Use of 6-Methylisoxanthopterin, a Fluorescent Guanine Analog, to Probe Fob1-Mediated Dynamics at the Stalling Fork Barrier DNA Sequences.

Fluorescent nucleic acid base mimics serve as excellent site-specific and real-time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6-methylisoxanthopterin (6-MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site-specific incorporation of this probe, we show that 6-MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double-stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double-stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6-MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double-stranded versus HJs. This work highlights the unique advantages of 6-MI as a probe when incorporated in nucleic acid frameworks.

Photophysical Characterization of Enhanced 6-Methylisoxanthopterin Fluorescence in Duplex DNA.

The structure and dynamic motions of bases in DNA duplexes and other constructs are important for understanding mechanisms of selectivity and recognition of DNA-binding proteins. The fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, is well suited to this purpose as it exhibits an unexpected 3- to 4-fold increase in relative quantum yield upon duplex formation when incorporated into the following sequences: ATFAA, AAFTA, or ATFTA (where F represents 6-MI). To better understand some of the factors leading to the 6-MI fluorescence increase upon duplex formation, we characterized the effect of local sequence and structural perturbations on 6-MI photophysics through temperature melts, quantum yield measurements, fluorescence quenching assays, and fluorescence lifetime measurements. By examining 21 sequences we have determined that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI and the presence of adenines at locations n ± 2 from the probe. Investigation of duplex stability and local solvent accessibility measurements support a model in which the DEF arises from a constrained geometry of 6-MI in the duplex, which remains H-bonded to cytosine, stacked with adjacent bases and inaccessible to quenchers. Perturbation of DNA structure through the introduction of an unpaired base 3' to 6-MI or a mismatched basepair increases 6-MI dynamic motion leading to fluorescence quenching and a reduction in quantum yield. Molecular dynamics simulations suggest the enhanced fluorescence results from a greater degree of twist at the X-F step relative to the quenched duplexes examined. These results point to a model where adenine residues located at n ± 2 from 6-MI induce a structural geometry with greater twist in the duplex that hinders local motion reducing dynamic quenching and producing an increase in 6-MI fluorescence.

Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6-Methyl Isoxanthopterin (6-MI) in DNA.

The local conformations of individual nucleic acid bases in DNA are important components in processes fundamental to gene regulation. Fluorescent nucleic acid base analogues, which can be substituted for natural bases in DNA, can serve as useful spectroscopic probes of average local base conformation and conformational heterogeneity. Here we report excitation-emission peak shift (EES) measurements of the fluorescent guanine (G) analogue 6-methyl isoxanthoptherin (6-MI), both as a ribonucleotide monophosphate (NMP) in solution and as a site-specific substituent for G in various DNA constructs. Changes in the peak positions of the fluorescence spectra as a function of excitation energy indicate that distinct subpopulations of conformational states exist in these samples on time scales longer than the fluorescence lifetime. Our pH-dependent measurements of the 6-MI NMP in solution show that these states can be identified as protonated and deprotonated forms of the 6-MI fluorescent probe. We implement a simple two-state model, which includes four vibrationally coupled electronic levels to estimate the free energy change, the free energy of activation, and the equilibrium constant for the proton transfer reaction. These parameters vary in single-stranded and duplex DNA constructs, and also depend on the sequence context of flanking bases. Our results suggest that proton transfer in 6-MI-substituted DNA constructs is coupled to conformational heterogeneity of the probe base, and can be interpreted to suggest that Watson-Crick base pairing between 6-MI and its complementary cytosine in duplex DNA involves a "low-barrier-hydrogen-bond". These findings may be important in using the 6-MI probe to understand local base conformational fluctuations, which likely play a central role in protein-DNA and ligand-DNA interactions.

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity.

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5'-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.

Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

Quadruplex priming amplification for the detection of mRNA from surrogate patient samples.

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

Isothermal quadruplex priming amplification for DNA-based diagnostics.

We previously developed a method, known as quadruplex priming amplification (QPA), which permits isothermal amplification of DNA. The assay is based on a DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence. G3T has three unique properties that are fundamental for QPA; (i) G3T forms a quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplexes; (ii) removal of guanines at the 3'-end inhibits quadruplex formation; and (iii) incorporated fluorescent nucleotides, such as 2-aminopurine (2AP) or 6-methylisoxanthopterin (6MI), which are quenched by neighboring nucleotides, regain maximum emission upon quadruplex formation. New model studies carried out here with primers missing one, two and three guanines reveal that the driving force for QPA comes from the difference in thermal stability between the primer/template and the product complexes. Primers missing one and two guanines are able to self-dissociate from the template upon elongation, whereas QPA is not observed when the primer lacks three 3'-nucleotides. QPA reaches its maximum rate at temperatures slightly higher than the T(m) of the primer/template complex and is more efficient in the presence of only dGTP. QPA-based assays also revealed that Taq is able to incorporate thymidines opposite template 2AP, while no significant incorporation was observed opposite template 6MI.

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs.

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K(+) ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.

Electronic transition moments of 6-methyl isoxanthopterin--a fluorescent analogue of the nucleic acid base guanine.

Fluorescent nucleic acid base analogues are important spectroscopic tools for understanding local structure and dynamics of DNA and RNA. We studied the orientations and magnitudes of the electric dipole transition moments (EDTMs) of 6-methyl isoxanthopterin (6-MI), a fluorescent analogue of guanine that has been particularly useful in biological studies. Using a combination of absorption spectroscopy, linear dichroism (LD) and quantum chemical calculations, we identified six electronic transitions that occur within the 25,000-50,000 cm(-1) spectral range. Our results indicate that the two experimentally observed lowest-energy transitions, which occur at 29,687 cm(-1) (337 nm) and 34,596 cm(-1) (289 nm), are each polarized within the plane of the 6-MI base. A third in-plane polarized transition is experimentally observed at 47,547 cm(-1) (210 nm). The theoretically predicted orientation of the lowest-energy transition moment agrees well with experiment. Based on these results, we constructed an exciton model to describe the absorption spectra of a 6-MI dinucleotide-substituted double-stranded DNA construct. This model is in good agreement with the experimental data. The orientations and intensities of the low-energy electronic transitions of 6-MI reported here should be useful for studying local conformations of DNA and RNA in biologically important complexes.

Applying 6-methylisoxanthopterin-enhanced fluorescence to examine protein-DNA interactions in the picomolar range.

Incorporation of fluorescent nucleoside analogues into duplex DNA usually leads to a reduction in quantum yield, which significantly limits their potential use and application. We have identified two pentamer DNA sequences containing 6-methylisoxanthopterin (6-MI) (ATFAA and AAFTA, where F is 6-MI) that exhibit significant enhancement of fluorescence upon formation of duplex DNA with quantum yields close to that of monomeric 6-MI. The enhanced fluorescence dramatically increases the utility and sensitivity of the probe and is used to study protein-DNA interactions of nanomolar specificity in this work. The increased sensitivity of 6-MI allows anisotropy binding measurements to be performed at DNA concentrations of 1 nM and fluorescence intensity measurements at 50 pM DNA. The ATFAA sequence was incorporated into DNA constructs to measure the binding affinity of four different protein-DNA interactions that exhibit sequence-specific and non-sequence-specific recognition. In all cases, the K(d) values obtained were consistent with previously reported values measured by other methods. Time-resolved and steady-state fluorescence measurements demonstrate that 6-MI fluorescence is very sensitive to local distortion and reports on different degrees of protein-induced perturbations with single-base resolution, where the largest changes occur at the site of protein binding.

Excited-state electronic properties of 6-methylisoxanthopterin (6-MI): an experimental and theoretical study.

6-Methylisoxanthopterin (6-MI) is a pteridine-based guanine analog that has a red-shifted absorption and high fluorescence quantum yield. Its Watson-Crick base-pairing and base stacking properties are similar to guanine. The fluorescence quantum yield of 6-MI is sensitive to its nearest neighbors and base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood. We have explored the excited-state electronic structure of the 6-MI in frozen 77 K LiCl glasses using Stark spectroscopy. These measurements yielded the direction and degree of charge redistribution for the S(0)→S(1) transition as manifested in the difference dipole moment, Δµ(01), and difference static polarizability, TrΔα. TDDFT (time-dependent density functional theory) was employed to calculate the transition energy, oscillator strength, and the dipole moments of the ground and lowest optically bright excited state of 6-MI (S(0)→S(1)). The direction of Δµ(01) was assigned in the molecular frame based on the Stark data and calculations. These results suggest that the C4═O and C2-NH(2) groups are electron-deficient in the excited state, a very different outcome compared with guanine. This implies that Watson-Crick hydrogen bonding in 6-MI may be modulated by absorption of a photon so as to strengthen base pairing, if only transiently. Solvatochromism was also obtained for the absorption and emission spectra of 6-MI in various solvents and compared with the Stark spectroscopic results using both the Lippert-Mataga and Bakhshiev models.

Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids.

We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base-base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA 'breathing' at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution.

Conformational heterogeneity and quasi-static self-quenching in DNA containing a fluorescent guanine analogue, 3MI or 6MI.

Two different microenvironments in the DNA sequence 5'-act aGa gat ccc tca gac cct ttt agt cag tGt gga-3' (in both single- and double-stranded forms) are explored using two similar fluorescent nucleoside analogues, 3MI and 6MI. Each probe was evaluated in two environments, one strand with the probe flanked by thymines (PTRT) and the other by adenines (PTRA) with positions indicated by G's in the sequence. Both time-resolved anisotropies and lifetimes of the probes depend upon local interactions, and these are altered by duplex formation. Integrals of lifetime curves compared with quantum yields reveal that each probe displays a "dark" component (below detection limits, with a lifetime of <70 ps). For 6MI in PTRA, this QSSQ "quasi-static self-quenching" or "dark" component represents approximately half the molecules, whether in single- or double-stranded form. In PTRT, 6MI displays an unusual increase in the quantum yield upon formation of the double strand (from 0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66 to 33%. This is also accompanied by doubling of steady-state anisotropy. Only 6MI in the PTRT duplex displays a rotational correlation time of >7 ns. In other words, the DS 6MI PTRA environment fails to constrain local motion and QSSQ remains the same as in the single strand; in contrast, the flanking T duplex environment restricts local motion and halves QSSQ. We collected both steady-state and time-resolved fluorescence quenching titrations of 3MI and 6MI in solution with the mononucleotides AMP, CMP, GMP, and TMP. The dynamic quenching rank of the free probes (quenching constant, kq: T > A > G > C) is totally different from that of incorporated probes. We hypothesize the production of weak 3MI.C or 6MI.C complexes that are somehow rendered less subject to dynamic quenching by collision with subsequent C molecules.

Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes.

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5' end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Multiphoton excitation of fluorescent DNA base analogs.

Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5 x 10(-50) cm(4)s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

Use of pteridine nucleoside analogs as hybridization probes.

The pteridine nucleoside analog 3-methyl isoxanthopterin (3-MI) is highly fluorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligonucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its fluorescence is variably quenched in a sequence-dependent manner. Bend ing, annealing, binding, digestion or cleavage of fluorophore-containing oligonucleotides can be detected by monitoring changes in fluorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the fluorophore as an insertion and when annealing occurs, the fluorophore bulges out from the double strand, resulting in increased fluorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple fluorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybridization technique can result in fluorescence intensity increases of up to 27-fold. Fluorescence intensity increases are only seen upon specific binding to bulge-generating complements, removing issues of high background from non-specific binding.

Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin.

The parallel (recombination) 'R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T.A) and 6MI*(C.G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative 'branch migration' and 'strand exchange' structures and discuss why these are less likely than the R-type triplex.

HU binding to DNA: evidence for multiple complex formation and DNA bending.

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.

6-Tetrahydrobiopterin functions as a UVB-light switch for de novo melanogenesis.

(6R)-L-erythro 5,6,7,8-tetrahydrobiopterin (6-BH4) and its 7-isomer (7-BH4) function as uncompetitive inhibitors of human and mushroom tyrosinases. Stoichiometry for the binding of [3H]-labeled 6-BH4 to both tyrosinases has been established as 1:1. Stable complexation of 6-BH4 to tyrosinase appears to involve a hydrophilic conserved glutamic acid (Glu131) with a pKa = 4.7. Photo-oxidation by UVB-light and O2 reverses the inhibition of tyrosinase by 6-BH4 and 7-BH4 with the 6-BH4/tyrosinase complex being four-fold more photolabile than 7-BH4/tyrosinase. The photo-oxidation of 6-BH4 by UVB-light can be assessed spectrophotometrically with this reaction yielding 7,8-dihydroxanthopterin as the final product, 7,8-Dihydroxanthopterin neither binds to nor inhibits tyrosinase. By contrast, UVA light does not catalyze the photodegradation of 6-BH4. Taken together, our results indicate that the photo-oxidation of the tetrahydrobiopterins by UVB may represent a photo-switch in the regulation of tyrosinase activity to promote de novo melanogenesis.