SYBR Green I: fluorescence properties and interaction with DNA.

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.

Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation.

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (~1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity.

Metal-enhanced PicoGreen fluorescence: application for double-stranded DNA quantification.

PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 microM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification.

Validation of peptide mapping with electrospray mass spectrometry for recombinant proteins of biopharmaceutical interest and its applications as an identity test and a characterization tool.

In this paper, the steps required to validate a liquid chromatography peptide mapping method with mass spectrometric detection (LC-MS) for use as an identity test and characterization tool are presented. All aspects of peptide mapping are evaluated and optimized, including protein sample preparation (protein reduction, alkylation and enzymatic digestion), high performance liquid chromatography (HPLC) separation of the resulting peptides, and the use of a mass spectrometric detection. In addition, the validation of a single quadruple MS detector is described and the implementation of on-line electrospray ionization MS (ESI-MS) as an adjunct detector to support the investigation of peak differences is presented. Applications of peptide mapping with tandem MS using an electrospray ion-trap instrument throughout the biopharmaceutical product development cycle are discussed, including assessing protein product heterogeneity derived from post-translational modifications (e.g. multiple N- or C-termini, deamidation, oxidation and glycosylation) and protein degradation.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Phosphorescence and optically detected magnetic resonance of HIV-1 nucleocapsid protein complexes with stem-loop sequences of the genomic Psi-recognition element.

The binding of NCp7, the nucleocapsid protein of human immunodeficiency virus type 1, to oligonucleotide stem--loop (SL) sequences of the genomic Psi-recognition element has been studied using fluorescence, phosphorescence, and optically detected magnetic resonance (ODMR). RNA SL2, SL3, and SL4 constructs bind with higher affinity than the corresponding DNAs. G to I substitutions in the SL3 DNA loop sequence lead to reduced binding affinity and significant changes in the triplet state properties of Trp37 of NCp7, implicating these bases in contacts with aromatic amino acid residues of the zinc finger domains of NCp7, in agreement with the NMR structure of the 1:1 complex of NCp7 and SL3 RNA [DeGuzman, R. N., Wu, Z. R., Stalling, C. C., Pappaladro, L., Borer, P. N., and Summers, M. F. (1998) Science 279, 384-388]. The NCp7 to SL binding stoichiometry is 2:1 for intact SL sequences but is reduced to 1:1 for SL variants with an abasic or hydrocarbon loop. It is proposed that Delta D/Delta E(0,0), where Delta D is the change in the zero-field splitting D parameter and Delta E(0,0) is the shift of the tryptophan phosphorescence origin, provides a measure of aromatic stacking interactions with nucleic acid bases. Values on the order of 10(-5) indicate significant stacking interactions, while values closer to 10(-6) result from interactions not involving aromatic stacking. Binding of NCp7 to oligonucleotide substrates produces shortened Trp37 triplet state lifetimes by enhancement of k(x) and an increase of the relative value of P(x), the intersystem crossing rate to the T(x) sublevel. These effects are attributed to a reduction in the degree of electronic symmetry of Trp37 in the complexes. Guanine and adenine triplet states produced by optical pumping of SL3 DNA are characterized. We find, as with tryptophan, that D < 3E.

Effect of nucleic acid binding on the triplet state properties of tetrapeptides containing tryptophan and 6-methyltryptophan: a study by phosphorescence and ODMR spectroscopy.

Complexes of four peptides [KWGK, KGWK, K(6MeW)GK, KG(6MeW)K] with the nucleic acids [poly(A), poly(C), poly(U), poly(I), and rG(8)] have been investigated by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The intrinsic spectroscopic probes used in these studies are tryptophan (W) and 6-methyltryptophan (6MeW). Binding to the nucleic acids results in a red-shift of the phosphorescence 0,0-band (delta E(0,0)) of the aromatic residue as well as a reduction of its zero-field splitting parameter (delta D). Results are compared with earlier studies of the HIV-1 nucleocapsid protein, NCp7, that contains a single tryptophan residue (Trp37) within a retroviral zinc finger sequence. Binding of poly(A) or poly(U) to the tetrapeptides induces larger delta E(0,0) and delta D than when bound to NCp7, indicating stronger stacking interactions. Poly(I), on the other hand, produces larger shifts in Trp37 of NCp7. Binding of rG(8) produces sequence-dependent effects in the peptides. When bound to NCp7, but in contrast with tetrapeptide binding, nucleic acids produce large changes in triplet state kinetics consistent with enhanced spin-orbit coupling. These results are discussed in terms of three limiting types of tryptophan-base interaction: intercalation, aromatic stacking, and edge-on interaction. These should have differing effects on the properties of the triplet state.

Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein.

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.

Role of the histidine residues of visna virus nucleocapsid protein in metal ion and DNA binding.

Zinc finger (ZF) domains in retroviral nucleocapsid proteins usually contain one histidine per metal ion coordination complex (Cys-X(2)-Cys-X(4)-His-X(4)-Cys). Visna virus nucleocapsid protein, p8, has two additional histidines (in the second of its two ZFs) that could potentially bind metal ions. Absorption spectra of cobalt-bound ZF2 peptides were altered by Cys alkylation and mutation, but not by mutation of the extra histidines. Our results show that visna p8 ZFs involve three Cys and one His in the canonical spacing in metal ion coordination, and that the two additional histidines appear to interact with nucleic acid bases in p8-DNA complexes.

Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8.

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.

Bisimidazoacridones: effect of molecular environment on conformation and photophysical properties.

Bisimidazoacridones (BIA) are highly selective antineoplastic and antiviral agents. Ultraviolet-visible spectroscopy and steady-state and time-resolved fluorescence spectroscopy studies were carried out to probe the behavior of BIA in aqueous and nonaqueous (organic solvents, colloid micelles) solutions. Three ranges of fluorescence lifetimes were revealed: approximately 0.2-0.5 ns (presumably reflecting the chromophore-chromophore interaction), approximately 1-5 ns (interpreted as linker-perturbed chromophore decay) and approximately 6-12 ns (nonperturbed chromophore decay). The pre-exponential and steady-state contributions of these components to the decay signal as well as the data on steady-state fluorescence intensities, wavelength maxima and bandwidths showed that the BIA conformations in solution were sensitive to the environment and influenced strongly by their propensity to minimize hydrophobic interactions. In water, the molecules tend to adopt condensed conformations that bring the two imidazoacridone moieties into close proximity (resulting in intramolecular fluorescence energy transfer), while in nonaqueous systems the conformations become more relaxed. The transfer from a polar to more lipophilic environment of macromolecules is suggested to be the main driving force for binding of BIA to biomacromolecules, such as nucleic acids.

Binding properties of the human immunodeficiency virus type 1 nucleocapsid protein p7 to a model RNA: elucidation of the structural determinants for function.

HIV-1 nucleocapsid protein (NCp7) is a double zinc-fingered protein that has been traditionally implicated in viral RNA recognition and packaging, in addition to its tight association with genomic RNA and tRNA primer within the virion nucleocapsid. The availability of large quantities of viral or recombinant wild-type NCp7 and mutant p7 has made possible the assignment of the different roles that structural motifs within the protein play during RNA binding. At low ionic strength binding to the homopolymeric fluorescent RNA, poly(epsilonA), is electrostatically driven and four sodium ions are displaced. Arg7 in the flanking N-terminal region, Lys20 and Lys26 in the first zinc finger and one positively charged residue (attributed to Lys41) in the second zinc finger are involved in electrostatic contacts with RNA. The p7 zinc fingers do not function independently but concomitantly. The first zinc finger (both isolated or in the context of the full-length protein) has a more prominent electrostatic interaction than the second one. The second zinc finger dominates the non-electrostatic stabilization of the binding to RNA due to stacking of its Trp residue with nucleic acid bases. Mutations in the highly conserved retroviral Zn-coordinating residues (CCHC) to steroid hormone receptor (CCCC) or transcription factor (CCHH) metal cluster types do not affect RNA binding. In spite of the limited impact in RNA binding affinity in vitro or RNA packaging in vivo that such mutations or structural alterations impart, they impair or abolish virus infectivity. It is likely that such an effect stems from the involvement of NCp7 in crucial steps of the virus life cycle other than RNA binding.

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity.

The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine chi1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 A for the backbone atom, and 1.68 A for all heavy atoms. For the well-defined region encompassing residues 2-12 and 18-27, the corresponding values were 0.71 and 1.66 A, respectively. Circulin A adopts a compact structure consisting of beta-turns and a distorted segment of triple-stranded beta-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a "cystine-knot" motif. This motif is present in a range of other biologically active proteins, including omega-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.

Sequence-specific binding of human immunodeficiency virus type 1 nucleocapsid protein to short oligonucleotides.

We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)2 was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.

Binding of the nucleocapsid protein of type 1 human immunodeficiency virus to nucleic acids studied using phosphorescence and optically detected magnetic resonance.

The binding of p7 nucleocapsid protein of type 1 human immunodeficiency virus (HIV-1) to various oligonucleotides and polynucleotides has been investigated by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The intrinsic spectroscopic probe used in these studies is the photoexcited triplet state of Trp37, which is associated with the C-terminal zinc finger of p7 and is its only tryptophan residue. Complex formation produces a red-shift of the phosphorescence 0, 0-band (DeltaE0,0) of Trp37 as well as a reduction of the zero field splitting (zfs) D parameter. Increases of -DeltaE0,0 (A < C < U < G

Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.

HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.

Fluorescence, phosphorescence, and optically detected magnetic resonance studies of the nucleic acid association of the nucleocapsid protein of the murine leukemia virus.

Fluorescence, phosphorescence, and optical detection of triplet state magnetic resonance (ODMR) are employed to investigate the interaction of p10, the nucleocapsid protein of the Moloney murine leukemia virus, with nucleic acids. p10 is a 55-amino acid protein containing a single zinc finger motif, C26C29H34C39, that includes Y at position 28 and W at position 35. In addition, the interactions of a zinc finger peptide, p10-ZF, comprising residues 24-41 of p10, and a doubly mutated 24-41 peptide, p10-ZF' in which the positions of Y and W are interchanged, also are reported. The measurements focus on the direct involvement of the sole W residue in the nucleic acid interaction. Fluorescence quenching and salt-back titrations indicate complex formation of p10 with several octanucleotides--(dT)8, (dI)8, (dU)7dT, and (5-BrdU)7dT--and with the polynucleotides poly(dT) and poly(dI). Poly(dI) binds with the highest affinity. Apparent binding constants and salt-back midpoints are reported. Neither p10-ZF nor p10-ZF' exhibits significant fluorescence quenching by these DNA substrates. Binding of p10-ZF to fluorescent poly(ethenoadenylic acid) was detected with greatly reduced affinity relative to p10, but binding of p10-ZF' was undetectable. These results are in general agreement with phosphorescence and ODMR measurements monitoring W. Addition of poly(I) to p10 leads to a phosphorescence red shift, reduction in the zero-field splitting (ZFS) parameters D and E, and a significantly reduced phosphorescence lifetime, each consistent with aromatic stacking interactions between W and the nucleobases. These effects are smaller with p10-ZF and undetectable with p10-ZF'. Poly(U) produces no significant changes in the triplet state parameters of W; no stacking interactions are observed even for p10. (5-BrdU)7dT yields large phosphorescence red shifts in p10 and p10-ZF, and reductions of D, but no significant heavy atom effects. These effects probably are due to enhanced local polarizability caused by Br, but any stacking interactions in these complexes would exclude van der Waals contacts between W and the Br atoms.

Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain.

We have analyzed two human immunodeficiency virus (HIV-1) reverse transcriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-primer. These enzymes, G262A and W266A, were previously shown to have greatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3'-azidodeoxythymidine 5'-triphosphate. Here we describe their processivity and error specificity. The results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence-dependent termination of processive synthesis and in error specificity, and (iii) the magnitude of the mutator effect relative to wild-type enzyme for deletions in homopolymeric sequences decreases as the length of the run increases. Thus amino acid substitutions in a subdomain thought to interact with the duplex template-primer confer a strand slippage mutator phenotype to a replicative DNA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity. These effects, obtained in aqueous solution during catalytic cycling, are consistent with and support the existing crystallographic structural model.