Abstracts for papers from the Bruist laboratory (1999 - 1989)

Guo, X., Liu, Z., Liu,S., Bentzley, C.M., and Bruist, M.F. (2006)  Structural Features of the L-Argininamide-Binding DNA Aptamer Studied with ESI-FTMS. Anal. Chem. 78(20):7259-66

The 24-mer DNA aptamer of Harada and Frankel (Harada, K.; Frankel, A. D. EMBO J. 1995, 14, 5798-5811) that binds L-argininamide (L-Arm) was studied by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). This DNA folds into a stem and loop such that the loop is able to engulf L-Arm. As controls, two derivatives of the same base composition, one with the same stem but a scrambled loop and the other with no ability to form a secondary structure, were studied. The two DNAs that could fold into stem-loop structures showed a more negatively charged distribution of ions than the linear control. This tendency was preserved in the presence of ligand; complexes expected to have more secondary structure had ions with more negative charges. Distinct species corresponding to no, one, and two bound L-Arm molecules were observed for each DNA. The fractional peak intensities were fit to a straightforward binding model and binding constants were obtained. Thus, ESI-FTMS can provide both qualitative and quantitative data regarding the structure of DNA and its interactions with noncovalent ligands.

Sha, R., Liu, F., Bruist, M.F., and Seeman, N.C. (1999)  Parallel helical domains in DNA branched junctions containing 5',5' and 3',3' linkages. Biochemistry 38:2832-41

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms that flank a branch point. In the presence of Mg2+, the four arms are known to stack in pairs forming two helical domains whose orientations are antiparallel but twisted by about 60 degrees. The basis for the antiparallel orientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these two possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the branch point, the strand opposite it contains a 5',5' linkage, and the other two strands contain conventional 3',5' linkages. Electrostatic effects are expected to lead to an antiparallel structure in this system. We have characterized the molecule in comparison with a conventional immobile branched junction by Ferguson analysis and by observing its thermal transition profile; the two molecules behave virtually identically in these assays. Hydroxyl radical autofootprinting has been used to establish that the unusual linkages occur at the branch point and that the arms stack to form the same domains as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. Remarkably, we find them to be closer to parallel than to antiparallel, suggesting that the preferred structure of the branch point dominates over electrostatic repulsion. We have controlled for the number of available bonds in the branch point, for gel concentration, and for the role of divalent cations. This finding suggests that control of branch point structure alone can lead to parallel domains, which are generally consistent with recombination models derived from genetic data.

Bruist, M.F.  (1998)  Use of a Spreadsheet to Simulate Enzyme Kinetics.  J. Chem. Ed.,  75: 372 375.

A spreadsheet with graphics capabilities can be used to illustrate enzyme kinetics and to explain how computer simulation of a reaction mechanism is accomplished.  The exercises described below help illuminate the link between chemical phenomena and their mathematical description.  I use these exercises in an advanced undergraduate biochemistry course.  They can be done in two or three 4-hour computer laboratory sessions.  The first session introduces students to spreadsheets; enzyme kinetics is developed in the remaining sessions.  These examples were created on Microsoft Excel 5.0.  However, they can be generated on any standard spreadsheet.

Bruist, M.F. (1998)  A Simple Demonstration of How Intramolecular ForcesMake DNA Helical.  J. Chem. Ed., 75: 53.

All of our students have heard that DNA is a double helix. Thisdouble helix provides a beautiful and easy to understand exampleof how intermolecular forces combine to determine macromolecularstructure. A simple consideration of hydrogen bonds, dispersionforces, and ionic interactions explains why DNA is most stableas a helix. A model easily made from boxes and string illustratesthe principles clearly. I present this demonstration to my generalchemistry students after intermolecular forces have been introduced.

Myers, E., and Bruist, M.F. (1997)  Why a Particle Physicist is Interested in DNA Branch Migration.  Nucl. Phys. B (Proc. Sup.) 35: 856-858.

We describe an explicitly discrete model of the process of DNA branch migration.  The model matches the existing data well, but we find that branch migration along long strands of DNA (N > 40 bp) is also well modeled by continuum diffusion.  The discrete model is still useful for guiding future experiments.

Kirby, A.W., Gaskin, M.N., Antezana, M.A., Goodman, S.J., Myers,E., and Bruist, M.F. (1997)  Triple-Helical DNA is a Reversible Blockof the Branch Point in a Partially Symmetric DNA Four-Arm Junctions. J. Mol. Bio. 271:349-361.

DNA branch migration is a fundamental process in genetic recombination.A new model system has been developed for studying branch migrationin a small synthetic four-arm junction. The key is the abilityto fix the location of the branch point during the assembly ofthe junction with a reversible block. The block is provided bya short oligonucleotide that forms triplex DNA adjacent to thebranch point at low pH. Raising the pH causes the triplex strandto dissociate, making the branch point free to migrate. Once mobile,the branch point can run off the end of the junction. The timecourse for this runoff is consistent with a random walk of thebranch point. If it is assumed that one migration step moves thebranch point one base pair, the time course gives a rate constantfor one step of 0.2 s-1 at 20?in 10 mM MgCl2, 200 mM NaCl, and1 mM spermine. These values are consistent with othermeasurements of nonenzymatic branch migration. Eco RI restrictionendonuclease binds to two sites on the junction and does not cleavein the absence of Mg++.The presence of the protein essentially blocks branch migrationthrough the binding site. In vivo there must be a specialprocess to get branch points to migrate past bound proteins. Amathematical method for describing a discrete-step model for branchpoint migration is presented and compared to a continuous diffusionmodel.
Keywords: branch migration; triplex DNA; kinetics; oligonucleotides;migration roadblocks

Patsey, R.L., and Bruist, M.F. (1995) Characterization of theInteraction between the Lambda Intasome and att B. J. Mol.Biol. 252:47-58.

Bacteriophage lambda DNA integrates intothe chromosome of Escherichia coli by first forming anintasome at the phage attachment site on the phage DNA with theintegrase Int and integration host factor. This intasome searchesthe host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactionsto achieve integration. This study characterizes the weak interactionof the intasome and att B. The hypothesis that all of theproteins necessary for integration are brought to the reactionsite by the intasome is given additional support by showing thatthe concentration of phage attachment site and not att Bdetermines the optimal concentration of proteins for integration.The value of the dissociation constant of the complex formed betweenthe intasome and att B is determined two different ways.First, the rate of the integration reaction is measured as a functionof the att B DNA concentration. The saturation constantreflects the dissociation constant of the complex. Second, a recombinationreaction is inhibited by the introduction of varying amounts ofa second att B with a sequence change that blocks recombinationwith this site. The inhibition constant reflects the dissociationconstant of the intasome and altered att B in this experiment.The two methods agree and give a dissociation constant of approximately300 nM. Att B contains two core binding sites for the intasome;it is shown that both are necessary for efficient capture. Thevalue of the dissociation constants are considerably lower whena mutant integrase, IntE174K, is used. This increased affinityfor core sites can explain how IntE174K can function in the absenceof integration host factor. The inhibition constants also showdependence on the exact sequence of the inhibiting att B.Possible implications of this dependence are discussed.
Keywords: site-specific recombination; saturation kinetics;inhibition kinetics; DNA binding; Holliday intermediate

Bruist, M.F. (1991) A Study of Ion Gradients across Membranes Using Sonicated Phospholipid Vesicles and Halobacterium halobium. in Pew Undergraduate Biology Laboratories (Heston, K.P.,and Stewart, B.Y., eds) Mid-Atlantic Cluster, Pew Science Programfor Undergraduate Education, Haverford, PA. Part 4, Chapter X.

Hydrogen ions gradients across membranes provide a crucial intermediatein bioenergetics. This lab has two parts. The first uses a modelmembrane system, sonicated phospholipid vesicles, to investigatethe properties of three ionophores: CCCP, valinomycin, and nigericin.The ability of these ionophores to transport or stimulate transportof hydrogens ions across the bilayer will be measured. In thesecond part, the bioenergetics of the salt-loving bacterium Halobacteriumhalobium will be investigated. Two retinal-containing proteins,bacteriorhodopsin and halorhodopsin, play important roles in thisprocess. They are light driven ion pumps. The uncouplers studiedin the first section will be used to investigate the potentialand hydrogen ion gradients which form across the cytoplasmic membraneof these bacteria when they are illuminated.

Johnson, R.C., and Bruist, M.F. (1989) Intermediates in Hin-MediatedDNA Inversion: A role for Fis and the recombinational enhancerin the strand exchange reaction. EMBO J. 8:1581-1590.

The site-specific inversion reaction controlling flagellin synthesisin Salmonella involves the function of three proteins:Hin, Fis, and HU. The DNA substrate must be supercoiled and containa recombinational enhancer in addition to the two recombinationsites. Using mutant recombination sites or modified reaction conditions,large amounts of complexes can be generated which are recognizedby double-stranded breaks within both recombination sites uponquenching. The cleaved molecules contain 2-bp staggered cuts withinthe central dinucleotide of the recombination site. Hin is covalentlyassociated with the 5' end, while the protruding 3' end containsa free hydroxyl. We demonstrate that complexes generated in thepresence of an active enhancer are intermediates that have advancedpast the major rate limiting step(s) of the reaction. In the absenceof a functional enhancer, Hin is also able to assemble and catalyzesite-specific cleavages within the two recombination sites. However,these complexes are kinetically distinct from complexes assembledwith a functional enhancer and cannot generate inversion withoutan active enhancer. The results suggest that strand exchange leadingto inversion is mediated by double-stranded cleavage of DNA atboth recombination sites followed by rotation of the strands toposition the DNA into the recombinant configuration. The roleof the enhancer and DNA supercoiling is discussed.
Key words: DNA cleavage/DNA supercoiling/nucleoproteincomplexes/recombinational enhancer/site-specific DNA recombination



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