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
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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