Biomedical subjects

G P Schroth

Publications and source records attributed to G P Schroth.

At least 19 recordsLinked to original sources

PubMed2001

Allosteric interaction of minor groove binding ligands with UL9-DNA complexes.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

Allosteric Regulation↗

PubMed2001

The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.

The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.

Base Pairing↗

PubMed1999

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.

Base Pairing↗

PubMed1996

Sequence-dependent effects of spermine on the thermodynamics of the B-DNA to Z-DNA transition.

Spermine has been shown to bind to and stabilize a number of altered DNA conformations, including left-handed Z-DNA. Here, we have quantitatively studied the effects of spermine on the negative supercoil-induced transition from B- to Z-DNA. We have determined the intrinsic association constants for and the effective number of ligands that bind to both B- and Z-DNA. The intrinsic affinity of spermine for Z-DNA is approximately 10 times higher for d(CA/TG) (KZP = 1.2 x 10(8) M-1) than for d(CG) dinucleotides (KZP = 1.5 x 10(7) M-1), and both are greater than that for B-DNA (KBP = 1.4 x 10(5) M-1). This accounts for the stabilization of Z-DNA by spermine. The number of spermine accommodated by Z-DNA (nZ) is sequence-dependent [nZ = 0.6 spermine per 18 d(CA/TG) dinucleotides and 2.3 for 12 d(CG) dinucleotides]. The value of nZ of < 1 was interpreted as evidence for negative cooperativity in spermine binding to d(CA/TG) dinucleotides. Thus, although d(CA/TG) sequences saturate at lower spermine concentrations, the ligand has an overall greater effect on the stability of d(CG) dinucleotides as Z-DNA. B-DNA accommodates more spermines per base pair than either sequence as Z-DNA. At higher concentrations (> 10 microM), spermine destabilizes Z-DNA. Using these parameters in a model for competitive spermine binding to B-DNA and Z-DNA, we can make predictions for how potential Z-DNA sequences found in the human genome are affected by cellular levels of superhelical density and spermine.

DNA↗

PubMed1996

Protein and DNA requirements for the transcription factor IIIA-induced distortion of the 5 S rRNA gene promoter.

Transcription factor-induced DNA distortion has become a common theme in eukaryotic gene regulation. A number of techniques have been applied to the study of transcription factor-induced DNA bending and flexibility including electron microscopy, circular permutation gel analysis, helical phasing gel analysis and cyclisation kinetics in solution. We have applied these techniques in order to assess the role that specific DNA sequences and protein domains of transcription factor IIIA (TFIIIA) play in the TFIIIA-induced distortion of the Xenopus 5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA:DNA complexes indicate that TFIIIA binding involves compaction of the 5 S promoter into a precise three-dimensional hairpin-shaped structure. This compaction can be detected utilising circular permutation gel analysis and the distortion results in an apparent bend angle of 55 to 60 degrees near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60 degrees bend angle as measured by circular permutation can be detected as a static bend directed towards the minor groove between bases +63 and +64 of the 5 S rRNA gene. The amplitude of the TFIIIA:5 S gene phasing signal is similar to the phasing signal obtained utilising bacterial CAP:DNA complexes with bend angles of approximately 90 degrees. These results are supported by phased ligase-mediated cyclisation kinetics in solution. Analysis of DNA deletion constructs indicate that the 5' A block of the internal 5 S gene promoter, which is required for transcriptional activity, is also required for TFIIIA-induced distortion of the 5 S gene promoter. Analysis of the N-terminal papain fragment of TFIIIA indicates that the 34 kDa zinc finger DNA binding domain is sufficient for compaction of the 5 S gene promoter. These results are discussed in relation to the modular model of TFIIIA:DNA interaction in which individual zinc fingers contribute to the protein-induced distortion of the DNA helix and overall DNA binding affinity in a complex, non-additive fashion.

Base Sequence↗

PubMed1995

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

Animals↗

PubMed1995

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Base Sequence↗

PubMed1995

Alternating and non-alternating dG-dC hexanucleotides crystallize as canonical A-DNA.

We have solved the single-crystal X-ray structures of two different hexanucleotides: the alternating sequence d(Gm5CGm5CGC), and the non-alternating sequence d(Gm5CCGGC). Both of these hexamers crystallize readily as A-DNA in the orthorhombic space group C222(1). Although hexanucleotides have been previously crystallized as Z-DNA, and in one case as B-DNA, this is the first time hexanucleotides have been crystallized as A-DNA. Both hexamers adopt a typical A-conformation, which is surprisingly more similar to the structure of A-DNA fibers than to other A-DNA single crystals. The structure of d(Gm5CGm5CGC) was solved to a resolution of 2.1 A (R-factor = 19.6%). This structure has all of the features characteristic of canonical A-DNA, including it's helical repeat (11.2 bp/turn), helical rise (2.6 A/bp), base-pair displacement (-4.7 A), base inclination angle (16.9 degrees), and sugar puckers that are predominantly 3'-endo. The lower resolution, non-alternating structure has similar overall average values for these parameters. We observed several sequence-dependent correlations in these parameters, especially in the d(CG) base step. These steps have lower twist and rise values, coupled with high roll angles as compared to d(GC) steps. The molecular interactions involved in crystal packing and the detailed structure of the bound water in the crystals, however, are similar to those of longer 8 and 10 bp A-DNA crystal structures. Although the structural effect of cytosine methylation on A-DNA appears to be minimal, this modification significantly affects the ability of these sequences to crystallize as A-DNA. In conclusion, we present the A-DNA forming class of hexanucleotides, a new crystallographic system for studying DNA structure at near atomic resolution.

Crystallization↗

PubMed1995

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.

Base Sequence↗

PubMed1994

Binding of histones H1 and H5 and their globular domains to four-way junction DNA.

We have compared chicken erythrocyte linker histones H1 and H5 binding to a synthetic four-way DNA junction. Each histone binds to form a single complex, with an affinity which permits competition against a large excess of linear duplex DNA. The affinity of H5 is higher than that of H1. The globular domain from either protein will also bind strongly, but in this case multiple binding occurs. Binding of intact H1 is inhibited by cations: Mg2+ and spermidine are very effective, Na+ much less so. This inhibition is not likely to be a general ion-competition effect, for Mg2+ is much less effective in inhibiting the binding of H1 to linear DNA. Instead, the inhibition of binding may be due to ion-dependent changes in the conformation of the four-way junction, which are known to occur under similar conditions. These results strongly suggest that the angle formed between the arms of the DNA junction could be a major determinant in the interaction of H1 with DNA crossovers.

Animals↗

PubMed1993

Structure and thermodynamics of nonalternating C.G base pairs in Z-DNA: the 1.3-A crystal structure of the asymmetric hexanucleotide d(m5CGGGm5CG).d(m5CGCCm5CG).

We have solved the single-crystal X-ray structure of the complementary hexanucleotides d(m5-CGGGm5CG) and d(m5CGCCm5CG). The hexamer duplex was crystallized as Z-DNA, but contains a single C.G base pair that does not follow the alternating pyrimidine/purine rule for Z-DNA formation. This is the first crystal structure which serves to illustrate the structural consequences of placing a cytosine in the sterically disfavored syn conformation. In addition, since these sequences are not self-complementary, the individual strands of this asymmetric hexamer are unique in sequence and therefore distinguishable in the crystal lattice. Nevertheless, the crystal of this duplex is isomorphous with other Z-DNA hexamer structures. The asymmetry of this hexamer sequence required that the structure be solved using two unique models, which are distinguished by the orientation of hexanucleotides in the crystal lattice. In one model (the GG model) the cytosine in the syn conformation is packed against the terminal guanine base of a symmetry-related hexamer, while in the alternative model (the CC model) this cytosine sits exposed in a solvent channel of the lattice. We find that neither model alone can completely account for the observed electron densities. The two models ultimately were refined together. A composite structure consisting of 65% GG model and 35% CC model refined to an R-factor of 19.3%, which was significantly lower than refinements using either model alone. A detailed analysis of these two structures shows that, in spite of the out-of-alternation C.G base pair, the features characteristic of Z-DNA have been maintained. Both models, however, show significant local structural adjustments to accommodate the single cytosine base which is forced to adopt the syn conformation in each hexamer. In general, it appears that in order to relieve the energetically unfavorable steric contacts between the cytosine base in the syn conformation and the deoxyribose sugar, the base is forced into a highly buckled conformation, and that this large buckle in turn alters the conformation of neighboring residues. This unusual conformation also significantly weakens base-stacking interactions between the cytosine in syn and the adjacent residues in the helix and affects the exposure of the bases to solvent. We conclude that this crystal structure provides a molecular rationale for why nonalternating bases are energetically disfavored in Z-DNA.

Base Composition↗

PubMed1993

Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA.

The effect of acetylation on the DNA binding properties of the rigidly conserved histone H4 amino-terminal tail has been studied in detail using the technique of thermal denaturation. The quantitative DNA-binding parameters for both the non- and fully acetylated H4 amino terminus have been determined from thermal denaturation data for complexes of the peptides bound to mixed sequence 146-base pair DNA. We find that under dilute buffer conditions (5 mM Tris-HCl) the binding constant for the non-acetylated peptide to double-stranded DNA is 5 x 10(11) M-1 and that acetylation of lysine residues in the peptide reduces the binding constant to 1 x 10(5) M-1. The dramatic differences observed in the binding constants for the non- and fully acetylated peptides are probably due to the effect of acetylation on the even distribution of positively charged residues in the H4 amino terminus. In other experiments, the binding of both peptides to a 30-base pair oligonucleotide has been studied in solution with varying concentrations of sodium, magnesium, and phosphate ions. These experiments demonstrate that both magnesium and phosphate ions have strong effects on the binding of the H4 tail to DNA, especially weakening the binding of the acetylated peptide. For instance, the dissociation of the non-acetylated peptide from DNA requires 6 mM magnesium, yet the binding of the acetylated peptide is abolished in only 30 microM magnesium. The modulation of the DNA binding interactions of the H4 amino terminus by physiologically relevant ionic conditions, in addition to the effect of acetylation, can be important in the regulation of chromatin structure and function.

Acetylation↗

PubMed1992

Mapping Z-DNA in the human genome. Computer-aided mapping reveals a nonrandom distribution of potential Z-DNA-forming sequences in human genes.

In this work, we have predicted and mapped the potential Z-DNA-forming sequences in over one million base pairs of human DNA, containing 137 complete genes. The computer program (Z-Hunt-II) developed for this study uses a rigorous thermodynamic search strategy to map the occurrence of left-handed Z-DNA in genomic sequences. The search algorithm has been optimized to search large sequences for the potential occurrence of Z-DNA, taking into account sequence type, length, and cooperativity for a given stretch of potential Z-DNA-forming nucleotides. In this extensive data set we have identified 329 potential Z-DNA-forming sequences. The exact locations of the potential Z-DNA-forming sequences in the data set have been mapped with respect to the location of structural features of the genes. This analysis reveals a distinctly nonrandom distribution of potential Z-DNA-forming sequences across human genes and, most notably, that strong Z-DNA-forming sequences are more commonly found near the 5' ends of genes. We find that 35% of the Z-DNA-forming sequences are located upstream of the first expressed exon, while only 3% of the sequences are located downstream of the last expressed exon. The remaining 62% of the Z-DNA-forming sequences, which are located either in introns (47.1%) or exons (14.9%), are also nonrandomly distributed, with a strong bias toward locations near the site of transcription initiation. We interpret this distribution of potential Z-DNA-forming sequences toward the 5' end of human genes in terms of the well established "twin-domain model" of transcription-induced supercoiling and the effect of this topological strain on Z-DNA formation in eukaryotic cells.

Base Sequence↗

PubMed1992

Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility.

We have identified two intrinsically bent regions of DNA which flank the transcription start site of the rRNA gene from Physarum polycephalum. DNA fragments from both regions were analyzed by circular permutation polyacrylamide gel electrophoresis assay and computer modeling. Both types of analysis indicate that one fragment contains a relatively simple bend centered about 160 base pairs (bp) upstream of the transcription start site while the other fragment contains multiple bends, the most prominent of which is centered about 150 bp downstream of the start site. According to both gel mobilities and computer modeling we estimate that the net bending in each is about 45 degrees. These fragments were studied in detail by varying parameters of electrophoresis that are known to affect bending. Previous work indicates that anomalous mobility should decrease when temperature or ethidium bromide concentration is increased, whereas anomalous mobility should increase when polyacrylamide gel percentage is increased. The anomalous mobility of both fragments decreases as temperature is raised from 4 to 65 degrees C, although the bent structure centered at -160 bp is more temperature labile than the bend at +150 bp. Strikingly different behavior was observed for the two fragments as the polyacrylamide concentrations was varied. As polyacrylamide concentrations are increased from 6 to 10%, the anomalous mobility of the bend centered at -160 bp increases while that of the bend centered at +150 bp decreases. The bend centered at +150 bp is "straightened" at all ethidium concentrations tested. In sharp contrast and unexpectedly, the anomalous migration of the bend centered at -160 bp increases dramatically in 0.1 micrograms/ml ethidium bromide. Many of the mobility differences we observe suggest that the two regions studied represent structurally distinct forms of bent DNA. The location of these strongly bent regions on either side of a RNA polymerase I transcription start site suggests important roles for such structures in chromatin structure and transcription initiation.

Animals↗

PubMed1991

Prediction of a crystallization pathway for Z-DNA hexanucleotides.

Crystallization of macromolecules for structural studies has long been a hit-or-miss process. The crystallization of hexanucleotides as Z-DNA was studied, and it was shown that the cation concentration for crystal formation could be predicted from solvation free energy (SFE) calculations. Solution studies on the conformation and solubilities of the hexanucleotides showed that a critical concentration of the DNA in the Z-conformation must be present in solution to effect crystallization. The SFE calculations therefore predict the propensity of the hexanucleotides to adopt the left-handed conformation and the driving force required to reach this critical concentration relative to the intrinsic solubility of Z-DNA for crystallization.

Base Sequence↗

PubMed1991

TFIIIA induced DNA bending: effect of low ionic strength electrophoresis buffer conditions.

We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of the Xenopus somatic-type gene [Schroth, G.P. et al. (1989) Nature 340, 487-488]. In addition, we show that TFIIIA induced DNA bending is dramatically affected by the ionic conditions used during gel electrophoresis. By modifying the conditions of the electrophoresis, we can detect two distinct conformations for the TFIIIA/DNA complex. In very low ionic strength buffers, the degree of DNA bending in the complex is estimated to be about 25 to 30 degrees, whereas in higher ionic strength buffers it is about 60 to 65 degrees. These data explain the apparent discrepancy between our results and the results of another study in which it was claimed that TFIIIA did not 'substantially' bend DNA [Zweib, C. and Brown, R.S. (1990) Nucleic Acid Res. 18, 583-587]. These results also demonstrate that the TFIIIA/DNA complex has a large degree of conformational flexibility. Both DNA bending and conformational flexibility are structural features which may provide a key insight into the function of TFIIIA as a positive transcription factor.

Animals↗

PubMed1990

Zinc-induced secondary structure transitions in human sperm protamines.

Using CD we show that human group II protamines undergo novel zinc-dependent secondary structure transitions. The CD spectra of protamine is characteristic of random coil proteins with a large minima at 197 nm. Upon the addition of 1 mM zinc, the magnitude of this minima is decreased by 44%. This spectral change is not induced by 1 mM calcium or magnesium. Cadmium, which has chemical properties similar to zinc, can also induce the structural transition although not as effectively as zinc. The spectral changes that accompany zinc binding are indicative of an increase in beta-turn and anti-parallel beta-sheet structures. This is consistent with the predicted secondary structure for protamines which is dominated by beta-turns. Our data support a model in which protamine adopts a folded structure in the presence of zinc. We propose that a zinc-modulated structure is physiologically significant considering the relatively high levels of zinc in human sperm.

Amino Acid Sequence↗

PubMed1990

A NMR study of mobility in the histone octamer.

The histone octamer from chicken erythrocytes was studied in 2 M NaCl using 500 mHz 1H NMR spectroscopy. We compared the spectrum of control octamers with that of octamers isolated from trypsinized nucleosome core particles. We observe that the sharp resonances found in the spectrum of the native octamer disappear completely after trypsinization. Therefore, within the time frame of the NMR experiment, all of the mobile amino acid residues in the histone octamer are found in the well defined trypsin sensitive domains. These results indicate that there is a very clear structural demarcation between the random coil N- and C-terminal tails and the globular domains of the histones.

Amino Acids↗