Search

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature ( T m ) of the limiting primer ( T m L ) relative to that of the excess primer ( T m X ) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when T m L - T m X ≥ 5°C. We also investigated how altering T m X relative to the higher T m of the double-stranded amplicon ( T m A ) affects the rate and extent of linear amplification. Excess primers with T m X closer to T m A yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator ( CFTR ) gene, mutations of which are responsible for cystic fibrosis.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.

Objetivos: Diseñar una estrategia alternativa por PCR para el genotipado de secuencias ricas en citosinas, basada en modificación nucleotídica. Material y métodos: Se modificó el gen FMR1 nativo de ocho individuos clínicamente no afectados por el Síndrome X frágil, cambiando las citosinas por uracilos, empleando bisulfito de sodio. El ADN modificado fue purificado y cuantificado por espectrofotometría. Las estructuras alternativas y potenciales islas CpG que adopta el microsatélite inestable fueron simuladas con los programas MFOLD y CpGplot. Se generaron cebadores específicos que hibriden tanto con el microsatélite modificado (Primer T) y con una secuencia modificada de las islas CpG (Primer M), utilizando el programa MethPrimer. Finalmente, ambas secuencias fueron amplificadas por PCR y los amplicones fueron separados por electroforesis en gel de poliacrilamida (PAGE por sus siglas en inglés) al 6% y visualizados con tinción de nitrato de plata. Resultados: La modificación del ADN fue evidenciada por espectrofotometría al uracilo. Las estructuras observadas en la simulación fueron las horquillas encontrándose dos potenciales islas CpG. La amplificación con los cebadores T, confirmó el diseño in silico desarrollado para abordar la estructura en horquillas. La amplificación con los cebadores M permitió detectar metilación de la primera isla CpG del gen FMR1.Conclusión: Se propone un diseño alternativo para amplificación de secuencias de microsatélite que contengan citosinas metiladas y no metiladas. Se requieren estudios posteriores con muestras de ADN que contengan microsatélites muy expandidos para validar su aplicación para diagnóstico molecular. Objectives: To design an alternative strategy for genotyping cytosine-rich sequences using PCR and nucleotide modification. Methods: The FMR1 gene wild type was modified in the DNA obtained from eight individuals clinically unaffected for Fragile X Syndrome; cytosines were replaced by uracils using sodium bisulfite. Modified DNA was purified and quantified by spectrophotometry. Alternative structures and potential CpG islands of the unstable microsatellite were simulated using MFOLD and CpGplot tools. Specific primers were generated to hybridize with both the modified microsatellite (Primer G) and a modified sequence of CpG islands (Primer M) using the MethPrimer software. Finally, both sequences were amplified by PCR and the amplicons were separated by electrophoresis in silver-stained PAGE 6% gels. Results: The DNA modification was evidenced by spectrophotometry to uracil. We found two potential CpG islands. The amplification with T primers confirmed the "in silico" design developed to engage hairpin structures. The amplification with M primers detected methylation of the first CpG island in the FMR1 gene. Conclusion: We propose an alternative design for amplifying microsatellite sequences that contain methylated and unmethylated cytosine bases. Further studies are required with DNA samples containing expanded microsatellites to validate its molecular diagnostic application

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

We hypothesized that the thermal stability of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) will increase with increases in its ability to bind with a template-primer (T/P). To test this hypothesis, we introduced positive charges into MMLV RT by site-directed mutagenesis at positions that have been implicated in the interaction with T/P. Thirty-six variants were constructed in which one of the twelve residues (Glu69, Gln84, Asp108, Asp114, Glu117, Glu123, Asp124, Glu286, Glu302, Trp313, Leu435, and Asn454) was replaced with Lys, Arg, or Ala, and these were expressed in Escherichia coli. In about half of these 36 variants, thermal inactivation at 50°C was reduced in the presence of the T/P, which suggested that this strategy was effective at stabilizing MMLV RT. We next combined three of the 36 mutations, Glu286→Ala, Glu302→Lys, and Leu435→Arg, and the mutation, Asp524→Ala, which is known to abolish the RNase H activity and increase the stability. Temperatures of 54 and 56°C reduced the initial reverse transcription activity by 50% over a 10-min incubation in the triple variant E286R/E302K/L435R and quadruple variant E286R/E302K/L435R/D524A, respectively. These temperatures were higher than that observed for WT (45°C). The highest temperature at which the triple and quadruple variants exhibited cDNA synthesis activity was 60°C, which was again higher than for WT (54°C). Thus, highly stable MMLV RT variants were generated by this mutation strategy.

Reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukaemia virus (MMLV) have been most extensively used as a tool for conversion of RNA to DNA. In this study, we compared the thermal stabilities of AMV RT and MMLV RT by observing their irreversible thermal inactivation. The temperatures reducing initial activity by 50% in 10-min incubation, T(50), of AMV RT were 47 degrees C without the template-primer (T/P), poly(rA)-p(dT)(12-18), and 52 degrees C with the T/P (28 microM). T(50) of MMLV RT were 44 degrees C without the T/P and 47 degrees C with the T/P (28 microM). Unexpectedly, AMV RT was considerably activated when incubated with the T/P at 45 and 48 degrees C. Such activation was not observed in MMLV RT. These results suggest that AMV RT and MMLV RT are different in the following: (i) The intrinsic thermal stability of AMV RT is higher than that of MMLV RT; (ii) AMV RT is activated by thermal treatment with the T/P at 45-48 degrees C; and (iii) AMV RT is stabilized by the T/P more potently than MMLV RT. Thermodynamic analysis indicates that thermal inactivation of AMV RT and MMLV RT is due to the large entropy change of activation for thermal inactivation.

Surface plasmon resonance (SPR) biosensor technique was applied to analyze the human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) binding to DNA template–primer (T–P) duplexes. The kinetics including the initial binding and the subsequent conformation change of the protein–DNA complex in the absence and presence of various chemicals were determined. The effects of three nucleoside-based reverse transcriptase inhibitors (NRTIs) and three non-nucleoside-based reverse transcriptase inhibitors (NNRTIs) on the RT–DNA interaction were analyzed based on the conformation change model. Efavirenz (EFV) slightly affected the binding kinetics of the RT–DNA interaction and the binding affinity ( K A = 2.99 × 10 8 M −1 ) is lower than that in the absence of the inhibitor ( K A = 5.61 × 10 8 M −1 ). Nevirapine (NVP) induced RT to bind tightly to DNA duplexes followed by a conformation change step. The action of an unsaturated fatty acid, nervonic acid (NA) was similar to NVP. The dideoxynucleosides, whether complementary (‘correct’) or non-complementary (‘incorrect’) to the bases of 3′-end of template–primer DNA, showed no effect on the binding of RT to DNA. However, the initial association and dissociation rates for the RT–DNA interaction are greater in the presence of ‘correct’ dideoxynucleosides than that of in the presence ‘incorrect’ ones, indicating slight preference of RT to ‘correct’ bases. The azido group of AZT (3′-azido-3′-deoxythymidine) enhanced the dissociation of RT from DNA in the initial binding step, where the absence of which resulted in a 34-fold increase in the overall binding affinity ( K A = 1.94 × 10 10 M −1 ).

Surface plasmon resonance biosensor technique was used to study the binding of Moloney murine leukemia virus reverse transcriptase without RNase H domain (MMLV RT-) with DNA in the absence and in the presence of inhibitors. Different DNA substrates, including single-stranded DNA (ssDNA), DNA template-primer (T-P) duplex and gapped DNA, were immobilized on the biosensor chip surface using streptavidin-biotin, and MMLV RT(-)-DNA binding kinetics were analyzed by different models. MMLV RT-; could bind with ssDNA and the binding was involved in conformation change. MMLV RT-; binding DNA T-P duplex and gapped DNA could be analyzed using the simple 1:1 Langmuir model. The lack of RNase H domain reduced the affinity between MMLV RT-; and T-P duplex. The effects of RT inhibitors, including efavirenz, nevirapine and quercetin, on the interaction between MMLV RT-; and gapped DNA were analyzed according to recovered kinetics parameters. Efavirenz slightly interfered with the binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.21 x 10(6) M(-1)) was lower than in the absence of the inhibitor (KA = 4.61 x 10(6) M(-1)). Nevirapine induced relatively tight binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.47 x 10(7) M(-1)) was approximately three folds higher than without nevirapine, mainly due to rapid association and slow dissociation. Quercetin, a flavonoid originating from plant which has previously shown strong inhibition of the activity of RT, was found to have minimal effect on the RT-DNA binding.

HIV-1 Reverse Transcriptase (RT) converts viral RNA into dsDNA that is integrated into the human genome. Nonnucleoside RT inhibitors (NNRTIs) are highly effective in the treatment and prevention of HIV. NNRTI resistance mutations arise from therapy, yet the mechanism(s) of how these mutations inhibit polymerization by RT is unclear. We examine the role of NNRTI on the dynamics between RT and its Template/Primer (T/P) substrate using a combination of molecular modeling, single-molecule and bulk fluorescence techniques and provide an unprecedented glimpse into the dynamics of RT-T/P interaction as well as the intramolecular conformation of RT itself while bound to its substrate. The data suggests a unique mechanism of resistance that is mediated by interplay between intramolecular conformational changes in RT and intermolecular dynamics of the RT-template/primer-dNTP complex.

Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by extension of a tRNA primer that is associated with the viral genome, and plus-strand DNA synthesis is initiated from an RNase H- resistant polypurine tract of the genomic RNA that remains bound to the newly synthesized minus-strand DNA. All other phases of reverse transcription represent elongation of a DNA primer. We demonstrate that the polymerase fidelity of RT enzymes is significantly higher in tRNA-primed reverse transcription compared with DNA-primed reactions. Two mechanistic explanations can be proposed. First, the type of template-primer (T- P) duplex (RNA-RNA versus RNA-DNA) may affect the RT enzyme conformation such that the discrimination against incorrect nucleotides is affected. Second, the tRNA primer may act as a fidelity co-factor through specific association with the RT enzyme. According to the latter hypothesis, the increased fidelity observed for an RNA-RNA T-P should persist at a distance from the initiation site, where the enzyme-bound nucleic acid duplex will consist of RNA-cDNA. However, we measured that the effect of tRNA on the fidelity is detectable only at a short distance from the initiation site. These results indicate that the type of T-P duplex influences the fidelity of reverse transcription, suggesting that two small segments of the viral genome downstream of the initiation sites for minus- and plus-strand DNA synthesis are copied with a fidelity that is greater than average.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).

In the fourth unit, students extract genomic DNA from uncultured bacterial populations from environments that they choose, and they use polymerase chain reaction (PCR) to amplify bacterial 16S ribosomal RNA (rRNA) genes. The PCR product synthesized will likely contain a mixture of amplicons derived from 16S rRNA genes from each of the bacterial species in the sample. The terminal restriction fragment length polymorphism (T-RFLP) method uses one fluorescently labeled primer and one unlabeled primer so that one end of the amplicon has a fluorescent tag. The students digest the amplicons with a restriction endonuclease and use capillary electrophoresis to detect restriction fragments containing the labeled primer. T-RFLP analysis indicates the complexity of a bacterial population, and differences (or similarities) between two bacterial populations may become obvious by comparing their T-RFLP patterns.

The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T·P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T·P binary complex (RTT·P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T·P (for single-nucleotide incorporation). To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3′-azido-3′-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T·P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Kmshould be equivalent to Ki, and since Kmfor AZTTP should be influenced by the dissociation rate constant for RTT·P, we examined the effect of altering RTT·Pdissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T·P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T·P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16was biphasic, with a burst followed by a slower steady-state phase representing kcat(0.42 min−1) which was similar to the rate constant for RTT·Pdissociation. Additionally, Kmfor AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (KiAZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT·Pdissociation rate constant and KiAZTTPwas not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT·Pexist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T·P where the binary RTT·Pcomplex undergoes an isomerization before binding of the deoxynucleotide substrate.

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.

We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K(d)) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k(pol)). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K(d) and k(pol) values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.

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.; Götte, M.; Heumann, H. Nucleic Acids Res. 1994, 22, 4625-4633). The sequence of this domain is homologous to 259KLVGKL-(X)16KLLR284 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, ka and kd, for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 x 10(3) M(-1) s(-1) and 1.22 x 10(3) s(-1), respectively.

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.

Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum, T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.

HIV-1 reverse transcriptase (RT) is entirely responsible for the conversion of the viral RNA genome into double stranded DNA. Due to its essential role in virus replication, it is a primary target for drug development. In order to rationally modulate RT binding to its Template/Primer (T/P) substrate using novel inhibitory compounds, an accurate description of DNA-protein binding kinetics in the absence of ensemble averaging is crucial. We are therefore developing a robust single-molecule assay to directly measure the association/dissociation rates of protein-nucleic acid complexes at equilibrium. Utilizing this method in comparison with bulk methods, we describe in detail the effects of various known RT inhibitors (RTIs) as well as novel RTIs under development in our laboratory on the binding kinetics of individual RT-T/P complexes.