Metal Ions in DNA Cruciforms: the effect of Zn2
on linear and supercoiled AGCT sequences
by Noelle Dwarzski
Abstract
The (AGCT)n sequences in negatively supercoiled DNA can adopt several noncanonical structures, including cruciforms. Zinc ions have been shown to stabilize these
cruciforms. To understand the role of zinc in the stabilization, the d(AGCT) tetramer was
synthesized and studied using NMR techniques. Zinc binding sites of the d(GC)
dinucleotide were also investigated. The NMR results for d(GC) indicate that zinc ions have
high affinity toward binding to the N3 position of the cytosine bases, followed by binding to
the N7 site of guanines. The zinc ions also introduced conformational changes in the
d(AGCT) sequence. The NMR results were used to design a model of the d(AGCT)14
cruciform by computational chemistry.
Introduction
Zinc is one of the essential elements in biology. Specifically numerous enzymes are
known to employ zinc ions in the processes of replication, transcription, and translation. In
addition, zinc ions play a role in the conformational changes that occur within the DNA
structure. (1) For example, several proteins, such as zinc fingers, use zinc ions to stabilize
the DNA structure.
Zinc is also known to interact directly with the genomic DNA. Zinc induces a bend
within the transcription factor IIIA binding region of the 5SRNA gene. (2) Structural
polymorphisms of some homopurine-homopyrimidine sequences are observed in the
presence of zinc ions. (3) Zinc ions also cause the isomerization of certain intramolecular
DNA triplexes. (4)
One of the most interesting recent developments is the finding that Zn2+, as well as
Co and Cd2+, selectively induces a conformational change in AGCT sequences in
negatively supercoiled plasmids. (5) Reactivity of several plasmids, including pUC19
(Figures 1 and 2), toward chloroactealdehyde (CAA) and bromoacetaldehyde (BAA) was
investigated. Both CAA and BAA react strongly with cytosine and adenine, while guanine
only shows a weak reaction. (6) The studies showed that CAA and BAA modified all
cytosines of the AGCT sequences in the plasmids following the addition of zinc ions.
Specifically, it is believed that the conformational changes induced by zinc cause a distortion
of the Watson-Crick guanine-cytosine base pair, possibly by direct binding of the Zn2+ to the
bases. (Figures 3 and 4) The changes induced by Zn2+ were found to be related to the
2+
negative supercoiling of the plasmids; however, linear analogs of supercoiled plasmids also
show the effect, although somewhat weaker.
The zinc-induced modifications are sequence dependent. Only the 5’-AGCT-3’
sequences are affected. The sequence 5’-TCGA-3’ does not undergo a conformational
change. Sequences with some of the bases switched such as the 5’-AGCT-3’ are not
affected. Furthermore, the 5’-GC-3’ sequences also show changes in conformation,
although these modifications are not as significant as those in the AGCT fragments.
All the observations suggest that the AGCT sequence is unique in its interaction with
Zn ions. Because of this uniqueness, the sequence could be applied as a marker in
genomic research. There could as well be some other potentially important applications,
taking into account that the Zn2+-AGCT interaction in the main sequences of plasmids is
different from that in the protruded sequences such as AGCT cruciforms. The study by
Kang and Wells (5) performed on the pRW2302 plasmid with and (AGCT)8 insert clearly
supports this idea.
2+
Structural biology of plasmids is still in its infancy, mainly because of the complexity
and dynamic behavior of these large systems. NMR and crystallographic studies of the
AGCT sequences in supercoiled plasmids could be very difficult due to the conformational
flexibility of the plasmid DNA. On the other hand, studies of the conformational changes
in the linear AGCT fragments seem quite feasible. We were particularly interested in
exploring Zn2+ binding sites in the AGCT sequence. Also the binding properties of the
central fragment of the sequence (5’-GC-3’) were of great interest. In order to understand
the dynamics of Zn2+ binding, NMR techniques were applied. To elucidate the differential
behavior of Zn2+ ions in the AGCT cruciforms, we embarked on a structural study of the
(AGCT)14 cruciform by employing the methods of molecular modeling.
Materials and Methods
Materials. The tetranucleotide was prepared using a Pharmacia Gene Assembler by
the phosphoramidite method (7,8) and purified using Waters HPLC. The DNA was
detritilated using an ether extraction, and the resulting solution was lyophilized into a dry
sample. The GpC dinucleotide was purchased from Sigma Co. and was used without further
purification.
Methods. NMR: The DNA samples were dissolved in D2O and 0.10 M NaCl and
the 1H NMR spectra were taken. 1H spectra of the AGCT and GpC nucleotides were first
obtained, then the Zn2+ titration was performed and the corresponding spectra were
recorded. All of the NMR spectra were recorded on a Bruker 400 MHz instrument at the
Shenandoah Valley Regional NMR Facility, JMU.
Modeling: All of the modeling was performed on a Silicone Graphics Octane
computer. The InsightII/Discover program was used to build the starting structures then
the Amber force field and the conjugate gradient method were employed to minimize the
structures to the lowest energy conformation.
Results and Discussion
Results. NMR. Changes in the chemical shifts of the base aromatic protons are often
used as a tool in the NMR studies of metal-nucleotide complexes (9). Therefore, our
analysis focused on the modifications involving the H8 of G, H8 and H2 of A, and H5 and
H6 of C. Figure 5A shows the 1H spectrum of the AGCT sequence without zinc added to
the solution. The aromatic protons resonate in the 7.0-8.0 ppm region, as previously
observed (10,11). The 1’Hs are found at approximately 6.1 ppm and the 5H of cytosine is
found at 5.9 ppm. The 3’H, 4’H, 5’H, and 5’’H are found in the 3.0-5.0 ppm region and
the 2’H and 2’’H are found in the 1.5-2.5 ppm region. Finally, the methyl group on the
thymine is observed at approximately 1.0-1.7 ppm. The complicated spectrum of 1H NMR
is a product of the coupling, the overlap of different peaks, and the added water interference,
which is normal for an experiment performed without water suppression. This is also the
case in spectrum of the GpC sample. (Figure 5C). Guanine’s H8 is observed at 7.9 ppm,
cytosine’s H6 appears at 7.7 ppm, the deoxyribose’s 1’s are observed at 5.8 ppm and are
followed by cytosine’s H5 at 5.6 ppm. Figure 5B illustrates the results of the AGCT titration
with zinc chloride. All of the aromatic protons undergo a shift in the presence of zinc ions.
Initially only the cytosine and adenine are significantly affected by the presence of the zinc
ions; however, at higher concentrations the guanine is also affected. Broadening of the
peaks observed upon addition of Zn2+ suggest a conformational change within the sequence.
This change seems to be quite significant at the R-value between 2 and 4.
Figure 5D displays the results of the zinc chloride titration of the GpC sequence and
the results are similar to the AGCT titration. Initially the cytosine is only affected by the
addition of zinc, the H5 and H6 clearly shifts downfield as a product of the zinc being
bound to the N3. Then, the guanine’s N7 binds the zinc and the H8 moves downfield.
Figure 6 illustrates the relationship between the chemical shifts of the aromatic
protons of the AGCT sequence and the [Zn2+]/[DNA] ratio. Initially, the cytosine and
adenine are the nucleotides affected by the presence of zinc ions, then once the
concentration of zinc increases the guanine also is affected. This figure shows that the
guanine requires higher concentrations of zinc ions to become saturated. Figure 7 presents
the chemical shifts of the aromatic protons of the GpC sequence in the presence of different
concentrations of zinc ions. This figure also demonstrates that the cytosine becomes
saturated first, then the guanine.
Modeling. Kang and Wells (5) observed that the (AGCT)n inserts of some plasmids
were involved in the formation of cruciforms. They also found that the nature of the
interaction between Zn2+ and the AGCT sequences depends on the location of these
sequences. The AGCT fragments of the cruciform stems are much less reactive than the
corresponding sequences in the main chains of supercoiled plasmids. Here, Zn2+ ions could
actually stabilize the GC base pairs instead of inducing the base pair disruption. In order to
elucidate structural details of the interaction, we built and optimized a model of the
(AGCT)14 cruciform. Schematic representation of this model is shown in Figure 8 and the
minimized structure is presented in Figure 9. Figure 10 shows the space filling model of the
cruciform. The stems of this cruciform consist of the AGCT sequences in B-DNA
conformation. The complementary strands of these sequences are connected by a AGCT
single-stranded loop. The cruciform junctions are at the C and G bases, leaving them open
for interaction with Zn2+ ions. The unpaired bases are involved in stacking with the flanking
bases. Interestingly, the unpaired guanines are also involved in a hydrogen bond to the
neighboring phosphate oxygens.
Discussion. NMR studies of the Zn2+ interaction with the AGCT and GC sequences
provide several interesting observations. Of the two bases constituting the GC dinucleotide,
the cytosine base seems to be the first to be involved in zinc binding. Most likely, this
binding happens at the N3 site of the ring. However, saturation of this site occurs quite
quickly with the increasing concentration of Zn2+ (Figure 7). On the other hand, continuing
changes in the chemical shifts of G-H8 suggest that, at higher Zn2+ concentration, guanine is
the main binding site.
The situation in the AGCT sequence is more complex. The aromatic protons of
guanine, adenine and cytosine are affected by the Zn2+ binding (Figure 6), while thymine’s
protons seem unperturbed. The interpretation of the shifts is obscured by peak broadening
occurring at higher Zn2+concentrations. This broadening suggests a conformational change
within the sequence. At present, the exact nature of the change is not well understood.
Previous studies (12) indicate that the best Zn2+ binding site could be N7 of guanine.
However, it is not clear how this binding would indicate conformational changes in the
sequence. On the other hand, crosslinking of two binding sites, leading to substantial
changes in the geometry of the sequences seems quite likely. It should also be noted, that
superhelical stress may produce several additional binding sites, usually not available in linear
DNA. In order to test this hypothesis, we constructed a model of the AGCT sequence in a
supercoiled plasmid. The geometry of the supercoiled AGCT sequence would be
characterized by smaller twist angles and increased roll angles (Figure 11) (6). Our
preliminary calculations suggest that under these conditions the N7 sites of guanine and
adenine approach each other, allowing a simultaneous metal binding to those coordination
centers.
Crosslinking of bases may also be expected in the AGCT cruciform. Figure 12
shows the center of the minimized model of the cruciform. This model describes the
conformation before the addition of Zn2+. Here the junction cytosine bases are available for
metal binding at their N3 sites. Furthermore, the flanking GC pairs are quite distorted
exposing the N3 sites of the cytosine rings. The distances between the neighboring N3 sites
are 4.08 and 4.11 A. Since an average Zn-N bond length is about 2 A, it seems quite likely
that the zinc center could crosslink the two N3 sites. Additional calculations and studies of
model compounds mimicking the crosslinking are presently under way.
Conclusions. Our experimental and computational studies of zinc binding to the linear
AGCT sequences indicate that this interaction may cause a distortion of the conformation of
the sequence. To model the behavior of AGCT under the superhelical stress we initiated a
series of calculations simulating the supercoiling environment. Further experiments and
theoretical consideration are necessary to characterize the exact nature of the Zn-AGCT
complexes. The results obtained so far indicate that, in addition to the structural studies,
calculations of the electronic properties of the AGCT sequence may be necessary.
Specifically, the electronic structure of this sequence could be an important factor in the
interaction with metal ions. Quantum-chemical ab initio calculations should provide a better
knowledge of the electron density distribution, electrostatic potentials and other electronic
properties. This in turn would allow a detailed comparison of the steric and electronic
factors.
Acknowledgements. We would like to thank Professor Cynthia Klevickis, CISAT, James
Madison University, and Mr. Thomas Gallaher, Department of Chemistry, James Madison
University for their excellent help with NMR and Professor Jill Granger, Sweet Briar College
for her assistance with the AGCT synthesis. Most of all, I would like to thank Dr. Sabat for
the many learning experiences I have had this summer.
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