Note: Descriptions are shown in the official language in which they were submitted.
CA 02218443 1998-10-26
M-DNA
Jererny S. ~.ee Biochemistry. U of S.
As described in the attached paper, M-DNA behaves as a molecular
wire. This property makes it ideal for the design of microelectronic circuits for
the nanotechnology of the future.
In addition, M-DNAis nuclease resistant and therefore it
will not be subject to rapid destruction as is the case with unmodified DNA.
As well, M-DNAis less negatively-charged than normal DNA and therefore
it will be able to penetrate the cell membrane more easily. Once inside the cellit will slowly convert back to normal DNA. These attributes would be useful
in the following technologies:
[a] DNA immunization. Plasmid DNA can be injected into the muscle of
animals either directly or with a gene gun. In the muscle cells, the genes
within the plasmid are expressed to produce proteins to which the animal
mounts an antibody response. Thus the vaccine is produced by the animal
itself. Because M-DNAis nuclease resistant it would be expected to produce a
more sustained immune response.
[b] Antisense or antigene technology. Short oligonucleotides which are
complimentary to a specific gene sequence have been shown to inhibit the
production of that gene product. However, the oligonucleotide must be able
to enter the cell and survive attack from ubiquitous n-l~leases. Therefore,
conversion of the oligonucleotide to M-DNA would serve as a very useful
but general delivery system.
CA 02218443 1998-10-26
It has been proposed that the stacked aromatic bases of DNA may act as a '~
way' for the efficient transfer of electrons 1,2,3, For example, it has been
demonstrated that photoinduced electron transfer occurred between two
metallointercalators tethered at either end of a 15-base pair duplex 4. On the other
hand, kinetic analysis of distance-dependent elect~on transfer in a DNA hairpin
sugges~ed that DNA is only somewhat more effective than proteins as a conductor
of electrons 5~6. We report here that M-DNA not only contains the ~ stack but also
an interchelated metal ion (Zn2+, Co2+, or Ni2+) 7, nlal~in~ it an excellent candidate
for the conductance of electrons. Efficient electron transfer is observed between two
fluorophores separated by 54 base pairs (over 150 A) in an M-DNA duplex.
Moreover, addition of a sequence-specific DNA-binding protein prevents the flow of
electrons. Therefore, M-DNA behaves as a molecular wire and could be
manipulated to prepare self-assembling electronic circuits.
M-DNA is formed at pHs above 8 in the presence of Zn2+, M2+, and Co2+ but
not Mg2+ or Ca2+ 7. All bacterial and synthetic DNA (except perhaps poly[d(AT)])dismutate to M-DNA under these conditions but the process is readily reversible by
lowering the pH and/or addition of EDTA. Unlike B-DNA, ethidium will not bind
to M-DNA and this forms the basis of a rapid and sensitive "ethidium fluorescence
assay" to monitor M-DNA formation. The mobility of linear or covalently closed
circular forms of M-DNA in agarose gels was only slightly less than that of B-DNA,
ruling out the possibility that the metal ions were causing condensation or
aggregation of the DNA. NMR studies showed that the imino protons of T (pKa 9.9)and G (pKa 9.4) were not present in M-DNA explaining the requirement for a high
pH and suggesting that they were replaced by the metal ion7. Alternatively, the
imino protons might be opaque to NMR due to rapid exchange with solvent. To
CA 02218443 1998-10-26
distinguish between these possibilities the release of protons was monitored during
the formation of M-DNA. As shown in Figure 1, M-DNA begins to form at about 0.7
mM NiCl2 (as judged from the ethidium fluorescence assay); there is a concomitant
release of protons so that KOH must be added to maintain the pH at 8.5. At 1.8 mM
NiCl2, M-DNA formation is virtually complete and the complex starts to precipitate.
Therefore, one proton is released per Ni2+ atom per base pair during the formation
of M-DNA. The Zn2+ and Co2+ isomers of M-DNA also release protons during
formation but precipitation of the complex occurs at a lower concentration of
divalent metal ion than with Ni2+. These results are only consistent with the metal
ion being coordinated to the N3 position of T and N1 of G in every base pair.
Based on these observations, a proposed structure for M-DNA can be
modelled as shown in Figure 2. The A-T and GC base pairs are isomorphous which
is a common feature of all stable helical nucleic acid structures8 9. Compared to a
Watson-Crick base pair, insertion of the metal ion with an imino N-metal bond of 2
A 10,11,12 requires a 20~-30~ rotation of the bases which opens up the minor groove.
One hydrogen bond is retained in both base pairs so that rapid reformation of
normal B-DNA without denaturation of the helix can occur on removal of the
metal ion. The coordination geometry of the metal ion is distorted square planarwith the solvent providing the fourth ligand. The W-Vis spectrum of the Co2+
and Ni2+ isomers of M-DNA have peaks in the visible with ~ of 20 and 60 mol-1
cm-1 respectively; an observation which is consistent with this geometry 13 In the
M-DNA duplex, the metal ion is buried within the helix and d-7~ bonding may occur
with the aromatic bases above and below although we have no evidence for this.
The helix can be considered as a distorted member of the B-type farnily in agreement
with the unremarkable CD spectrum 7. On average the metal-metal distance is 4 A
suggesting that electron transfer within the helix could occur efficiently.
CA 02218443 1998-10-26
This was investigated by preparing duplexes of 20 base pairs with fluorescein
(the donor) and rhodamine (the acceptor) at opposite ends. Under conditions which
favour B-DNA the fluorescence of the donor is quenched and the fluorescence of
the acceptor is enhanced. This is an example of through space energy transfer
(Forster resonance energy transfer or FRET) which has been well-documented in a
number of different laboratories 14,15. The degree of quenching is due to dipole-
dipole interactions and is distance dependent (1/r6); the value of 25% measured in
our experiments is appropriate for this length of helix15. As shown in Figure 3a, the
fluorescence intensity is relatively stable at pH 9 although at long times there is
some loss due to photobleaching. On addition of Zn2+ the fluorescence is quenched
up to 95% over a period of 1 hr, the rate of which mirrors the rate of formation of
M-DNA under these conditions 7. As a control, the 20-mer duplex with only a
fluorescein label shows a small decrease in intensity due to photobleaching as above
(Figure 3a) . Similarly a mixture of two duplexes, one with fluorescein and one with
rhodamine at one end, show minimal quenching either as B-DNA or M-DNA
(Table 1). Upon reformation of B-DNA by addition of an excess of EDTA after 4,000
sec, the quenching is rapidly reversed. These results are summarized in Table 1. The
simplest explanation is that the excited electron on the fluorescein is rapidly
transmitted down the M-DNA helix to the rhodamine; or, in other words, rapid andvery efficient electron transfer must be occurring. The Co2+ and Ni2+ isomers of M-
DNA show quenching of the fluorescein by up to 95% even in the absence of the
rhodamine acceptor (Table 1). One must conclude that the Co2+ and Ni2+
chromophores can themselves act as electron acceptors since the overlap with thefluorescein emmission spectrum is minimal.
The electron transfer in the Zn2+ isomer of M-DNA was investigated in a
much longer helix of 54 base pairs with an estimated length of over 150A. The 54-
mer also contained the recognition site for the D-site binding protein in the middle
CA 02218443 1998-10-26
of the sequencel6. As shown in Figure 3b, there is no quenching due to FRET
because the fluorophores are now well separated. However, upon addition of Zn2+
to form M-DNA, the fluorescent intensity rapidly drops to 25% of the initial value.
In the presence of the D-site binding protein, the fluorescence intensity only drops
slowly. However, as judged from the ethidium fluorescence assay 7, the majority of
the DNA is still converted into M-DNA. Therefore, the DNA-binding protein is
interrupting the flow of electrons by preventing interchelation of the metal ions to
the central portion of the 54-mer. As a control, the D-site binding protein has no
effect on the quenching of the 20-mer (Table 1). On addition of protease at 3000 sec,
the protein is cleaved and the fluorescence intensity begins to drop, eventuallyreaching the minimum value of 25% as above. This experiment is a simple example
of a bioreactive electronic switch.
M-DNA behaves as a molecular wire with a thickness of one atom. It is
surrounded by a negatively-charged organic sheath which acts as an insulator andallows for its manipulation in electric and possibly magnetic fields. It is readily
interconverted with B-DNA; therefore, the rules for cutting and splicing, and for the
self-assembly of a variety of structures such as two and three-way junctions are well-
documented 17,18. The binding of sequence-specific proteins can be manipulated to
mimic electric switches and resistors. Even the manufacture of capacitors can beenvisaged by inserting a backbone-modified, uncharged stretch of M-DNA into a
membrane. These are all ideal properties for the design of microelectronic circuits
for the nanotechnology of the future.
Correspondence and requests for materials to J.S.L. at leejs~sask.usask.ca
Acknowledgements. This work was funded by MRC by grants to J.S.L., L.J.T.D. and
W.J.R., by HSURC by a post doctoral fellowship to P.A. and.by an MRC fellowship to
L.T.
CA 02218443 1998-10-26
References
1. Dand~ker, P. J., Holn~in, R. E. & Barton, J. K. Science 275,1465-1468 (1997) .
2. Hall, D. B., Holn~in, R. E. & Barton, J. K. Nature 382, 731-735 (1996).
3. Arkin, M. R., Stemp, E. D. A., Holmlin, R. E., Barton, J. K., Hormann, A., Olson, E.
J. C. ~ Barbara, P. F. Science 273,475479 (1996).
4. Murphy, C. J., Arkin, M. R., Jenkins, Y., Ghatlia, N. D., Bossmann, S. H., Turro, N.
J. & Barton, J. K. Science 262,1025-1029 (1993).
5. Lewis, F. D., Wu, T., Zhang, Y., Letsinger, R. L., Greenfield, S. R., & Wasielewski,
M. R.Science 277,673-676 (1997) .
6. Taubes, G. Science 275,1420-1421 (1997).
7. Lee, J. S., Latimer, L. J. P. & Reid, R. S. Bio~hem. Cell Biol. 71, 162-168 (1993).
8. Palecek, E. CRC Crit. Rev. Biochem. Mol. Biol. 26,151-226 (l99l).
9. Yagil, G. CRC Crit. Rev. Biochem. Mol. Biol. 26, 475-559 (1991).
10. Swaminathan, V. & Sundralingham, M. CRC Crit. Rev. Biochem. Mol.
Biol. 14, 245-336 (1979).
11. DeMeester, P., Goodgame, D. M. L., Skapski, A. C. & Warnke, Z. Biochem.
Biophys. Acta 324,301-303 (1973)
12. McGall, M. J. & Taylor, M. R.Biochem. Biophys. Acta 390,137-139 (1973)
13. Lever, A. B. P. "Inorganic Electronic Spectroscopy" (Elsevier, Amsterdam) (1988).
14. Cheung, H. C. in "Topics in Fluorescence Spectroscopy" pp 128-171, ed. Lakowicz,
J. R. (Plenum, New York) (1991).
15. Clegg, R. M. Methods in Enzymology 211,353-371 (1992).
16. Roesler, W. J., McFie, P. J. & Dauvin, C. J. Biol. Chem. 267, 21235-21243 (1992).
17. Lilley, D. M. J. & Clegg, R. M. Ann. Rev. Biophys. Biomol. Str. 22, 299-328 (1993).
18. Seeman, N. C. & Kallenbach, N. R.Ann. Re~. Biophys. Biomol. Str. 23, 53-86
(1994).
CA 02218443 1998-10-26
19. Brunger, A. T. X-PLOR Manual, Version 3.1 (Yale University Press, New Haven
USA (1993).
Figure Legends
Figure 1. Release of protons on formation of M-DNA. Upon addition of NiCl2
protons are released and KOH was added (left axis) to maintain the pH at 8.5. After
each addition 10 Ill was removed to assess the formation of M-DNA by the ethidium
fluorescence assay7 (right axis). The experiment was performed in a 10 mL volume,
with 1.1 rnM in base pairs of calf thymus DNA. The DNA was dialyzed against
water and sheared by passing through a 30 gauge needle five times. Arrow (a)
indicates the point at which M-DNA formation began. This lag phase is
proportional to the DNA concentration (data not shown) and is presumably due to
the initial binding of the metal ion to the outside of the helix. Arrow (b) indicates
the point at which 1.1 mM of H+ had been released, beyond which precipitation was
observed.
Figure 2. Modelled structure of M-DNA. (a) Stereo diagram of a mixed-sequence 12base pair helix. Zinc ions are shown as green spheres and are buried in the centre of
the helix. Overall, the structure is similar to that of B-DNA with about 11 basepairs/turn. The model was generated from a canonical ~DNA helix by replacing theimino protons of guanine and thymine with zinc ions and then empirical energy
minimization was performed with X-PLORl9. Parameters describing the
coordination of Zn2+ were included in the minimization together with a water as
the fourth ligand. (b) G~ and A-T base pairs from the modelled structure. Hydrogen
bonds and interactions between Zn2+ and its coordinating groups are shown as
dotted lines. To accommodate the metal ion the base pairs open towards the minorgroove with an additional shearing of the purine towards the same groove. In the
CA 02218443 1998-10-26
G~ base pair, the coordinated water molecule mediates the interaction and helps to
neutralize the electrostatic repulsion between the metal ion and the 2-amino group
of guanine. In the isomorphous A-T base pair the water molecule occupies a sirnilar
position.
Figure 3. The fluorescence of fluorescein-labelled oligonucleotides during the
formation of M-DNA (see Table 1 for the sequences of the 20-mer and 54-mer) (a)
Effect of Zn2+ on the 20-mer duplex. (i) Fl-20-mer duplex without Zn2+; (ii) Fl-20-
mer duplex with Zn2+; (iii) Fl-20-mer-Rh duplex in the absence of Zn2+; (iv) F1-20-
mer-Rh duplex in the presence of Zn2+; (v) addition of EDTA after the formation of
M-DNA. (b) Effect of Zn2+ on the 54-mer duplex. (i) Fl-54-mer-Rh with D-site
binding protein (1~g/ml) (the site is located at the centre of the 54-mer duplex) in the
presence of Zn2+; (ii) addition of proteinase K (50~Lg/ml) after 3,000 sec; (iii) F1-54-
mer-Rh duplex with Zn2+. The experiments were performed in 20mM NaBO3
buffer, pH 9.0 at 20 ~C with 10mM NaCI and 1 mM Zn2+ as appropriate.
Fluorescence intensities are normalized with respect to the fluorescence intensity of
the Fl-20-mer-duplex either in the absence or presence of Zn2+.
CA 02218443 1998-10-26
Table 1. Normalized Fluorescence of the Fluorescein-labelled oligonucleotides
Oligonucleotide Treatment Fluorescence
Fl-20-mer duplex ~--
.. +zn2+ 0.98
+Zn2+ at pH 8.0 0.92
Fl-20-mer single strand ---- 0.87
Fl-20-mer duplex + ~-- 0.97
Rh-20-mer duplex
Fl-20-mer-Rh duplex ---- 0.73
.. +zn2+ 0.05
+Zn2+ + EDTA 0.87
+Zn2+ at pH 8.0 0.92
+Co2+ 0.05
+Co2+ +EDTA 0.7
.. +Ni2+ 0.06
" +Ni2+ +EDTA 0-7
.. +Mg2+ 0.83
" +D-site binding protein 0.06
+zn2+
Fl-54-mer-Rh "
.~ +zn2+ 0.21
All experiments were performed in 20mM NaBO3 buffer, pH 9.0 (or 20 mM
Tris pH 8.0) with 10mM NaCl at 20 ~C and 1 mM Zn2+ or 0.2 mM Co2+ or 0.2
mM Ni2+ or 2 mM EDTA as appropriate. Excitation was at 490 nrn with
emmision at 520 nm. Fluorescence intensities are normalized with respect to
the fluorescence intensity of the Fl-20-mer-duplex either in the absence or
presence of Zn2+ and were measured after 3,000 sec. Sequences and
nomenclature:
The oligonucleotides were labelled 5' with Fluorescein (Fl~ or Rhodamine
(Rh) and were obtained from the Calgary regional DNA synthesis laboratory.
Fl-20-mer:-F1-5'-d(GTC ACG ATG GCC CAG TAG TT)
Rh-20-mer:-Rh-5'-d(AAC TAC TGG GCC ATC GTG AC) and the same
unlabelled sequence was used to produce the Fl-20-mer-duplex.
Fl-54-mer:-Fl-5'-d(GCT ATG ATC CAA AGG CCG GCC CCT TAC GTC AGA
GGC GAG CCT CCA GGT CCA GCT) (The ~site is underlined)
Rh-54m:-Rh-5'-d(AGC TGG ACC TGG AGG CTC GCC TCT GAC GTA AGG
GGC CGG CCT TTG GAT CAT AGC) and the same unlabelled sequence was
used to produce the Fl-54-mer duplex.
CA 02218443 1998-10-26
2.0
.o~4 ~ ~ 100
1.5 -- ~ O
~-
1.0- ~_ 50
a ~ 25 ~
v~a
0.000~ ' ' O
0.0 0.5 1.0 1.5 2.0
[NiCI 2]~ (mM)
CA 02218443 1998-10-26
H\ I =\
/N H-- - -O ~N_ R
R O
~ 2~,
H ~1~
~_</~ --H N~N~ R
N--( ~n+
H
~ ,.
~ ~b =~c
CA 02218443 1998-10-26
(a)
. ~ (i)
08 -
~ ~ ~ (V)
0.6 - ~ i)
_ 0.4 - \
C (iv) \
0.2 - \
~. I ~ I
0 1000 2000 3000 4000 5000
C ~ ~_ (i) (b)
- 0.8 - I ~
' I ~ (ii)
Z 0.6 - ~ \
I (iii) \
0.4 - ~ \
0 2
-
O
0 2000 4000 6000 8000 1 0000
Time (sec~nd~)
CA 02218443 1998-10-26
162
A cooper~tive conformational change in duplex DNA induced by Zn2+ and other divalent metal ions
IEREMY S. LEE ~ND LAUR~ J. P. LATIMER
..~ Or Bloc' r J~, University of Soskat~hewan, Sn~ a,., Sask., C~nada S7N OW0
AND
R. STEPHE~ REID
Dt, , Jl of Chemisrry, Universi~y of S ' ~ ~Ic~.. ., Sasko~oon, Sosk., Conado S7N OW0
Received November 13, 1992
LEE, J. S., LATlMER, L. J. P., and RElD, R. S. 1993. A COO~...I;.~COnrOrmatjOnal change in duplex DNA induced
by Zn2~ and other divalent melal ions. ~iochem. Cell Biol. 71: 162-168.
Zn2~ and some othcr divalcnt metal ions bind to duplex DNA at pHs above 8 and cause ~ confG- -' change.
This new structure does not bind ethidium, allowing the dc~.'.,, - of a rapid r ~ - assay. All duplex DNAs,
regardless of sequence or G-C content, can form this structure. The rate of formation shows a strong d~, ~
on ~ , pH, and Zn2~ r .,Lon, at 20~C, I mM Zn2~, and pH 8.6 the ~' is half complete in
30 min. Addition of EDTA causes rapid reversion to ~B~ DNA, showing that the ncw conformation retains two strands
that are ~ " ' Unlike the ultraviolet or circular dichroism speara, the nucl~ar magnetic resonance spectrum was
informative since thc imino protons of both A ~ T and G C base pairs are lost upon addition of a 5~ ;C amount
of Zn2~ . The pitch of the helix was estimated from gel clc.l.oph ~;a of circular DNAs in the presence of Zn2~ and
it contains at least 5q. fewer base pairs per turn than ~B' DNA. The transformation is coo~.at;~e and shows hysteresis,
suggesting that this is a distinct structure and not simply a minor vuiant of ~B~ DNA. It is proposed to call this ncw
struaure 'M' DNA because of the intimate i...ol.. of metal ions.
Ke,v words: DNA . ~o., COv~ dti.~ trulsition, ahidium binding, divalent maal ions, proton nuclear magneaic
LEE, J. S., L~TIMER, L. 1. P., et RElD, R. S. 1993. A coOp~ confor ?I ' change in duplex DNA induced
by ZD2~ and other divalent metal ions. Biochem. Cell B;Ol.71:162-168.
Lc zinc (Zn2 ~ ) et d'autres ions l ~ " ,_ divalents se lient à l'ADN b;c~~' - e lorsque le pH est supérieur à 8
et ils p.- ' un ' _ de conformation. Cette nouvelle struaurc ne lie pas l'ethidium, ce qui a permis la
mise au point d'une méthode de dosage rapide par lluv-~ ~ Tous Ies ADN bir~;- ., peuvent forma cette struc-
ture peu impone lcur sequence ou leur contenu en G ~ C. La vitesse de ce c'~ _ de conformation est fonement
~;F ' edelai .' _ ~,dupHetdela,;~n~ - a dcZn2~;1a ' - - n. fft àmoitiécomplétéeen30min
a 20~C, Zn2~ I mM et pH 8,6. L~addition d'EDTA entral'ne un retour rapide à la conformation '8' de I'ADN, ce
gui démontre que la nouvelle conformation est formée des dew~ chaines a~ni~Ja~ "'' CO..nail. aux speares
ultraviolct ou de ~I;.h.,: - circulaire, le spectre de résonance ~ L. nucléaire est informatif puisque les protons
des e~ ~ _, imino des deux paires de bases A T a G C sont perdus lors de I'addition d'une quantité s~ e
d~ Zn2 ~ . L'~ - du pas de I'hélice par cl.~llvt~hv.~se en gel d'ADN circulaires en presence de Zn2~ indique
que Ie nombre de paires de bases par tour qu'elle contient est au moins 5q inferieur à celui de l'ADN de conformation
'B'. Cette transformation est ~OVP~aI;~eeteIIe montre une hystéresis, ce qui sug8ère que cette conformation est une
structure distincte et non seulement une variante mineure de la conformation ~B' de I'ADN. Nous proposons de nom-
mcr cette nouvelle structure, I'ADN de conformation ~M', à cause du rôle esscntiel d'ions, ~
Mors clés: conformation de I~ADN, transition .oope. ~ , liaison d'éthidium, ions ': "; divaknts, résonance
~, -, nucléaire.
Traduit par la rédactionl
~ I ~d ~I ~r Divalent cations are perhaps more interesting because a
The binding of metal ions to DNA has been studied exten- series can be written in decreasing order of DNA stabiliza-
sivdy for nearly 40 years. In genaal, cations that bind tion i.e., {Mg, Co, Ni, Mn, Zn, Cd, Cu~ (Eichorn 1962;
primarily to the phosph~e backbone will stabilize the duplex Eichorn and Shin 1968). Thus Mg2+ increases the Tm at all
conformation, whereas those that bind to the bases will tend ~o~ .àlions, wher~as sufficientlY high col-ntl alions of
to denaturc the duplex. Thcse effects are readily demon- Cu2+ will lead to ,i al~,lation of the duplex at room tem-
strated with thermal denaturation profiles ( Tm measure- perature (Eichorn and Shin 1968). This series also correlates
ments). Thus most l-.ono-a' cations such as Na + with thc ability of the ions to bind to the bascs (Hodgson
stabilizc thc duplcx and there is about a 12~C increase in 1977; Sw~nin~h~n and Sullda.àli~.,,l.all. 1979).
Tm for cach l~fold increase in conc~ lion (Marrnur ant As well as influencing thc hclix to coil l-an~ilion, cations
Doty 1962). An important cxception is Ag +, which binds are also involved in pl- e several other structural tran-
tightly to thc bases and therefore decreases the Tm (Guay sitions and ~ nc First, Mg2+ and pol~ ~ both
and B~ 1979)- Similarly multivalent ions, particu- favour the ~ ion of pyr ~ pur DNAs to triplexes (Htun
larly thc pol~ , arc very cffectivc duplex stabilizers. and Dahlberg 1988; Maher e~ al. 1990; Haner and Dervan
A8BRE~IIATIONS: pyr, ~" " ~ pur, purinc, NMR, nucl~ar 1990;Hampeletal. 1991).Thiseffectcanbequitedramatic,
ma~a;ic r~ . MES, ~ ' - .-' '' acid- CHES since the presence of spermine can obviate thc rC~ui~ nl
Ie ~ . '', -acid;CD,circulardichroisrn;DSS, for low pH in the formation of pyr-pur-pyr triplexes.
3~(n '~' 'yl)-l-p.o~~ 'fonic acid. Second, L~lca.ing the ionic strcngth pa~ la~ly with
c~ ~ Imvrm~ c~
CA 02218443 l99X-10-26
LEr ET ~L 163
multivalent cations c.. ~l.. agcs thc formation of Icft-handed 80
'Z' DNA from dl,propriatc alternating pyr-pur s~q. n~
(Rich e~ ol. 1984; Palecck 1991). Third, a r~ ca~d DNA ~ A
structur~ which occurs in (C-A),~ tracts and rcquircs sper- ~ ~Q
mine for stability has bcen dcscribcd recently (Timsit et ol. I ~ pH=6 5
1991). It seems that all thrce of thcse structurcs can occur 70 - ¦ ~ ~
undcr p~ aiO~ cC~ nc and thcy may have ,~ x . ~r,t ~ ~
bi -'~, ' roles (Rich el al. 1984; Lee et at. 1987). Mctal ions ' ~ \
are also found in some DNA-binding agents such as cis- ~ pH=9
plalinum t~ ~c;-- and Fe(lI)-EDTA (Rosenbcrg et al. 'o1969; D'Andrea and Haseltine 1978; Latham and Cech 60
1989), and many DNA-binding proteins rcquire specific 0 100 200300 400 500 600
cations for activity (Mildvan and Loeb 1979). For examplc, Zinc concenl,dllon (IlM)
the 3 ',5 ' -cxonuclcase of DNA polymerasc I from FIG I EffectofZnl~onthcmeltingi a~ (Tm)ofcalf
F~ . 7 CO/; has two metal ions in thc actlvc slt~ (B~sc ~hymus DNA at pH 6.S (_) or 9 (~).
and Steitz 199t).
In this paper we describe the discovery and p. Cli~ ar~ Once formcd. thc ncw conformation is stable in this buffer, but
characterization of a novcl DNA conformaIIon In which th~ Zn2~ c~r - ,~ion is too low to allow the ' - on of 'B'
divalent metal ions play a very Intlmate rolc- Thls lln~ of DNA Addition of 2 rnM EDTA to thc ethidium-zinc buffer
research arose quite fortuitously. Originally, Tm mcasure- rapidiy chclat~s th~ Zn2~ and conv~rts it to the cquivalcnt of ~he
ments wcre being performed to investigate thc cffects of standard ~thidium nUO- . ~e assay containing 0.1 mM EDTA,
Zn2+ and Mg2+ on the stability of pur-pur-pyr triplcxes but no Zn2~, as described earlier ~Morgan er ol. 1979). With
(Kohwi and Kohwi-Shieem~)su 1988; Lyamichev et al. excitation at 525 nm and cmission at 600 nm, 0.5 1lg of duplcx calf
1991). It was dccided to use a pH of 8 ot greater to prevent thymus DNA gives 70 fl n.. - units and thc scalc is linear in
intcrferencc from triplexes of th~ pyr-pur-pyr type (Lce thc range of 0-200 ~ ~;cs units.
et ol. 1984). It soon became apparent that a different struc- Kinel~cs
ture which was dcpPn~l~r~ on Zn2~, but not Mg2+, was Standardrcaaionswercpcrformedat20~Cin300~Lofasolu-
being formed. This new struaurc was availablc to all DNA tion containing 75 ~-M DNA, 15 mM NaCI, 10 mM Tris-HCI
duplexcs rc~Sa. .~ of sequcnce. Furthermore, it did not (pH 8) (from the DNA solution), and 20 mM CHES (pH 9). The
bind ahidium, so that a rapid fluorescence assay WâS quickly final pH was found to bc 8.5. Other buffers uscd were 20 mM
developed (Morgan e~ al. 1979). In this wây, most of the CHES (pH 9.5) (final pH 9.0), 20 mM Tris-HCI (pH 8.0) (final
pleL,~ a.~ charaacrization of this structure could be pH 8.0), and 20 mM MES (pH 6.5) (final pH ~.0). Thesc solutions
pcrformed . Later, NMR studies confirmcd that a Zn 2 ~ w~r~ ~ with ZnC12, which was always added last. At
atom was replacing the imino proton common to both G ~ C vari~Us timcs 3~.L al~qUbcutfSfcr and tPhc rcading was takcn within
and A-T base pa~rs. 5 min.
Malerials snd me~hods Circular Dichroism
~ucleic ocids CD spcctra werc kindly performed by A.R. Morgan ~F.' -
Synthctic duplcx DNA was preparcd by ~~ of an appro- in a buffer of 20 mM sodium borate (pH 9.0) with or without
priate templatc with E. coli or ~f;.,~cocc~.s lu~eus DNA pOIy. I mM ZnC12. Thc calf th~nus DNA ~ ~ r 1. was 150 ~.M.
mert, sc I as described previously (Evans et ol. 1982). Open circular Nuclear Magnetic Resonance
pUCI9 was prepared by r-irradiation with a dose of 8 x 10 rad Spcara werc measured on a Bruker Am 300 , . .d~
(I rad = 10 mGy). Relaxed pUCI9 was prepared by inr bmi-. magnet s~ a at a frequency of 300.13 MHz. Chemical
with 2 units/~g of calf thymus t, p 2 ~ ~ ~ I (BRL) for I h, shifts ar~ quoted in ppm relative to DSS as an internal standard
followcd by deprot~ with phenol. Calf thymus DNA and with a probe ~- , c of 2S~C. Samplc volume was 500 I-L con-
bacterial DNAs were purchased from Sigma. All DNAs were taining 5 mM DNA (F~s ' ~) in 90qo H2O- lOqo D20. The
dialysed into a buffa of 10 mM Tris-HCI (pH 8) and stored frozcn Hore binomial pulse scquence ' 1331 ' was employed for water sup-
at -20Cc. pression(Horel983).Aninterpulsedelayofl85~s~o-,~r.,d g
Thermal ~1 ~ _ io prof les to an excitation maximum at 13.5 ppm was used. Repetition times
~ - - were made on a Gilford 600 sp~ctro- was 5.2 s. Between 1000 and 50Q0 transients were a - ' ~ for
phntc r equipped with a thermo~"o~l_ in either each CO~l~Im~liOfl of ZnC12 depending on the magnitude of the
(a) 20 mM sodium boraIe (pH 9.0) or (b) ~0 mM MES (pH 6.5) peaks in the aromatic reg~on.
,, ~.1 with ZnC12. At pH 9 the solubility limit of ZnC12 Agarose gel ~ rOp,~
is about 500 ~-M increasing to about 5 mM at pH 8.S. Addition Gels (I qo agarose) were run at 60 v for 5 h in a buffer of 20 mM
of 15 mM NaCI to some buffers also incr~as~s the solublllty of sodium borat~ (pH 9) ~u~F'~ '~ with up to 0-5 mM ZnC17
ZnC12. Thus, in general, great care had to be t?ken in making up The plasmid pUCI9 (0.5 ~.g) was incubated for 30 min with the
buffers a~ - 2 Zn-~ to prevent ~)r~ a. It should also ~, ., atiol~ of ZnC12 before addition to the gel.
be noted that DNA itself acts as a chelating agent and therefore
can increase the apparent solubility of ZnC12 quite conside.ibl~. Resulls
Figure I shows the effect of i.~ ashlg Zn2+ c~,..c~ ,a
All fluorescence - I - were performed on a Turner
modd 430 ~c.t.~,f' - ~ :u in a buffer con~ 5 mM Tris- tions on the Tm of calf thymus DNA at pH 6.5 and 9. At
HCI (pH 8.0), 200~M ZnC12 and 0.S ~-i/mL of ethidium pH 6.5, the mqYjmllm inctease in Tm was only 3~C. This
(ethidium-zinc buffa). This buf;er was chosen to take advantage agrees with earlier work by Eichorn's group performed at
of the hysteresis in the d - - n.~ (see below). Iow pH, which had sugg~s~ed that Zn2~ was mostly
CA 02218443 1998-10-26
164 BIOCHEM. CELL BIOL, VOL. 71, 1993
120 ~ ~ 120 ~ ~ ~ ' ~ '
(A) Zlnc cDl,esl at'~n ~ ~ (~) ph
'~ 1 00C~ - l oo~
80 ~ ~ ~02~M' 80 ~--~7
. 60'~ u\ ~ 60
'o aos,ru' 40 ~ ~ ~
20 ~ A 20 ~ ~5
~G 05~ o '~? ~ . Q 9 .
0 501 001 50 0 20 40 60 80
. , 120 ~ ~ ~ ' ~ ~ 120 ~ ~
(C) Te.. , Iralul~ L (D) Metal lons
100D 100~ , o u~u~
80 ~ ' 80
~o~, ' 60
~e 20~ It U207~ 40 ~
0 ' ' ' ' ' ' ' ' ' ' ' ' " 0~--~
~ 50 1 00 1 50 0 20 40 60 80
Time (min) Time (min)
FIG.2. (A) Thc d ~, at different Zn2+ con.~.. t,~ - and pH 8.5 as mcasurcd by loss of ethidium fl e : ~, 200 IIM;
', 500 IIM; ~, I mM; _, 5 mM. (B) Th~ at differcnt pHs and I mM Zn2~ as m~asurcd by loss of ethidium lluor~ -~
-, pH 7; ~, pH 8; ~, pH 8.5; ., pH 9. (C) Th~ ~' - ). at differ~nt ~ ~ albl.~ and I mM Zn2~ and pH 8.5 as measured by
hss of ethidium fluorc~,c.. ~e. o, 0~C; o, 20~C; 3, 37~C. (D) Th~ at pH 8 5 with I mM of various divalent metal ions.
~, Mg2+; ~ Mn2+; ~, Cu2+; ~ Zn2+; ~ Ni2~; ~ Co2+
destabilizing (Eichorn and Shin 1968). On the other hand On the other hand, if poly[d(G-A)] was added to a solution
at high pH, Zn2+ stabilized the DNA duplex by as much of polyld(T-C)] under these con~litions (i.e., with EDTA),
as 12~C. The effect of pH was most unP~cte~l but sug- no [luo-~cace could be detected (data not shown). Previous
gested that some form of specific metal ion complex or workhasd~..lor~lraledthatthisabsenceofr~ndlu-dtionwas
structural rearrangPmPnt was occurring. In either event it due to the very dilute DNA :r ations and the low ionic
seemed rP~con~k to propose that ethidium might not bind strength of the nuolcs;..lcc assay buffers (Morgan et al.
to the new structure. This led to the devcloF of a rapid 1979). Overall, this is an ~ cl~ revealing result because
n ~ ~ence assay, since indeed the new conformation does it shows that this ~ n;on does not involve separation
not enhance the lluol.sc ~: of ethidium of the strands as occurs in dc.,~lu.alion or in triplex for-
lt was found by trial and error that pH of 8 and 200 ~lM mation (I.ee et ol. 1984).
Zn2~ were convenient for tluo,~scen.e ~..ea~u-~.n...ts, since The effect of pH is investigated in Fig. 2B. Above pH 8.5
the new structure would not form under these conditions; the riismlJ~ation occurred rapidly, whereas below pH 8 very
but once formed, it remained stable for at least 30 min. Iittle transformation was detectable. Again addition of
Thus, the ~-I.asu~.d fluol~sccll.c could be used to estimate EDTA increased the fluorescence back to 100qo. Therefore,
accurately the amount of unmodified DNA in a sample even at pH 9 the observed fluorescence loss was not due to
undergoing the transformation. denaturation of the DNA. The rate of formation was
Tbe kinetics of formation as a function of the Zn2+ con- increased with increasing t~l~.pe.alulc (Fig. 2C). However,
ccntration at pH 8.5 is shown in Fig. 2A. At co---e.-tl.-tions even at 0~C the conversion would go to co "1:~ on
above I mM Zn2+, the conversion was very rapid, while eventuaUy.
below SOO ~M formation of the new structure did not go Several other kinetic CA~.illllts were performed to
to co F ' M 3 n even after prolonged incubation. This narrow elucidate the new structure. The ~licmnr~ n of calf thymus
conc~ la~ion range is suggestive of a coo~lalive transition. DNA was measured in the presence of hl;ltd~;ug concen-
Again this is typical of other conformational changes such trations of ethidium. At I mM ethidium the conversion was
as 'Z' DNA (Rich et al. 1984). Another attractive feature c~ Iy inhibited (data not shown). Therefore, the lack
of this fluorescence assay is that e.~.h..-.lls can be per- of ethidiumfluorclc~..cewasmostlikelYduetoweakbind-
formed directly in the ethidium buffer solution. For exam- ing co...ydrcd with 'B' DNA. Thc ~licn~ ion of poly-
plc,additionofanexcessofEDTArestoredthefluo.eî.~.l.e ld(T~)]-polyld(G-A)l was measured in the presence of
immr~li?trly (within I or 2 s) to lOOqo of the value that a~ ion~l polyld(T~)] and polyld(G-A)I (data not shown).
would be expected for the buffer in the absence of Zn2+. Neither had a significant cffect on the rate of formation
CA 02218443 1998-10-26
16
LEE ET ~L. 165
again, s~gR-sting a two-s~randed structure. A-iso consistent 120 - ' ' ' ' '
with this result was the lack of a significant concentration 100O (A) ~aeterlal DNA~
effect. For example, lowering the DNA conce.ll-dtion by
10-fold did not change the rate of transformation, ~3 ' 2 80
an intra-, rather than an inter-mol~ular .c~. ~. ~--e~ ~ The
effect of base composition and sequence on the rate was 60
investigated (Fig. 3). As shown in Fig. 3A bacterial DNAs
ranging from 72~70 G + C (M. Iuteus), through 50qo G + C
(E. coli), to 32qo G + C (Clostridium perfringens), all 20 '
J- ~ed atequivalent rates. Itwastherefore l-nPYpected , ~ , ~
to find that synthetic DNAs showed large differences ~0 20 40 60 80
(Fig. 3B). Under these conditions poly[d(A-T)] was
l, whereas poly(dA) - poly(dT) readjly dicmu~20
and polyId(G-C)l was intermediate in rate; poly[d~T-G)]~ ) Synth-tic DNAr
polyld(C-A)I appeared to be panicularly willing to ~ 100~,
~licm~ , 'Finally, synth~tic DNAs con~ining modified <~ 80 G Cl
bases are shown in Fig. 3C. Unlike polyld(A-T)I, both
polyld(A-U)I and polyld(z7A-T)I formed the new structure ~ 60 ~--
slowly. Other DNAs containing 1, m6A, and z'A all o ~ ~,~
~u~ed rapidly. These last two modifications were ~ 40
imponant because they eliminated the possibility that ~L 20 ~ L
Hoogsteen pairing was involved in the structure.
Other divalent metal ions were also evaluated (Fig. 2D). ~ ' ' ~ '
Ni2+ and Co2+ behaved likeZn2' but Mg2+, Mn2+, and ~ 20 40 60 80
Cu2~ wcre ineffcctive. Thus there appeared to be no cor-
relation between the ability to bind to the bases and the 120 ~
ability to form this new structure. Ag+ was also tested, 100~ (C) Modined DNAs
since it is known to form stable co 1" ~ with the bases
(Guay and E~eauch~F 1979). It was found that under the 80
con~ ionc of Fig. 2D, addition of I mM Ag~ immer~ y r~
led to a 100qo loss of fluorescence. However, addition of 60 ~r~
EDTA caused no increase in fluorescence, demonstrating
that the DNA had been denatured. This served as an excel-
lent control to contrast with the effect of Zn2+. 20 .
Spectroscopic characterization of this structure was ' ~
invfftigated The UV absorption spectrum showed little ~o 20 40 60 80
change. Compared with ordinary 'B' DNA, there was a Time (min)
small h~luc,..~ (about 10%) at 260 nrn, but in general
this was insufficient to allow easy detection or analysis (data FIG.3. E~fect of DNA sequence on thc rate of the ~" '
not shown). The CD spectrum was compared to that of 'B' (ANA - ~ ! """"5 'lu~ s DNA ~Bj~Sy'nthaic DNAs- O poly-
DNA ffig. 4). Sul~ inglj, for a structure that did not bind [d(A~ , polyld(G C)l; ~, poly[d(T-T C)l-poly[d(G-A-A)I; c,
ethidium, the spectrum showed only small chang~s- In poly[d(T C-C)I poly[d(G-G-A)l; ~. poly(dA) poly(dT); ~. Poly-
general the depression in intensity of both the positive and [d(T-G)I poly[d(C-A)l. (C) Modified DNAs: ~, poly[d(A-U)I; ~,
negative bands was similar to that of DNA in the presence poly[d(z7A-T)I; ~, poly[d(m6A-T)I; o, poly[d(T-C)I-poly-
of high concc.-lraIions of cesium (Zimmer and Luck 1974; [d(G-m6A)I; ~, poly[d(T C)l-poly[d(l-A)l; ~, poly[d(T4)1-poly-
Chan et a/. 1979). [d(G-z~A)I.
The NMR spectra of d(T-G)~5-d(C-A)~5 in the absence
and presence of Zn2' is shown in Fig. 5. The imino pro- to an imino proton which is adjacent to a metal ion.
tons of both the A-T and G-C base pairs at 13.4 and If thenewstructurecontainsaZn2+ atomforeverybase
12.3 ppm, respectively (Sklenar and Feigon 1990), appeared pair, the winding of the heli~ might be expected to be rather
abscnt in the spectrum of the new structure (Fig. 5C). Large different. Thus the final series of e,~. ile.lIs were designed
changes were not observed in the rest of the spectrum. At to investigate the topal~ PI properties of circular DNAs
intermediate Zn2+ COIlCe"~rdlions (Fig. 5B) the peaks at in the presence of Zn2+. Supercoiled, relaxed, and nicked
high field were d.";e~cd and a small peak appeared at plasmid DNA was electrophorcseJ on Iqo agarose gels at
12.9 ppm. ' pH 9 wjth in.l~ ' G con.~ lions of Zn2+ (Fig. 7). Elec-
Titration of the imino protons is shown in Fig. 6. The I-opho..;.is under these cor-1:lionc led to more diffuse bands
A-T imino proton was lost before the G-C proton which may with some smearing . - - cd with clc.IIophorcsis at pH 8.
explain some of the sequence effects described above. Com- The control with no znf+ showed that relaxed and nicked
pl~te loss of the signal occurred at about 2.4 mM Zn2~ DNAs (both l-.ono...c. and dimer plasmids) had thc same
which is equivalent to one metal ion per base pair. This is mobility, and some t"F ~ , wcre also di~c~ in the
co~ en~ with every imino proton being replaced by Zn2+ relaxed sample. At 0.5 mM Zn2+, on the other hand, the
or a rapid exchange process. The proton at 12.9 ppm that relaxed and ,~ DNAs had the same .,.ot "- and
appeared at intermediate Zn2+ conc~.lIl.-tions may be due no tOpO;s~,..lc" could be seen in the relaxed DNA. As well,
CA 02218443 1998-10-26
166 BIOCHEM. CELL CIOL. VOL. 71, 1993
~0 220 2~0 260 ~B0 3
WAVELENGTH (nm)
FIG. 4. Circular dichroism Or calf thymus DNA at pH 9.0 in thc abscnce ( ~ - ) or presence of l mM Zn
lD~
0.8-
0~ - o
OL - _
I
o
' ~ ~ 0.2- ~ -
'. t. h ~ W, ~1 ~ 8
0.0 ' ~ ' '
(A) ~) (C) 0 0 0.5 1.0 1.5 2.0 2.5
Zinc Concentr~tion tn~M)
.0 12.0 13.0 12.0 13.012.0
FIG. 6. ~J~ integrals v~sus ZD2 , . for th~
ppm imino protons of 5 mM d(T-G)l,-d(C-A)". ~, 12.4 ppm; o,
FIG. 5. Irnino p~oton NMR of d(T-G),5-d(C-A),5 at pH 9.0 13.4 ppm; ~, 12.9 ppm. This l_st signal is only observed at inter-
with (A) 0, (8) 1.2, and (C) 2.4 mM Zn2~. mediate Zn2~ ~c ~i -
the presence of Zn2+ tended to decrcase the sharpness of Discussion
the bands. The sudden change in mobilities at 0.4 mM again It is clear that the new structure does not represent a minor
demonstrated that the transition was coop~.a~Bc. Here the structural variation along the continuum of 'B' DNA struc-
mobility of the relaxed sample, particularly the relaxed tures. First, it does not bind ethidium well, as this drug
dimer, was interrnediate between the nicked and supercoiled inhibits the dismutation. 'A' and 'B' DNA, as well as RNA
DNAs. Thus, the dismutation induced negative ~u~.coils and even some triplexes, bind ethidium. Second, it demon-
and the helix contains fewer base pairs per turn than 'B' strates hysteresis since it remains stable under ~on~ onc in
DNA. Attempts to measure the overwinding accurately by which it will not form ffig. 2). In other words, the structure
two~; n~ n~ onq~ cl~I~opho~csis (Haniford and Pulleyblank formed under a certain set of con~itionC depends on the
1983; Yagil l991) were u -ncc~rul, because the presence route taken to achieve those cond;t;ons. This manif~station
of Zn2 always inl-odu.ed a level of smearing which of hysteresis is often seen in other ~lluaulal transitions of
obscured the t . pois ~. However, from Fig. 7 it can be DNA (Rich et ol. 1984; Lee er al. 1984; Hampel et a/. 1991).
cstimated that the new structure contains at least 5~o fewer Third, the transition is coopc.ali~c as judged from the
base pairs per turn than 'B' DNA. It is also apparent from effects of iuer ~ e Zn2~ conce.-l-aIions in the kinaic
Fig. 7 that there is an increased mobility and that the separa- t,~.h-IS (Fig. 2) and in the agarose gel cI~rophOlctic
tion of nicked and supercoiled species is reduced. Both of studies ffig. 7). None of these p-o~. Iic~ would be expected
these faas would be q-~icipat~d if the new helix is shorter of a minor variant of duplex DNA, but are more typical
and more compact than in 'B' DNA (Mickel et al. 1977). of the behaviour of a distinct structure such as 'Z' DNA.
CA 02218443 1998-10-26
18
LEE ET AL. 167
OmM0.2 mM 0.3mM 0.4 mM Q5 mM
~ 1 2 3 ~ U 1 2 3 ~ ~ 1 2 a ~ ~ 1 2 3 ~ ~ 1 2 3 .t
FIG 7. Agarosc gel ek.ll~ph~,.c~ at pH 9.0 with 0, 0.2, 0.3, 0.4, and 0.5 mM Zn2+. M, ~ molecular weight markers; lane I, ccc
plasmid; lanc 2, topn'S_ aae-relal~ed plasmid; lane 3, oc plasmid. In lanes I and 3, at 0 and 0.5 mM Zn2+, 'M' and 'D' show the
position of the plasmid monomer and dimer, lea~~
It is proposed to call this new structure 'M' DNA because than the unmodified polymers; the relevant pKas are U
of the intimate h~vol~e.. l.. ll of metal ions. (9.3) and 1(8.8). The metal ions which favour 'M' DNA;
Many potential structures which might be assigned to 'M' that is Zn2+, Ni2+, and Co'+ all have ionic radii of about
DNA can be eliminated on the basis of a few key observa- 0.70 A or less, whereas Mn2+ and Cu2+ which are innef-
tions. First, rapid restoration of normal duplex DNA upon fective have radii of 0.80 and 0.92 A (I A = o.l nm),
addition of EDTA to the ethidium fluorescence buffer lea~ l),(CottonandWilkinsonl966).Theoneexception
cannot be eYp!qin~d by denaturation, a triplex structure, or is Mg2+ with an ionic radius of 0.65 A, but Mg2+ does
one Co~ ;..g parallel strands. Second, since 'M' DNA can not form stable complexes with nitrogen bases. Ag + was
be formed by all DNAs regardless of sequence, then A must of interest because it is known to form stable ~opl.,~ with
still pair with T and G with C. Third, Hoogsteen or other the bases in which the imino protons are lost (Guay and
types of hydrogen bonding can be ~limin~ted because m6A Be~ homr 1979). However, it will not form 'M' DNA, but
and z7A do not inhibit the tlicmut~ n. Therefore, these instead denatures the DNA. Its ionic radius is l.l3 A, which
results imply that some features of the usual Watson-Crick may be too large to be accommo~ ed within the helix. Spe-
base pairs are retained in the structure of 'M' DNA. Fourth, cific cation effects have also been noted in the formation
the NMR clearly shows that the signal for the imino protons of tetraplex structures where again a metal ion is bound
has been elimin~ed and that there is one Zn2+ for each tightly to the bases (Lee 1990; Zahler et al. 1991).
base pair. This can be interpreted either as a replacement Binding of the Zn + undoubtedly leads to distortion of
of the imino protons by Zn2+ or the formation of a com- the helix. For example, imino-Zn2+-imino bonds are
plex which allows for their rapid exchange with solvent. usually about 4 A compared with 3 A for the Watson-
Since Zn2+ usually forms tetrahedal complexes, several Crick base pair (sw~min~th~n and Sundralingham 1979).
functional groups on the bases or the sugar-phosphate The presence of the metal ion would also reduce the total
backbone are probably involved. charge on the helix. This, in turn, would tend to compact
Whatever the structure, the tight association of Zn2+ the heliY~ and decrease the number of base pairs per turn as
with every base pair explains many of îhe properties of ~M' was observed in Fig. 7. Further details of the structure of
DNA. For example, ethidium does not intercalate because ~M' DNA will have to await X-ray crystallographic or two-
of charge repulsion; triplexes containing C-G-C+ base dimensional NMR studies.
triads will not accommodate ethidium for the same reason Zn2+ is one of the few metal ions which can coordinate
(Morgan et al. 1979; Lee et al. 1984; Scaria and Shafer 1991). well to both oxygen and nitrogen. Therefore, together with
Addition of EDTA rapidly restores 'B' DNA because no its small ionic radius, it is ideally suited for specific inter-
rearrangements of the strands are necessary. The require- action with nucleic acids. It is intriguing that many nucleases
ment for an increased pH reflects the pK, of G (9.4) and and polymerases contain Zn2+ (Mildvan and Loeb 1979).
T (9.9) because the imino protons must be removed or be As well, Zn2+ is a good catalyst in the nonbiological
eY- h ~-.g~ ~hle (Saenger 1984). As well, some of the sequence poly,i~alion of nucleic acids, especially since it favours
effects on the rates of dismutation may be related to the the correct 3~-5~ orientation of the phospho~iester
pK,s of the bases. For example, polyld(A-U)I and backbone (Bridson and Orgel 1980). Therefore, even though
polyld(T C)]-poly[d(l-A)] both transform more rapidly a role for ~M~ DNA may appear to be unlikely in vivo
CA 02218443 1998-10-26
19
168 FIIOCHE!U. CELL BIOL VOL. 71, 1~93
b(eeause of the requirement for high pH and millimol~r Lee, J.S., Woodswonh, M.L., Latima, L.J.P., and Morgan, A.R.
Zn~t, its structure may ye~ provide clues to understanding 1984. Pyr-Pur DNAs containing 5-methylcytosine form stable
the prebiotic chemistry of nucleic acids. triplexes at neutral pH. Nucleic Acids Res. 12: 6603-6613.
Lee. J.S., Burkholder, G.D., Latimet, L.J.P., Haug, 8.L., and
Beese, L.S., and Steitz, T.A. 1991. Structural basis for the 3'-5' Braun, R.P 1987. A ~ ~' antibody to tripkx DNA binds
~Y- I ~ activity of E ~oli DNA f~l~.. aae l . a two metal to eukaryotlc .h.c,.. oso Nucleic Acids Res. 15: 1047-1061.
ion r-~~~ ' EM80 J. 10: 25-33 Lyamichev, V.l., Voloshin, O.N., Frank-tC: ~ ' '' M.D., and
Bridson, P.K., and Orgel, L.E. 1980. Catalysis of accurate poly(C} Soyfer, V.N. 1991. Photofc~ty.' " a Of DNA triplexes. Nucleic
directed synthesis of 3'-5' linked ~'i o -~ es by Znl~. Ac~ds Res. 19: 1633-1638.
J Mol. Biol. 144 567-577 '~ v Maher, L.l., Dervan, P.B., and Wold, B.l. 1990. Analysis of
Chan, A., Kilkuskie, R., and Hanlon, S. 1979. Cc",elations promota-specificrepressionbyt.i~ " 'DNAcomplexesof
between the duplex winding angle and the circular dichroism eukaryotic cell-free tIal~S~ . system. r - ~ - r, 29:
spectrum of calf thymus DNA. 13ic-' .1, 18: 84-91. 882~8826.
Cottoo, F., and Wilkinson, G. 1966. Advanced inorganic Marmur, 1., and Doty, P. 1962. D te. -' ~r of the base com-
chemistry. 2nt ed. 1- -- :' , New York, London, Sydney. posltion of DNA from its thermal d - ation ~ f~ aIh.C.
D'Andrea, D.A., and Haseltine, W.A 1978 Sequence specific . Mo. io. 5: 1 - 18.
cleavage of DNA by the -- antibiotlcs nc ~ ~ - Mickel, S., Atena, V., and Bauer, W. 1977. Physical properties
and bleomycin. Proc. Natl. Acad. Sci. U.S.A. 15. 3608-3612. and gel ele.l.ù,)l ~ ;, behaviour of R-12 derived plasmids.
Eichhorn G.L. 1962. Metal ions as stabili_~rs or d~t~ s of Nuclelc Acids Res. 4: 1465-1479.
DNA. Nature (Loodon), 194: 474-475 Mildvan, A.S., and Loeb, L.A. 1979. The role of metal ions in
Eichhoro G.L., and Shin, Y.A. 1968. Ibteraction of metal ions the ~ - ' ' of DNA and RNA POIJ ~;. CRC Crit. Rev.
wnh~l~ andrelatedv~ L ' Xll l-Am-Chem- Mo8r~gCahnemARM~LeeB~~ll's6 F21~9~ jc414 Ik DE Murray NL and
Evans, D.H., Lee, I.S., Morgan, A.R., and Olsen, R.K. 1982. Evans,D.E. 1979.Ethidiumbromidelluu., ,~ assays,Part 1.
uâing th~ A-T sp~cific ~ 1. iodn ~tfbjpolyld(A T)] synthesis Nucleic Acids Res 7 547-569.
1. 8iochem. 60: 131-136. Crit. Rev. Biochem. Mol. 8iol. 26: 151-226.
Guay, F., and E! . ' - ., A.L. 1979. Model ~ for the Rich, A., No-l" ' , A., and Wang, A.H.I. 1984. The chemistry
' - ~aion of silver I with pol~ '' - 1. Am. Chem. Soc. 101: and biology of left-handed DNA. Annu. Rev. 8iochem. 53:
626~6263
Hatopel, K.l, Crosson, P., and Lee, J S. 1991 Pol~ favor Rosenberg, 8, van Camp, L., Trosko, J.E., and Mansour, V.H.
DNA triplex formation at neutral pH. 8iochemistry 30 1969. Platinum cu~po- ' . a new class of potent ~ u
4455_4459 ' ' agents. Nature (London), 222: 385-386.
Haner, R., and Dervan, P.8. 1990. Single-strand DNA triple-helix Sa nger, W. 1984. Principles of nuckic acid structure. Springer-
formation. P:c-'- ' - ~ 29. 9761-9765 erag, ew or .
Haniford, D.8., and P~ , D.E. 1983. Facil~ transition of Scaria, P-Vj, and Shaf~r, R.H. 1991. 8inding of ethidium to a
poly[d(TG)] poly[d(CA)I into a left handed helix in ph~,iolo~J DNA tnp e e tx. . 8iol. Chem. 266: 5417-5423.
''-'- Nature (London), 302. 632-634 Sklenar, V., and Feigon, 1. 1990. Formation of a stabk tripla~
Hodgson, D J. 1977. The ste.. ' '- ~ of metal complexes of from a single DNA strand. Nature (London), 34S: 836-838.
nucleic acid ~ - . Prog. Inorg Chem 23- 211 -254 S~ ' ' ~ n, V, and S ' ' _' M. 1979. The crystal struc-
Hore, P.l. 1983. 8inomial composite pulse sequences for water tures of nuclelc acids and their CO~ CRC Crit. Rev.
suyy.c ' ~ in NMR speara. I. Magn. Reson. 54. 539-542 8iochem. Mol. 8iol. 6: 245-336.
Htun, H and Dahlberg, I.EM988s. Single strands triple strands Timsit, Y, Vilbois, E., and Moras, D. 1991. 8as~ pairing shift
and kinks in H-DNA. Science (Washington D.C.), 241: m the ma30r groove of (CA)1 traas by 8-DNA crystal struc-
1791-1796 tures. ature ( on on), 354: 167-1 0.
Kohwi, Y., and Kohwi-S~ '_ . T 1988 M~ ion- Yagil, G- 1991. Paranemic structures of DNA and their role in
dependent triple-hclix struaure formed by pyr-pur xquences DNA unwinding. CRC Crit. Rev. 8iochem. Mol. 8iol. 26:
in supercoiled DNA. Proc. Natl. Acad. Sci. 85: 3781-3785. ' . .
Latham~ I A, and Cech, t R. 1989. Defining the insid~ and out- Zahler, A.M., Wllllamson, J.R., C~ch, T.R., and Pr~scott, D.M.
side of a cata3ytic RNA molecule. Science (Washington, D.C.) 1991. Inhlbition of tel~. . ~e by G~uartet DNA structures.
245: 276-282. Nature (London), 350: 718-720.
Lee, J.S. 1990. Thc stability of polypurine tetraplexes in the Zimmer, C., and Luck, G. 1974. Conformation and reaaivity of
pre~ence of mono and divalent ions. Nucleic Acids Res. 18: DNA Clrcular dichroism studies, Vl. 8iochem. 8iophys. Acta,
6057-6060.
CA 02218443 1998-10-26
M-DNA
Plea~e ~ind the ~ ched flgure demon~tra~ng the nudease re~i6tance of
M-DNA. Ihe ~mount of duplex DNA remalnlng ~~ a func~on of ~me ~a~
~6~et by ~e e~idium fluor~ ,ce a~say (under cont~tion~ where M-DN~
rapidl~r reve~s to 8-DNA so ~t e~h~ can blnd). The d~ on wa~
p~,fo...~ed at 37 ~C ln 10 mM Tns-Hcl pH 7.4, 5 mM MgC12, lmM N~C12,
1mg/ml g~ , and 0.2 ~g/~l DNase I. Ihe M-l)~ s~mple wa~ prefarmet
~t pH 9 bef~ adding ~o tht dige~on buffer whereas ~e ~DNA W,~18 ~dtled
~rectly. ~he M-ON~ 18 completely res~ while ~e ~DNA ~9 di8e~ted ln
~bout 10 m~n. Note ~at ~i~ expedment ~ hows th~t ~e Ni fonn of M-
DNA is ~te st~le under ph~iologlcal condl~ which retate~ to ih utility
in DNA lmmw~ sense technologle~.
.
o~
C~ 1.2 ~ DN~
t
0.
~0.0
~0.~
0.2 -
O
0 6 10 15 20 25
~ (~)
