimeric primers having tRNA(super Lys3) linked to U and T residues of various lengths were synthesized. Results showed that the affinity of the oligonucleotide primers for HIV-1 RT is drastically enhanced when linked to primer tRNA.

The reason for the seemingly slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and the template are made of RNA, was investigated through single-turnover and equilibrium measurements. Results showed that binding and not catalytic characteristics of the initiation p/ts cause the overall slow, nonprocessive polymerization rate during initiation.

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 reverse transcriptase (HIV-1 RT) and bacteriophage T7 exo- (T7-) were investigated. The goal was to examine the misincorporation potential for 8-oxo-dGTP and to study the function of base pairing symmetry in DNA polymerase fidelity. Results indicated that polymerase fidelity does not rely only on the overall geometry of Watson-Crick base pairs and the enzyme binding site is asymmetrical.

A new study compares the differing modes of DNA synthesis used by heterodimeric equine infectious anemia virus (EIAV) p66/51 and homodimeric EIAV p51/51. Analysing the dimerization behaviour shows that the dimer stability of HIV-1 reverse transcriptase enzyme is lower than that of EIAV transcriptase. It is suggested that the homodimetric enzyme forms a conformation similar to p66/51.

RNA primers initiate plus-strand synthesis and minus-strand synthesis of HIV replication. A new study analyses the structure of the 3'-DNA-RNA-5' junction formed during the commencement of minus-strand synthesis of HIV replication. It is shown that the hybrid octamer which corresponds to the DNA-RNA junction adopts a global A-type conformation throughout its length.

A study was conducted to examine the possible function of the p51 subunit of HIV-1 reverse transcriptase (RT) in the template-primer binding process. The effects of acetonitrile addition on the polymerase activity of HIV-1 RT were also studied before and after the templaten primer binding step. Results indicated that acetonitrile dissociated the subunits of the HIV-1 RT into catalytically inactive monomers.

A study was conducted to analyze the fidelity of mutant HIV-1 reverse transcriptases using random sequence mutagenesis and complementation in a bacterial system. A complementation system in Escherichia coli that allowed genetic selection for DNA-dependent DNA polymerase activity of HIV RT was used in the study. Results suggested mechanisms controlling the replication accuracy of HIV RT based on drug-resistant mutants.

A study was conducted to examine the ability of 5'-triphosphates to inhibit HIV reverse transcriptase (RT) to analyze the preference of 3'-azido-3'-deoxythimidine (AZT) for the extreme (sub 3)E conformation. Conformational preferences were corroborated by X-ray analysis. Results indicated that the inhibition of RT only occurred with the conformotionally locked (sub 2)E (N)-methano-carba-AZT 5'-triphosphate.

The effect of RNA secondary structure switching in in vitro replication of the human immunodeficiency virus reverse transcriptase was investigated through the use of rapid kinetic methods. Results obtained showed that the RNA secondary structure switching mediated the translational regulation, protein synthesis and mRNA splicing. Also, the switching played an important role in replication catalyzed by the HIV-RT in vitro.

It is possible that the RNA dependent DNA replication event plays a role in the generation of 3'-azido-3'-deoxythymidine (AZT) resistance. Research indicates that RNA dependent DNA polymerization function of reverse transcriptase (RT) may play a more significant role in the generation of resistance to AZTTP than DNA dependent DNA polymerization. No differences were seen in the cleavage patterns of the degraded RNA template for mutant RT and wild type. This finding does not support the possibility of an altered binding mode for the mutant compared to wild type RT.

Research designed to compare replication fidelity of HIV-1 reverse transcriptase (RT1) using DNA/DNA and RNA/DNA template-primer substrates has established that there are differences between DNA and RNA substrates. RT showed high fidelity, indicating that the high degree of genomic variation of HIV-1 may be attributable more to a defense mechanism of the virus to escape the surveillance of the host immune system than to insufficient regulation on the part of RT.

The thiocarboxanilide UC781 was studied to determine its use in the retrovirucidal/microbiocidal formulations in preventing HIV sexual transmission from infected individuals to noninfected individuals. Results reveal that thiocarboxanilde UC781 is a tight-binding inhibitor of HIV-1 RT. The treatment of UC781 with isolated HIV-1 virions leads to virus ineffectivity. This suggests that the compound is useful in the formulation of retrovirucidal.

The nucleotide conformational alteration in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was studied using nondenaturing gel electrophoresis. Dead-end complex was formed in the presence of deoxynucleotide triphosphate (dNTP) between dideoxynucleotide chain-terminated primer, HIV-1 RT and DNA template. Dead-end complex formation by a complementary nucleotide was a minimum of 2000-times more efficient than with ribonucleotides or noncomplementary dNTP's.

The contribution of primer grip residues of human immunodeficiency virus type 1 reverse transcriptase to its palm subdomain architecture and nearby structural elements was studied by analyzing the ribonuclease H and DNA polymerase activities of enzymes where aromatic substitutions had been made at Tyr232 and Trp229. Research suggests that oligoribo- and oligodeoxyribonucleic acid primers may be discriminated between as a result of small changes to primer grip architecture.

Enzymatic profiles of recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with five mutations (A62V, V75I, F77L, F116Y and Q151M) which confers resistance to multiple 2',3'-dideoxynucleosides (ddNs) on HIV-1 were examined. Findings indicated that RT(sub A62V/V75I/F77L/F116Y/Q151M) is insensitive to ddATP, AZTTP, D4TTP, FddATP and ddCTP but is insensitive to PMEApp, 3TCTP and FddCTP.

Site directed mutagenesis of Arg 110 was performed, replacing it with Ala, Lys and Glu to determine whether Arg 110 of murine leukemia virus (MuLV) reverse transcriptase (RT) is a significant contributor to the catalytic mechanism of MuLV RT. Findings indicated that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation and that Arg 110 of MuLV RT contributes to the conformational change steps before and after the chemical step of polymerase reaction.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin were screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase on a poly(rA)*oligo template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin and trans-vinyl-linked bisdistamycin, indicated improved inhibition in reactions started with pre-incubated enzyme template-primer and reduced affinity for a free TP analog, when compared with the parent drug distamycin.

Amplification of oligonucleotide repeats may be due to an aberration in the copying process of polymerases. It is shown that in the presence of MnCl2, mismatch formation commits HIV-1 reverse transcriptase to a new mode of DNA synthesis which yields repetitive products. This activity is distinct from terminal transferase since it needs specific DNA motifs in the template. This mechanism also works on homologous RNA templates where it produces reiterative products more than 150 nucleotides long.

Alterations in Leu(super 234) in the primer grip of the p66 subunit of the human immunodeficiency virus 1 reverse transcriptase produces a subunit that fails to associate into a heterodimer. Mutations in any residue between Trp(super 229) and Tyr(super 232) change the functions associated with DNA polymerase and ribonuclease H domains. An analysis of retroviral sequences indicates that aromatic residues are preferred at positions 229 and 232. The residues probably interact and position the template-primer, and supply the architecture necessary for primer grip.

Reverse transcriptase enzyme derived from the human immunodeficiency virus type 1 can initiate transcription on any site along the human DNA strand as soon as it recognize a threshold concentration of substrate. The enzyme binds to its DNA template in two phases in which it forms an outer binary complex and then undergoes conformation changes into an inner complex. The transition from inner to outer complex becomes the rate-determining step for synthesis.

The specific binding of the human tRNA3Lys, prepared in vitro with no base modifications, to human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), was investigated relative to primer/template (p/t) binding, and verified by extrinsic fluorescence titrations. Affinity calculations indicate the nonselective binding of HIV-1 RT to tRNA3Lys which increases under low ionic strength conditions. This implies that the structure and charge of the tRNA3Lys are the major contributing factors in domain identification prior to binding with HIV-1 RT.

The replacement of 184 methionine of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) with valine at codon 184 adds to the fidelity and efficiency of DNA synthesis. M184 is the second residue of the conserved YXDD motif, located in RNA dependent DNA polymerases. The mutants M184A and M184V in which the Met residue is replaced with Ala or Val are catalytically more efficient than wild type enzyme. A three dimensional molecular model of HIV-1 RT ternary complex reveals that the position of M184 permits interaction with sugar motif of the primer nucleotide or dNTP substrate.

The p68/p58 heterodimer of the recombinant human immunodeficiency virus type 2 reverse transcriptase (RT) shows RT and ribonuclease H activities. The heterodimer obeys Michaelis-Menten kinetics, and its activity is suppressed by the DNA chain terminator dd thymidine triphosphate. The heterodimer has 574 amino acids and a molecular weight of 68 kDa. It is enzymatically different from the p68/p68 homodimer. The crystallization of the heterodimer opens up possibilities for producing drugs targeted against the replicative processes of HIV-2.

The role of the region in reverse transcriptase (RT) containing the tyrosine to cysteine mutation at position 181 (Y181) in the polymerization mechanism of RT was investigated using pre-steady-state techniques. The effect of Y181 mutation on the affinity of the nonnucleoside inhibitor Nevirapine to the enzyme, on nucleotide equilibrium and catalytic turnover of the mutant E.DNA complex was also evaluated. A biochemical analysis of the Y181 mutant is provided.

Single stranded DNA (ssDNA) molecules can be used as inhibitors or models for making small molecule inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) in vivo. The RT of HIV-1 plays an important role in the synthesis of a double-stranded DNA replica of the RNA genome to be integrated into the host chromosome. Using a method known as systematic evolution of ligands by exponential enrichment (SELEX) on an ssDNA library, binding of the selected ssDNA ligands to HIV-1 RT occurs, which causes inhibition of RNA-dependent DNA-polymerase activity. Inhibition is specific for HIV-1 RT, with little effect on other RTs.

Two non-nucleoside inhibitors of the HIV reverse transcriptase (RT) enzyme bind to different forms of the enzyme-substrate complex. Oxathiin carboxanilide (UC84) and a second-generation thiocarboxanilide (UC38) can inhibit HIV-1 RT in vitro. UC84 binds to the free enzyme and the enzyme-primer/template (P/T) complex, but not to the enzyme-P/T-dNTP complex. UC38 binds to the enzyme-P/T-dNTP complex but not to the free enzyme or the enzyme-P/T complex.

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.

Restriction of HIV-1 reverse transcriptase, mediated by 2'5'-Oligoadenylates (2-5A) is assessed. Metabolically steady 2-5A and its derivatives are a unique group of restrictors in the HIV-1 life cycle. 2', 5'-phosphorothioates which stimulate the natural cellular antiretroviral defense process and restrict HIV-1 duplication may be very useful for treatment of HIV-1 diseases.

The inhibitory activity of Bis (heteroaryl) piperazine U-88204 E on HIV-1 reverse transcriptase (RT) is analogous to that of AZT, ddI, ddC is useful in the treatment of AIDS patients. Thus, there is a great need for research involving RT inhibitors. U-88204E exhibits antiviral activity at nontoxic doses in the HIV-1 populated lymphocytes in cultured tissues.

The study of the activities of AKR leukemia virus enzymes reveals the functional relationship between the polymerase and RNase H domains of reverse transcriptase. The RNase H domain functions independently of the polymerase domain. Mutation between the two active centers alters the spatial relationship between them. An enzyme with two interdependent domains in the alteration of the normal polymerase RNase H ratio can affect RNase H cleavage and polymerase activities.

Affinity labeling of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by oligothymidylate derivatives was described. The derivatives contain chemically reactive groups either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside thiophosphate group of the oligonucleotide. The results suggest that the mechanism of primer binding to HIV-1 RT is different in the presence or absence of template. However, the 3' position of the primer interacts with the same specific domain of the enzyme active site in both cases.

The fidelity of reverse transcritase (RT) 1 and 2 from the HIV and murine leukemia virus RT (MLV RT) were compared using a primer extension assay using ribosomal RNA as primer. Results show that all three RTs are efficient extensors of the 3' end of mispaired bases. However, HIV RTs were more prone to qualitative as well as quantitative errors compared to MLV RT. This may account for the high frequency of mutation among HIVs.

Two-dimensional inverse-detected heteronuclear 15N-1H nuclear magnetic resonance spectroscopy was used to study the backbone dynamics of 15N-labeled ribonuclease H (RNase) domain of human immunodeficiency virus (HIV-1) reverse transcriptase. The RNase H domain was found to exhibit mobility throughout its structure. In addition, the C-terminal residues from Tyr-110 to Leu138 and the loops between beta-strands beta1 and beta2 and between alpha-helix alphaB and beta-strand beta4 showed increased overall mobility relative to the remainder of the protein. These results suggest a substrate binding site conformation that explains the lack of enzymatic activity of RNase H domain.

Human immunodefficiency virus reverse transcriptase is synthesized as a 66kDa protein, proteolytically cleaved and processed to produce a heterodimer consisting of a 66kDa- and a processed 51kDa-subunit. The single subunits and the heterodimer were subjected to circular dichroism spectroscopy to determine conformational alterations resulting from the association. Results indicate that much conformational change results from the association of the two subunits. Further studies revealed that the heterodimer has a catalytic advantage when enzyme and template concentrations are below optimum levels.

The search for a cure to acquired immunodeficiency syndrome (AIDS) inspires a kinetics study of reverse transcriptase inhibitors. Reverse transcriptase is responsible for changing thesingle-strand ribonucleic acid (RNA) of the human immunodeficiency virus (HIV) to a double-strand deoxyribonucleic acid (DNA). Four natural deoxynucleotidetriphosphate substrates and a series of analogues are tested fortheir inhibition efficacy. Results show that slow dissociation of the enzyme-template-primer complex saturates and thus inhibits the enzyme at low substrate concentrations. Other results and their analysis are presented.

The contribution of 3'-azido-3'deoxythymidine 5'-triophosphate (AZTTP) to inhibition against human immunodeficiency virus-reverse transcriptase (HIV-RT) was studied. The findings show that the low constant of inhibition, Ki, value for AZTTP against HIV-RT is due to the use of a homotemplate-primer /poly(rA)-oligo(dT)~. Ki was found to be dependent on the length of the poly(rA) region of the RNA template and inversely proportional to the number of adenosyl residues available for incorporation of thymidine monophosphate residues.

An assay for measuring the fidelity of reverse transcription during RNA- and DNA-dependent polymerization in vitro was developed. A T3 RNA polymerase-transcribed portion of lacZalpha RNA was used tomimic first-strand reverse transcription, while its DNA template was used to analyze reverse transcriptase errors during second-strand DNA synthesis. Results indicate that, compared with the reverse transcriptase from Moloney murine leukemia virus or Escherichia coli DNA polymerase I's Klenow fragment, reverse transcriptase obtained from HIV-1 showed less accuracy on the RNA or DNA templates.