DIMACS Workshop on DNA Sequence and Topology
April 19-20, 2001
DIMACS Center, Rutgers University
Presented under the auspices of the Special Focus on Computational Molecular Biology.
- Wilma K. Olson,, Rutgers University, email@example.com
- Bernard D. Coleman, Rutgers University, firstname.lastname@example.org
- Victor B. Zhurkin, NIH, email@example.com
Large-Scale Chromatin Structure and Dynamics
Andrew Belmont, University of Illinois
Over the past several years, we have developed a method for
labeling specific chromosome sites using the lac repressor/
operator interaction. This method allows direct in vivo
visualization of chromosome dynamics. In addition it can
be combined with gene amplification to create labeled,
amplified chromosome regions, ranging in size from chromosome
bands to entire chromosome arms, with homegeneous folding
patterns. Using immunogold labeling, the folding motifs of
these specifically labeled chromosome regions can be analyzed
by electron microscopy reconstruction methods. Here we review
some of the progress made using this technology. We demonstrate
large-scale chromatic fibers, representing a level of chromatin
folding beyond the 300 nm chromatin fiber, within mammalian
interphase nuclei. The compaction and orientation of these
fibers is generally stable over a period of hours. Over a
shorter time of seconds, rapid but constrained mobility is
observed and over longer periods, a reproducible pattern of
conformational changes occurs during cell cycle progression,
including changes associated with DBNA replication initiation.
Despite the general, long-term immobility of chromosome regions,
several examples of long-range motion within the nucleus of
specific chromosome regions have been observed which are linked
to distinct stages of the cell cycle. Targeting of
transcriptional activators to these labeled chromosome regions
results in dramatic unfolding of large-scale chromatin structure,
which may be related to targeting of HAT and remodeling complexes
to these chromosome regions; changes in intranuclear positioning
are also observed. Mitotic chromosome condensation is accompanied
by highly reproducible by dynamic positioning of specific sequences
within the condensed chromosone. EM reconstructions of labeled
chromosome regions of varying size during chromosome condensation
/decondensation are in progress to reveal the underlying folding
motifs for this large-scale chromatin structure.
Topology Driven Transitions in DNA Structure:
Their Mathematical Analysis and Roles in Biology
Craig J. Benham, Mount Sinai School of Medicine
This talk will briefly describe a formally exact mathematical
method for analyzing the topologically driven destabilization
of the DNA duplex. Our method calcuated the statistical-
mechanical equilibrium distribution of a population of
identical DNA molecules among all the available conformational
states. The global character of the topological constraint
couples together the transition behaviors of all regions within
the domain. When applied to the analysis of specific DNAs,
its results agree in a quantitatively precise manner with
experimental measurements of the locations and extents of
local strand separations as functions of the imposed linking
deficiency. This high degree of accuracy justifies its use
to predict the duplex destabilization properties of other
DNA base sequences, on which experiments have not been performed.
The sites of predicted duplex destabilization within natural DNA
sequences do not occur at random, but instead are closely
associated with specific types of DNA regulatory elements.
Examples include sites controlling the initiation and
termination of gene expression, origins of replication,
and positions where DNA is attached to the nuclear scaffold.
This work has implicated topology induced DNA duplex
destabilization in the mechanisms of funtion of several
types of regulatory elements. Examples will be presented
which illustrate how these analyses have helped elucidate
several mechanisms of DNA regulation, with emphasis on
DNA Sequence-Structure Relationships
Christopher A. Hunter, University of Sheffield
The structural properties of double helical DNA are sensitive
to the base sequence, and this plays an important role in
determining the way in which this molecule can be manipulated.
Sequence-dependent structure is utilised by proteins for
sequence specific DNA recognition and controls the positioning
of DNA in nucleosomes. The molecular basis of sequence-structure
relationships lies in the aromatic base stacking interactions.
We have used a synthetic chemical system to develop a reliable
computational model for these interactions. This model in
conjuction with analyses of X-ray crystal structures of DNA
oligomers has enabled us to produce a fast and accurate
computational method for predicting the sequence-dependent
structural properties of DNA. This presentation will outline
the approach and illustrate the problems which can be tackled
with these computational methods.
Probing the Architecture of the Crossover Site
Synapsis Promoted by gd Resolvase.
Andres E. Leschziner, Yale University,
gd resolvase catalyzes the recombination between the
two copies of the gd transposon that are found in a plasmid
after transposition, generating two plasmids containing
one transposon each . Recombination takes place
within an elaborate nucleoprotein complex containing
twelve resolvase monomers plus two 120 bp DNA sites
(the res sites) and has stringent topological
Crystal structures have been solved of resolvase both
in the absence [2, 3] and presence  of DNA. Although
a wealth of information was obtained from these structures,
nothing was learned concerning the interaction of the
resolvase dimers responsible for the actual strand exchange
- those bound at site I.
Ideally, to address this question one would like to be
able to study site I in isolation, thus bypassing many
of the complexities of the system. This has recently become
possible thanks to a novel resolvase mutant (; M. Boocock,
personal communication) that is able to catalyze recombination
between two isolated site I's in linear DNA. Given that
the structure of a resolvase dimer bound to a site I is known,
one can envision a system where the relative orientation of
two site I's (and therefore that of the dimers bound to them)
is fixed and can be set experimentally. In such a system, one
could probe for recombination to test which specific arrangement
of resolvase dimers is a favorable one.
We achieved this by taking advantage of another crystal
structure: that of the IHF-DNA complex . IHF binds and
bends DNA by about 180=B0, effectively creating a U-shaped DNA.
In a linear piece of DNA containing two site I's and an
IHF-binding site between them, the addition of IHF should
bring the two site I's in close proximity. If the substrates
are used at low enough concentration, intermolecular reactions
should be eliminated. The relative orientation of the two site
I-bound resolvase dimers can be adjusted by varying the number
of base pairs between the site I's and the IHF-binding site.
We generated a series of substrates spanning over a full B-DNA
turn. The results from this series indicate that the favored
orientation is one where the two dimers are aligned more or
less perpendicularly to the plane of the IHF-induced bend.
The data also suggest that the synapsis involves a crossing of
the crossover sites (as opposed to a parallel arrangement of
the two DNA=92s) but do not distinguish between the two
possible crossings (a positive or a negative node) and therefore
did not allow us to determine whether the two dimers interacted
through their catalytic or DNA-binding domains.
In order to address this question, we generated a substrate
(of known topology) consisting of two catenated circles of
3 kb and 200 bp. Each circle contains a site I. Depending
on the writhe of the crossover during the resolvase-catalyzed
recombination, the two circles would be fused together in one
of the two possible orientations and these could be easily
probed by restriction digests. Reactions using this substrate
showed a clear bias in favor of the product resulting from
resolvase acting on a crossing over, corresponding to that
having a negative writhe in the "IHF substrates" and indicating
that the resolvase dimers bound at site I interact through
their catalytic domains.
1. Grindley, N.D.F., Resolvase-mediated site-specific recombination,
in Nucleic Acids and Molecular Biology, Eckstein, F. and
Lilley, D.M.J., Editors. 1994, Springer-Verlag: Berlin. p. 236-267.
2. Rice, P.A. and T.A. Steitz, Model for a DNA-mediated synaptic
complex suggested by crystal packing of gamma delta resolvase
subunits. EMBO J, 1994. 13(7): p. 1514-24.
3. Sanderson, M.R., et al., The crystal structure of the catalytic
domain of the site-specific recombination enzyme gamma delta
resolvase at 2.7 A resolution. Cell, 1990. 63(6): p. 1323-9.
4. Yang, W. and T.A. Steitz, Crystal structure of the site-specific
recombinase gamma delta resolvase complexed with a 34 bp cleavage
site. Cell, 1995. 82(2): p. 193-207.
5. Arnold, P.H., et al., Mutants of Tn3 resolvase which do not require
accessory binding sites for recombination activity. EMBO J, 1999.
6. Rice, P.A., et al., Crystal structure of an IHF-DNA complex: a
protein-induced DNA U-turn. Cell, 1996. 87(7): p. 1295-306.
Ab Initio Quantum Mechanical Analysis of Nucleic
Alexander MacKerell, University of Maryland
Obigonucleotide structure is dictated by a balance of intrinsic
energetics and environmental contributions. Ab initio quantum
mechanical (QM) calculations are an ideal tool to systematically
investigate the intrinsic conformational energetic contributions
to DNA and RNA structure. Approaches used and results from QM
calculations on a variety of model compounds representative of
nucleic acid moieties will be presented. Highlights include the
relationship of the intrinsic conformational energetics of
rotatable bonds in the phosphodiester backbone to the conformation
of duplex DNA and RNA and the influence of cytosine on the A to
B form DNA equilibrium.
Extracting Localized Properties of the TATA-box Via
Continuum Modeling of Cyclization Experiments
Robert Manning, Haverford College
Continuum rod models have been used to extract long-scale
properties of DNA, such as persistence length, from
experimental measurements. On the other hand, many important
features of DNA are quite localized; for example, the
localized kinking of the TATA-box sequence is presumed to
play a role in the initiation of transcription. We will
present a hybrid mathematical model that inserts localized
kinks and/or hinges into a standard continuum rod model,
with the purpose of extracting quantitative measurements
of the local shape and flexibility of the TATA-box from
cyclization experiments made by Jason Kahn (University
of Maryland College Park).
Structure of a Flp Recombinase DNA Complex
Phoebe A. Rice, University of Chicago
Flp is a very distant member of the lambda integrase family
of tyrosine-based site-specific recombinases. Flp catalyzes
the inversion of a segment of DNA in the yeast m plasmid, a
process that permits the amplification of plasmid copy number.
The reaction involves a covalent protein-DBA intermediate and
the sequential exchange of pairs of strands. Using suicide
substrates based on mechanistic studies from several laboratories,
we have crystallized an intermediate of this reaction. The
asymmetric unit of the crystals contains a tetrameric complex,
with pseudo fourfold symmetry, and the Holliday junction
intermediate is in a nearly square planar conformation.
The nearly square planar geometry of the Holliday junction
and the fold of the C-terminal catalytic domain are similar
to that of the Cre-lox complex determined by the Van Duyne
group, but other important features of the complexes are
different. Among these are the protein-protein contacts
that mediate communication among monomers and enforce
the half-of-the sites activity of the tetramer, and the
connectivity of the helix bearing the active site tyrosine,
which is domain-swapped in Flp but not in Cre.
Dynamic Simulations of Regulatory DNA/Protein Systems
Tamar Schlick, New York University
One of the current challenges in macromolecular structure
is to bidge the all-atom level of resolution with the
macroscopic view of biological networks in the cell.
Computational work can help bridge these levels of
modeling, as well as provide insights into structural
and functional processes studied by experiment. Recent
work on using dynamic simulations and molecular modeling
in our laboratory to study TATA-element/TBP association
(role of DNA-sequence flexibility on transcription
interaction and activity), and polymerase/DNA function
(kinetics of DBA polymerase catalysis, including fidelity
mechanism) will be presented.
The TATA DNA studies reveal, by vigorous analysis, tailoring
sequence-dependent structural, energetic, and flexibility
properties for TBP interaction: overall deformability, minor
groove widening (with roll, rise, and shift increases at the
TATA ends), untwisting within the TATA element (with large
rolling at the ends) and relatively low maximal water densities
around the DNA. These properties are likely related to the
association/dissociation interactions within the eukaryotic
transcription assembly. The polymerase DBA studies have
delineated a sequence of local conformational events involved
in the large-scale opening motion of the polymerase and
suggested a rate-limiting step (Arg258 rotation and release
of the catalytic magnesium) that may explain the maintenance
of polymerase's selectivity for the correct incoming partner
to the template base.
Single-Molecule Analysis of DNA Uncoiling by a Type
Terence Strick, Cold Spring Harbor Labs
Type II DNA Topoisomerases (topo) are essential ATP-
dependent enzymes capable of transporting a DNA through
a transient double-strand break in a second DNA segment.
This enables the enzymes to untangle replicated
chromosomes and relax the interwound supercoils
(plectonemes) which arise in twisted DNA. Here we present
a micromanipulation experiment in which we follow in
real-time the action of a single D. melanogaster topo
II acting on a linear DNA which is mechanically stretched
and supercoiled. By monitoring the DNA's extension, we
directly observe the relaxation in the presence of ATP
of two supercoils during a single catalytic turnover.
By controlling the force pulling on the molecule, we
find that enzyme turnover decreases as the force acting
on the DNA increases. This suggests that resealing the
cleaved DNA is a rate-limiting step in the enzymatic
cycle. Finally, in the absence of ATP, we observe the
clamping of a DNA crossover by a single topo II on
at least two different time scales, i.e., configurations.
These results demonstrate that single molecule experiments
are a powerful new tool for the study of topoisomerases
and other proteins which interact with DNA.
DNA Conformation and Genomic Organization
Masashi Suzuki, AIST-NIBHT Structural Biology Centre, Japan
The complte necleotide sequences of a number of genomes have been
determined. The ultimate goal of genome science is to understand
the whole system of life on the basis of these sequences, but
not only to understand its individual components separately.
Analysis of genes coded by genomes appears to be of the central
focus of many researchers. However, understanding of a genome
will not be completed without studying its other active
component, the genomic DNA molecule. In fact, precise
identification of protein genes becomes possible only by
understanding physical characteristics of the DNA. A genomic
DNA molecule interacts with its gene products in many important
biological processes. Thus, gene-coding regions and non-coding
regions are designed, so that these could differentiate from
each other in terms of their physical characteristics, for
the efficient discrimination of the two by transcription
and translation machinery. Such differentiation will be most
important in the human genome, in which over 90% do not code
for a single gene. In fact, it seems difficult to explain
this high rate without assuming some active roles for
non-coding regions. In order to maintain interaction of
the DBNA with proteins inside organisms living at different
temperatures, nucleotide combinations change, so that an
appropriate level of flexibility of genomic DNA molecules
will be induced at these different temperatures. More
than one nucleotide combination can be used for the coding
of the same amino acid sequence, and this redundancy is
used for designing genomic DNA molecules. A genomic DNA
molecule is not an abstract medium designed only for
installation of information on protein sequences.
Importantly, a DNA molecule has physical reality. Physical
characteristics of DNA are coded by the nucleotide
sequence. This fact is comparable with another fact that
the 3D structure and function of a protein are coded by
its amino acid sequence. Physical characteristics of
protein and DNA have enabled life to evolve to the forms
that we see now. Also they have molded life into its
basic design which we now see, and maybe have restricted
life inside the limitation which we have now.
1. Kawashima, T. et al. (2000) Archaeal adaptation to
higher temperatures revealed by genomic sequence of
Thermoplasma volcanium. Proc. Natl. Acad. Sci.
USA 97, 14257-14262.
2. Makino, S., Amano, N., and Suzuki, M. (1999) Visual
presentation of complete genomic DNA sequences, and
its application to identification of gene-coding
regions. Proc. Jpn. Acad. 75B, 311-316.
3. Sucko, J. M. et al. (1998) A transcription frame-based
analysis of the genomic DNA sequence of a hyper-
thermophilic archaeon for the identification of genes,
pseudo-genes and operon structures. FEBS Lett. 426, 86-92.
4. Suzuki, M., Amano, N., Kakinuma, J., and Tateno, M. (1997)
Use of a 3D structural data base for understanding sequence-
dependent conformational aspects of DNA. J Mol. Biol. 274,
421-435.5. Amano, N., Ohfuku, Y., and Suzuki, M. (1997)
Genomes and DNA conformation. Biol. Chem. 378, 1397-1404.
A Theory of DNA Elasticity that takes into Account the
Dependence of Deformability on the Base-Pair Sequence.
David Swigon, Rutgers University
In recent colloborative research, B.D. Coleman, W.K. Olson,
and the speaker have demonstrated the feasibility of calculating
equilibrium configurations in the theory of a base-pair
level model for the elastic properties of DNA. In this
"naturally discrete model", base pairs are treated as flat
rectangular objects and the configuration of a DNA segment
with N+1 base pairs is specified by giving, for each of its
N base-pairs, 6 kinematical variables that measure the tilt,
roll, twist, shift, slide, and rise of a base pair relative
to its predecessor. The elastic energy of the segment is the
sum over the base-pair steps of functions of the 6 variables.
From fluctuations and average values of structural parameters
in crystals of pure DNA and DNA-protein complexes, Olson and
co-workers* have obtained empirical representations for 10
independent functions that quantify the sequence-dependent
elastic behavior of duplex DNA. Each function is determined
by the necleic acid sequence of the step and contains 6
intrinsic kinematical parameters and 21 elastic parameters,
or "moduli". (Some of these 27 parameters vanish when a
base-pair step is self-complementary). As one can calculate
the dependence of configurations on end conditions for any
specification of the elastic and kinematical parameters,
we now have in hand the possibility of adjusting these
parameters so as to maximize the agreement of the theory
with experiments on DNA in solution. Presented will be
the theory of the model and examples of its applications.
We have found that, even when the theory is applied to
idealized hypothetical examples, numerical solution of its
governing equations can give insight into the influences
of intrinisic curvature and twist on the changes in global
DNA configurations caused by intercalating agents and
* W.K. Olson, A.A. Gorin, X.-J. Lu, L.M. Hock, &
V.B. Zhurkin, "DNA sequence-dependent deformability
deduced from protein-DNA crystal complexes,"
Proc. Natl. Acad. Sci., USA, 95, 11163-11168 (1998)
Earliest Evolutionary Signatures in DNA Sequences
Edward N. Trifonov, The Weizmann Institute of Science,
University of Haifa
Modern nucleotide and amino-acid sequences stated their
evolutionary history from very simple patterns.
Reconstruction of the amino-acid and codon chronology
and full genome sequence analysis indicate that the
very first minigenes had the duplex structure
(GCC)6*(GGC), presumably RNA,
coding for Gly6 and Ala6,
respectively. The 3-base periodicity of guanines in
the modern protein-coding sequences and expansion of
triplets GCC and GCU in neurodegenerative diseases
reflect this earliest stage of sequence evolution.
The size (period) of 18 bases (6 aa) is detected so far
only at the protein sequence level. The next characteristic
size, 75-90 bp, comes from recently discovered basic loop
structure of proteins, and is observed in the actual
sizes of the closed loops, and in autocorrelation patterns
of protein sequences. The proteins, apparently, went through
the 25-30 aa loop closure stage in their evolution. Modern
proteins are largely built from consecutively connected
loops of that size. With the increase in protein chain
lengths respective genes soon reached their own optimal
circularization size, 350-450 pb, coding for the proteins
of typical fold size 120-150 aa. The well known
~ 10.5 bp periodicity in
DNA sequences is likely to appear first at this stage of
the sequence evolution. Both DNA curvature and anysotropic
bendability associated with this period would facilitate the
DNA ring closure. Thus, the consecutive stages in the evolution
of the nucleotide sequences have imposed on them the periodicities
of 3,~ 10.5, (18), (~80), and ~400 bases, of which the
bracketed ones are detected so far at the protein sequence level.
Measurement of DNA Intrinsic Curvature in Solution
Alexander V. Vologodskii, New York University
Exceptional accuracy fo DNA persistence lengh determination
can be achieved by the measurement of the cyclization
efficiency of short DNA fragments, about 200 bp in lengh.
Such accuracy results from the very strong dependence of
the cyclization efficiency of the short DNA fragments on
the persistence length. This accuracy and the possibility
to work with a desired DNA sequence allowed us to study the
sequence-dependent intrinsic curvature of DNA molecules.
The measurable persistence length of DNA molecules with
typical sequences depends both on the intrinsic curvature
and on the thermal fluctuations of the angles between
adjacent base pairs. Thus, to address the problem we
designed an "intrinsically straight" DNA molecule that
consists of 10 bp segments, in which the sequence of the
first five bases is repeated by the sequence of the second
five bases only. The persistence length of the first
five bases is repeated by the sequence of the second five
bases only. The persistence length of such DNA depends,
to a good approximation, on thermal fluctuations. We
measured the persistence length of this "intrinsically
straight" DNA and compared it with the persistence length
of regular DNA molecules. By this way we evaluated the
contribution of intrinsic curvature to the measurable
DNA persistence length.
Nucleosome Positioning DNA Sequences
Jonathan Widom, Northwestern University
Certain DNA sequences strongly bias the positioning of the
histone octamer (i.e., a nucleosome) to a particular location
along DNA. Conversely, given a strongly biased nucleosome
position, particular DNA sequences can strongly affect the
dynamic equilibrium accessibility of buried regions of the
nucleosomal DNA. The present talk will explore the DNA
sequence motifs responsible for such behavior. We will discuss
the results of selection experiment for nucleosome positioning
sequences, the basis of DNA mechanics of the selected sequences'
properties, and analysis of signals for nucleosome positioning
that are present in natural genomes.
Antiparallel DNA Loop in the Gal Repressosome
Victor B. Zhurkin, Natinal Cancer Institute, NIH
Gal repressosome assembly and repression of the gal operon in
E. coli occurs when two dimeric GalR proteins and the histone-
like HU protein bind to cognate sites causing DNA looping.
Negatively supercoiled DNA is absolutely required for the
repressosome formation, which prevents analysis of this 113
bp loop by concentional techniques. Structure-based genetic
analysis defined the GalR surfaces interacting to form a
stacked, V-shaped, tetrameric structure (similar to that of
the LacI tetramer). In principle, this tetrameric structure
is compatible with four different looping modes of DNA, two
parallel and two antiparallel. We constructed stereochemical
models for all the four possible DNA loops. The modeling was
based on the sequence-dependent structural parameters of the
interoperator DNA and conformation changes caused by GalR and
HU binding. The harmonic energy functions included in this
model account for the sequence-specific anisotripic bending
and twisting of the duplex at the level of dimeric steps (1).
They also incorporate the strong cross-correlation between
the DNA bending and twisting. Evaluation of the DNA elastic
energies gave unambiguous preference to a novel loop structure
in which the two gal operators adopt an antiparallel orientation
causintg undertwisting of DNA. The calculated DNA trajectory
is consistent with the AFM images of the Gal loop. The 92 bp
lac loop can also adopt a similar trajectory. We suggest that
the antiparallel Gal-like DNA loops may serve as putative
elementary units of prokaryotic chromatin architecture.
1. Olson, W.K., Gorin, A.A., Lu, X., Hock, L. & Zhurkin,
V.B. (1998) Proc. Natl. Acad. Sci., USA 95, 11163-11168.
Contacting the Center
Document last modified on April 6, 2001.