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K J Hampel

Publications and source records attributed to K J Hampel.

15 recordsLinked to original sources
PubMed2001

Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate-pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2'-OH and protonation of the 5'-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto-enol tautomerization.

Base Sequence↗
PubMed2001

A conformational change in the "loop E-like" motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis.

The catalysis of site-specific RNA cleavage and ligation by the hairpin ribozyme requires the formation of a tertiary interaction between two independently folded internal loop domains, A and B. Within the B domain, a tertiary structure has been identified, known as the loop E motif, that has been observed in many naturally occurring RNAs. One characteristic of this motif is a partial cross-strand stack of a G residue on a U residue. In a few cases, including loop B of the hairpin ribozyme, this unusual arrangement gives rise to photoreactivity. In the hairpin, G21 and U42 can be UV cross-linked. Here we show that docking of the two domains correlates very strongly with a loss of UV reactivity of these bases. The rate of the loss of photoreactivity during folding is in close agreement with the kinetics of interdomain docking as determined by hydroxyl-radical footprinting and fluorescence resonance energy transfer (FRET). Fixing the structure of the complex in the cross-linked form results in an inability of the two domains to dock and catalyze the cleavage reaction, suggesting that the conformational change is essential for catalysis.

Catalysis↗
PubMed2001

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation.

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.

Base Sequence↗
PubMed2001

A base change in the catalytic core of the hairpin ribozyme perturbs function but not domain docking.

The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5'-GU-3' immediately 5' of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a common interdomain distance as measured by time-resolved fluorescence resonance energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.

2-Aminopurine↗
PubMed2001

Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA.

Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min(-1). Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.

DNA Footprinting↗
PubMed1998

The solvent-protected core of the hairpin ribozyme-substrate complex.

The complex between the hairpin ribozyme and its substrate consists of two domains that must interact in order to form a catalytic complex, yet experimental evidence concerning the points of interaction between the two domains has been lacking. Here, we report the use of hydroxyl radical footprinting to define the interface between the two domains. Cations that support very efficient ribozyme catalysis (magnesium and cobalt(III) hexammine) lead to the formation of a docked complex that features several regions of protection, indicating a solvent-inaccessible core within the tertiary structure of the complex. Cations that are suboptimal in cleavage reactions do not produce complexes with regions of reduced solvent accessibility. Nucleotides encompassing the substrate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected, suggesting their internalization into the catalytic core. Four distinct segments of the ribozyme are protected, including G11-A14, C25-C27, A38, and U42-A43. Protection of these sites is eliminated when g+1, an essential base at the cleavage site, is replaced by A. In addition, mutations which are known to decrease the fraction of docked complexes decrease or eliminate formation of a solvent-inaccessible core. Taken together, these observations demonstrate that we have identified the catalytic core of the active hairpin ribozyme-substrate complex.

Base Sequence↗
PubMed1998

Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer.

The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.

Base Composition↗
PubMed1995

A stable interaction between separated pyrimidine.purine tracts in circular DNA.

Pyrimidine.purine tracts are widespread in eukaryotic genomes and have the potential to form a number of unusual structures including triplexes. Two such tracts, which could form triplexes with each other but not with themselves, were cloned into a plasmid at separate sites. Upon lowering the pH, linear, open circular and relaxed plasmid molecules formed a number of novel structures that were observed on agarose gels and directly by electron microscopy. In open circles a stable join was formed between the two Pyr.Pur tracts giving rise to molecules resembling dumbells, trefoils and tetrafoils, which collectively are termed T-loops. The structure was stable at pH 8 and contains a single-stranded region that was sensitive to P1 nuclease. Thus, there is no apparent topological impediment to the formation of triplex-mediated loops in circular molecules. These structures may be important for gene regulation and chromosome condensation.

Base Sequence↗
PubMed1995

Specificity of monoclonal antibodies produced against phosphorothioate and ribo modified DNAs.

A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their immunogenicity was studied in mice. Only those polymers which were nuclease-resistant were immunogenic and in these cases monoclonal antibodies were prepared. The specificity of the antibodies was measured by direct and competitive Solid Phase Radioimmune Assay (SPRIA) and on this basis four types of antibody could be identified. Type I antibodies are specific for the immunizing polymer and show very limited crossreactivity. For example, Jel 384 binds only to poly(dsA).poly(dT); Jel 453 and 462 bind only to poly(dsG).poly(dC) and poly(dsG).poly(dm5C). Type II antibodies bind to most polymers containing the appropriate modification but will not bind to unmodified DNAs. For example, Jel 343 binds to most thio DNAs regardless of sequence; Jel 346 binds well to most ribose-containing polymers and may be a useful reagent for the detection of the 'A' family of conformations. Type III antibodies bind to most nucleic acids whether modified or not. Their specificities are similar to autoimmune antibodies. Type IV antibodies are single strand-specific such as Jel 383 which binds to poly(dT). There were no examples of antibodies which bound specifically to the immunizing DNA and the unmodified polymer. Thus, modified DNAs cannot be used to prepare sequence-specific reagents. Also, the immunogenicity of modified nucleic acids may limit their usefulness in antisense technologies.

Animals↗
PubMed1994

Kilobase-range communication between polypurine.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes.

Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb. The tracts [d(TCCTTC)n and d(CTTCCT)n where n = 6 or 12] were designed so as to be able to form triplexes with each other but not with themselves. Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels. A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine. In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine. Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr.pur.pyr type. The extent of dimer and loop formation was dependent on the length of the pyrimidine tract: dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required. Hysteresis was also evident to a remarkable extent. Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped. However, the structures were resolved into the component linear plasmids by incubation with nuclease P1. This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation.

Base Sequence↗
PubMed1993

Coralyne binds tightly to both T.A.T- and C.G.C(+)-containing DNA triplexes.

Coralyne is a DNA-binding antitumor antibiotic whose structure contains four fused aromatic rings. The interaction of coralyne with the DNA triplexes poly(dT).poly(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[d(C+T)] was investigated by using three techniques. First, Tm values were measured by thermal denaturation analysis. Upon binding coralyne, both triplexes showed Tm values that were increased more than those of the corresponding duplexes. A related drug, berberinium, in which one of the aromatic rings is partially saturated, gave much smaller changes in Tm. Second, the fluorescence of coralyne is quenched in the presence of DNA, allowing the measurement of binding parameters by Scatchard analysis. The binding isotherms were biphasic, which was interpreted in terms of strong intercalative binding and much weaker stacking interactions. In the presence of 2 mM Mg2+, the binding constants to poly(dT).poly-(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[(C+T)] were 3.5 x 10(6) M-1 and 1.5 x 10(6) M-1, respectively, while the affinity to the parent duplexes was at least 2 orders of magnitude lower. In the absence of 2 mM Mg2+, the binding constants to poly[d(TC)].poly[d(GA)].poly[d(C+T)] and poly-[d(TC)].poly[d(GA)] were 40 x 10(6) M-1 and 15 x 10(6) M-1, respectively. Thus coralyne shows considerable preference for the triplex structure but little sequence specificity, unlike ethidium, which will only bind to poly(dT).poly(dA).poly(dT). Further evidence for intercalation of coralyne was provided by an increase in the relative fluorescence quantum yield at 260 nm upon binding of coralyne to triplexes as well as an absence of quenching of fluorescence in the presence of Fe[(CN)6]4-.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1993

Plasmid dimerization mediated by triplex formation between polypyrimidine-polypurine repeats.

The ability of independent pyr.pur tracts to participate in triplex formation has been investigated in linear plasmids. The pyr.pur tract could be positioned at the ends of the plasmids or internally by a suitable choice of restriction enzyme. Dimer formation between plasmids was monitored by mobility shifts on agarose gels as well as by direct visualization in the electron microscope. Linear dimers and X and Y structures were observed. Control experiments showed that a pyr.pur tract was essential and was consistent with triplex formation in which the two pyrimidine strands were antiparallel. These structures were formed at pHs between 4 and 6, but once formed they remained stable up to pH 7. Spermine was required for formation of dimers at low ionic strength, but once formed the dimers remained stable in the absence of spermine. Additional linear plasmids were constructed with pyr.pur tracts at both ends; these formed structures at pH 4 which had mobilities identical to those of open circles. Triplex formation of this type may serve as a good model for loop formation in eukaryotic chromosomes.

Base Sequence↗
PubMed1991

Polyamines favor DNA triplex formation at neutral pH.

The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly[d(GA)] showed that at low ionic strength and pH 7, 3 microM spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500 microM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurred at a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500 microM spermine. Fourth, intermolecular triplexes can also form in plasmids that contain purine.pyrimidine inserts by the addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 microM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex conformation in vivo.

DNA↗
PubMedDate not supplied

Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation.

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 micrograms/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 microM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.

Chromosomes, Fungal↗