|
|
|
|||||||
|
|||||||||
(Received for publication, November 28,
1995; and in revised form, February 7, 1996) From the
To evaluate the relative importance of alternating d(CG)
sequence length, DNA supercoiling, and salt in left-handed Z-DNA
formation, plasmids containing short d(CG)
Experiments in the early 1970s showed that exposure of
poly(d(CG)) DNA sequences to high salt concentrations resulted in
inversion of the circular dichroism spectrum due to the formation of an
unusual DNA structure(1) . Subsequently, detailed structural
analysis of a d(GC) hexamer in 4 M NaCl by x-ray
crystallography (2) showed that the DNA forms a left-handed
helical conformation, with alternating glycosidic bonds in anti and syn conformation and zigzag tracking of the phosphate
backbone (hence, the name, Z-DNA). Since these initial studies, many
other investigations have confirmed the occurrence of left-handed Z-DNA
using a variety of approaches, including immunological(3) ,
enzymatic(4) , chemical(5) , and spectroscopic methods
(for review, see (6) ). The factors that influence the
equilibrium between B- and Z-DNA in vitro have also been
studied. The nucleotide sequence is important for Z-DNA
formation(7, 8, 9) ; Z-DNA is generally
adopted by alternating purine-pyrimidine sequences. Among these
sequences, the d(CG) repeat has been shown to be the most favorable,
the d(CA) repeat is intermediate, and the d(AT) repeat is the least
favorable sequence for Z-DNA formation. Negative supercoiling of DNA is
also important for Z-DNA
formation(4, 10, 11) , as the higher energy
status of negatively supercoiled DNA may be relieved by opposite handed
helix formation. Under high superhelical density, some sequences with
imperfect purine-pyrimidine repeats adopt Z-DNA
conformation(9, 12) . The presence of salts and some
small molecules also affects Z-DNA formation (1) . High salt
concentration is thought to stabilize Z-DNA conformation by shielding
the repulsion of negatively charged phosphate groups, which are closer
in Z-DNA than in B-DNA. Finally, chemical modifications such as
methylation (13) and bromination (14) also affect Z-DNA
formation due to steric effects. Studies directed at in vivo Z-DNA formation have provided evidence for left-handed DNA in the
genomes of a variety of organisms, including plants, animals, and
microorganisms(15, 16, 17, 18, 19, 20, 21, 22) .
No studies, however, have been directed at the genome of extremely
halophilic microorganisms, for example Halobacterium halobium,
which are classified as archaea and which grow at 3-5 M NaCl. It seemed likely that H. halobium may harbor a
significant fraction of Z-DNA in its genome since its cytoplasm
contains nearly 5 M salts. Moreover, its genomic DNA has a
high GC content, 67%, and therefore, a high statistical likelihood of
alternating d(CG) repeat sequences(23) . We have also found, as
part of this study, that plasmid DNA in H. halobium has a
greater linking deficiency (and presumably negative supercoiling) than
in Escherichia coli(24) . These three factors together
suggested that Z-DNA in the H. halobium genome may be quite
prevalent and perhaps provides significant challenges and opportunities
to normal genetic processes. As an initial step in evaluating this
hypothesis, we constructed a plasmid series containing short d(CG)
repeats and capable of replicating in H. halobium and E.
coli, and we systematically studied the importance of d(CG) repeat
length, superhelical density, and NaCl concentration in Z-DNA
formation. As documented in this report, increasing the length of d(CG)
repeats and DNA supercoiling, but surprisingly not salt concentration,
were found to promote Z-DNA formation.
H. halobium NRC-1, a wild-type
strain, was cultured in a medium containing 4.5 M NaCl at 37
°C with illumination and shaking at 200 rpm(27) .
Transformation of H. halobium was done by the polyethylene
glycol-EDTA procedure of Cline and Doolittle(28) .
Transformants were selected by plating on agar plates containing 10
µg/ml mevinolin (a gift of Merck, Sharp, and Dohme Research
Laboratory, Rahway, NJ). Mevinolin was added at the same concentration
for growth in liquid culture.
For construction of E. coli-H. halobium shuttle vectors containing
d(CG)
Using the ZIBS assay, we examined Z-DNA formation in
d(CG)
Figure 1:
ZIBS assay using pUC12
and pJKCGn series containing d(CG)
Figure 2:
S1 nuclease-primer extension analysis for
assaying B-Z junctions. Plasmids analyzed were pUC12 (lane 1),
pJKCG3 (lane 2), pJKCG4 (lane 3), pJKCG5 (lane
4), and pJKCG6 (lane 5) using the forward sequencing
primer. The lane labeled M contains the ddGTP
sequencing reaction products using the forward sequencing primer and
pJKCG5 template, and the bar indicates the extent of the
d(CG)
Figure 3:
BssHII restriction inhibition
assay of pJKCGn plasmid series. Plasmids pJKCG4, pJKCG5,
pJKCG6, pJKCG8, and pJKCG11 (1.5 µg) isolated from E. coli DH5
Figure 4:
Effect of NaCl concentration on Z-DNA
formation. Supercoiled pJKCG3 (panel A, lanes
1-5), pJKCG4 (panel A, lanes 6-10),
pJKCG11 (panel B, lanes 1-5), and a HinfI-digested pJKCG11 (panel B, lanes
6-10) were incubated in NaCl at 0.1 M (lanes 1 and 6), 1 M (lanes 2 and 7), 2 M (lanes 3 and 8), 3 M (lanes 4 and 9), and 4 M (lanes 5 and 10) and then subjected to the ZIBS
assay.
To examine the effect of salt on d(CG) repeats in linear
DNA fragments, pJKCGn plasmids were digested with HinfI before antibody binding, incubated with various
concentrations of NaCl, and then assayed for Z-DNA formation using the
band shift assay (Fig. 4B). None of the d(CG)
Figure 5:
Z-DNA
formation in pJKCG4 of different superhelical densities. Panel
A, plasmids isolated from E. coli topA
The effect of supercoiling on Z-DNA formation
in the pJKCGn plasmids was also analyzed by BssHII
restriction inhibition analysis. Plasmids isolated from E. coli JTT (topA
Figure 6:
Restriction inhibition analysis of
pJKCGn plasmids isolated from E. coli topA
Figure 7:
DNA supercoiling density of plasmid pCY1
from H. halobium NRC-1. Topoisomers of pCY1 were produced by
treatment of the plasmid with topoisomerase I in the presence of
ethidium bromide at concentrations (in µg/ml) of 0.5 (lane
1), 1 (lane 2), 2 (lane 3), 3 (lane 4),
4 (lane 5), 5 (lane 6), 6 (lane 7), 7 (lane 8), 8 (lane 9), 9 (lane 10), and 10 (lane 11). Plasmid topoisomers generated in vitro were compared with the natural plasmid isolated from an NRC-1
culture (lane 12) on 1% agarose gels in the absence (panel
A) or presence of chloroquine at 2 µM (panel
B), 4 µM (panel C), and 6 µM (panel D). The position of migration of nicked DNA is
marked OC, and the number of superhelical turns in each
topoisomer is indicated at the left.
Compared with
Figure 8:
Comparison of Z-DNA formation in
d(CG)
In this study, the requirements for Z-DNA formation were
systematically examined using a plasmid series containing short
stretches of alternating d(CG) sequence in supercoiled plasmids. First,
we determined the minimum d(CG) Second, we determined that DNA supercoiling
is necessary for Z-DNA formation in short alternating d(CG) sequences.
Although d(CG) Third, the importance of
salt concentration in promoting Z-DNA formation in alternating d(CG)
sequences in supercoiled plasmids was studied. We focused on pJKCG4 and
pJKCG5, which were known to contain sequences that partly adopt Z-DNA
conformation at a low salt concentration and wild-type superhelical
density. However, no enhancement of Z-DNA in supercoiled pJKCG4 and
pJKCG5 was detected in NaCl concentrations up to 4 M using the
ZIBS assay (Fig. 4). The lack of a measurable salt effect is not
likely due to inhibition of Z22 antibody binding(6) . No
salt-enhanced Z-DNA formation was detected in highly supercoiled
topoisomers of pJKCG4 or pJKCG5 isolated from the topA The lack of a measurable effect of salt on Z-formation was
surprising given that d(CG) homopolymer (47) and d(CG) The
effect of polycations on Z-DNA formation in linear polymers (50) was also examined in this study using the ZIBS assay (data
not shown). However, we found no measurable increase in left-handed
conformation in d(CG) Since the ZIBS assay was
carried out in the presence of a 10-fold molar excess of Z22 antibody,
we were concerned about the antibody inducing or stabilizing Z-DNA
conformation in pJKCGn plasmids. To determine if Z-DNA can
form in the absence of antibody, several alternate Z-DNA detection
methods not involving Z22 antibody were used. S1 nuclease (Fig. 2) and OsO Z-DNA conformation in d(CG) Restriction inhibition analysis of pJKCG4, pJKCG5, and pJKCG6
provided a rough estimate of the rates of B-Z transitions. The
approximate time for 50% cleavage of the plasmids is in the range of 5
min to 2 h, which is consistent with the rate of B-Z transitions
measured in vivo by Lukomski and Wells using a methylation
assay(51) . In contrast, the resistance to cleavage shown by
plasmids containing longer than d(CG) In summary, we have carefully examined the relative
role of d(CG)
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9340-9346
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequences in Escherichia coli and Halobacterium halobium Plasmids
STABILIZATION BY INCREASING REPEAT LENGTH AND DNA SUPERCOILING BUT
NOT SALINITY (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
sequences (n = 3-17) with the capability of
replicating in either Escherichia coli or the halophilic
archaeum Halobacterium halobium were constructed. Z-DNA
conformation in the d(CG) sequences was assayed by
(i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody
(ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to
map B-Z junctions; and (iii) a BssHII restriction inhibition
assay. Using the ZIBS assay on plasmids purified from E. coli,
the transition from B-DNA to Z-DNA occurred from d(CG)
to
d(CG)
, with 20% of d(CG) and 90% of d(CG) in Z-DNA conformation. These findings were consistent with the
results of S1 nuclease cleavage observed at B-Z junctions flanking
d(CG) and d(CG) sequences. Resistance to BssHII restriction endonuclease digestion was observed only in
supercoiled plasmids containing d(CG)
or longer sequences,
indicating that shorter d(CG) sequences are in
dynamic equilibrium between B- and Z-DNA conformations. When a plasmid
containing d(CG) was isolated from a topA mutant
of E. coli, it contained 25% greater linking deficiency and
40% greater Z-DNA conformation in the alternating d(CG) region. In
plasmids purified from H. halobium, which showed 30% greater
linking deficiency than from E. coli, 20-40% greater
Z-DNA formation was found in d(CG)
sequences.
Surprisingly, no significant difference in Z-DNA formation could be
detected in d(CG)
sequences in plasmids from
either E. coli or H. halobium in the NaCl
concentration range of 0.1-4 M.
E. coli and H. halobium Strains, Culturing, and
Transformation
E. coli strain DH5
was employed for
most constructions and amplifications of plasmids. For isolation of
plasmids with differing superhelical densities, E. coli strains JTT (topA
) and RS2 (topA
) (gifts of Dr. K. Drlica, Public
Health Research Institute, New York) were used(25) . Culturing
and transformation with Ca
were done by standard
procedures(26) .Construction of d(CG)
Polydeoxyribonucleotide d(CG)
-containing
Plasmids (Sigma) was
partially digested with BssHII restriction endonuclease (which
cleaves at the recognition sequence G`CGCGC) (restriction enzymes and
polymerases were purchased from New England Biolabs or Bethesda
Research Laboratories), and the 5`-overhanging ends were then converted
to blunt ends by filling in with the large fragment of E. coli DNA polymerase I in the presence of dGTP and dCTP. After ligation
with SmaI-digested pUC12, E. coli strain DH5 was
transformed with the reaction mixture. Plasmids purified from
transformants were digested with HindIII and EcoRI
restriction endonucleases, and the approximate sizes of inserts were
estimated by electrophoresis on 15% polyacrylamide gels. Several
plasmids containing d(CG) inserts of different sizes were chosen for
further analysis by DNA sequencing. DNA sequencing was carried out
using the Sequenase kit (U. S. Biochemical Corp.) with the universal
sequencing primer and dITP label mix to determine the exact length of
the d(CG) stretch present in inserts. Plasmids containing various sizes
of d(CG) stretches from n = 5 to 17 were
obtained (29) and designated as pJKCGn, with n representing the number of d(CG) repeats. To construct a shorter
d(CG) length insert, pJKCG5 was digested with BssHII, treated
with S1 nuclease, and self-ligated to form pJKCG3, and pJKCG8 was
digested with BssHII, and the larger fragment was self-ligated
to form pJKCG4. Plasmid DNAs were isolated by the alkaline lysis method
followed by cesium chloride density gradient centrifugation in the
presence of ethidium bromide(26) . sequences, pUC12 and pJKCGn (n = 3-7) plasmids were digested with HindIII,
and the 5`-overhanging ends were filled in by the Klenow fragment of E. coli DNA polymerase I followed by SalI digestion.
The 2.65-kb (
)fragments were isolated by electroelution
after electrophoresis on a 0.8% agarose gel, ligated with SmaI-XhoI-digested pCY3(24) , and transformed
into E. coli DH5. Plasmids isolated from E. coli transformants were analyzed by restriction mapping and transformed
into H. halobium. Plasmids were purified from H. halobium by a previously published procedure(30) . These plasmids
were designated as pJKSUC and pJKSCG3, 4, 5, 6, and 7. Recombinant DNA
procedures were carried out as described in Sambrook et
al.(26) .Z-DNA Immunoband Shift (ZIBS) Assay
This assay
utilized the well characterized Z22 monoclonal antibody to detect
left-handed Z-DNA (a gift of Prof. B. D. Stollar, Tufts University
Medical School, Boston)(31) . The standard procedure involved
incubation of plasmid (0.1 µg) with 0.2 µg of Z22 antibody for
2 h at 37 °C in 100 µl of 50 mM Tris
HCl (pH
7.5), 1 mM EDTA, and 0.1 M NaCl. After sequential
addition of 2.25 µl of formaldehyde and 2 µl of glutaraldehyde (32) and incubation on ice for 15 min each, the reaction
mixture was passed through a Sephadex G-50 spin column and lyophilized
to remove aldehydes. The DNA-antibody complex was incubated with 10
units of HinfI in a 20-µl reaction volume at 37 °C for
2 h. The 3`-recessed ends were labeled by the addition of 10 µl of
1 nmol of dGTP, dCTP, and dTTP; 1 pmol of
[-
P]dATP (10 mCi/ml, 3,000 Ci/mmol;
Amersham Corp.); and 0.2 unit of Klenow fragment of E. coli DNA polymerase I. After incubation at 37 °C for 1 h, the
labeled restriction fragments were resolved by electrophoresis on 4%
polyacrylamide gels and detected by autoradiography on x-ray film. The
bands containing Z-DNA were shifted mostly to the origin of the gel,
and the decrease of band intensity was used as the criterion for Z-DNA
formation. Band intensity was quantified using a PDI densitometry
system (Huntington Station, NY) running on a SUN Sparcstation IPC
(Mountain View, CA). These results were confirmed in duplicate or
triplicate.S1 Nuclease Assays
For S1 nuclease assays, plasmid
DNAs (1 µg) were treated with 0.1 unit of S1 nuclease in 20-µl
reactions containing 50 mM sodium acetate (pH 4.6), 50 mM NaCl, and 1 mM zinc acetate at 37 °C for 10 min.
After extraction with phenol twice and with chloroform once, DNA was
concentrated by ethanol precipitation. DNA was dissolved in 10 µl
of deionized water, denatured by the addition of 10 µl of 0.8 M NaOH and incubation at room temperature for 10 min, neutralized by
the addition of sodium acetate (pH 5.2) to 0.45 M, and
precipitated with ethanol. Primer extension was carried out at 37
°C for 1 h in 20-µl reaction mixtures containing 0.5 pmol of
primer (either the forward universal primer or the reverse primer,
previously labeled at the 5`-end with T4 polynucleotide kinase and
[
-P]ATP), 40 mM Tris-HCl (pH 7.5),
20 mM MgCl
, 50 mM NaCl, 0.05 mM dNTPs, and 0.5 unit of Klenow fragment. Reaction products were
fractionated on 8% polyacrylamide, 8.3 M urea gels and
subjected to autoradiography.Restriction Inhibition Assay
For the standard
restriction inhibition assay, 1.5 µg of pJKCGn plasmid was
incubated with 6 units of BssHII restriction endonuclease at
37 °C in a 50-µl reaction volume. Aliquots were removed at
various time intervals, and the reaction was stopped by the addition of
50 mM EDTA and 0.1% sodium dodecyl sulfate. The digestion
mixtures were fractionated by electrophoresis on 0.7% agarose gels and
visualized by staining with ethidium bromide(26) . The
intensity of supercoiled DNA bands was quantitated using the PDI
densitometry system.DNA Supercoiling Assays
DNA supercoiling assays
were carried out using pCY1, a 5.3-kb plasmid that had been constructed
by ligation of two fragments, the mev gene fragment obtained
from pNGMEV100 as a 3.5-kb BamHI fragment (33) and the SalI fragment of a 1.8-kb Halobacterium plasmid
pGRB1(34) , and transformation of H. halobium. Plasmid
DNA was first purified by CsCl ethidium bromide gradient centrifugation
and agarose gel electrophoresis. Plasmid topoisomers were generated by
treatment of pCY1 with wheat germ topoisomerase I (Promega, Madison,
WI) in the presence of ethidium bromide. The reactions contained 4
µg of pCY1, 4 units of topoisomerase I, 50 mM Tris-HCl (pH
7.6), 50 mM NaCl, 10 mM MgCl, 0.1 mM EDTA, and 0-12 µg/ml ethidium bromide and incubated for
6 h at 30 °C. The reaction mixtures were extracted three times with
(10 mM TrisHCl (pH 7.5) and 1 mM EDTA saturated
with phenol and twice with chloroform, ethanol precipitated, and
dissolved in HO. Plasmid was electrophoresed on 1.0%
agarose gels containing 0-6 µM chloroquine(35, 36) . Electrophoresis was carried
out for 6-18 h at 3-4 volts/cm with circulation of buffer
(0.1 mM Tris base, 0.1 M boric acid, and 2
mM EDTA), and gels were stained with 0.5 µg/ml ethidium
bromide and destained before photography under uv illumination.
ZIBS Assay and Effect of d(CG)
For
quantitative analysis of Z-DNA formation within alternating d(CG)
sequences, a plasmid series containing d(CG) Length on
Z-DNA Formation in Supercoiled DNA Isolated from E. coli stretches of
various lengths was examined by the ZIBS assay. For this assay,
plasmids were incubated with Z22 monoclonal antibody specific for
Z-DNA, and antibody-DNA complexes were cross-linked by treatment with
aldehydes. The plasmids were then digested with HinfI
restriction endonuclease, end-labeled using the Klenow fragment of DNA
polymerase I and [-P]dATP, and
electrophoresed on polyacrylamide gels. If there was Z-DNA formation in
d(CG) stretches, antibody-HinfI fragments of 218 ±
d(CG)-bp size complexes were shifted up to the origin of
the gel. Reduction of band intensity was quantified by densitometry,
and the change was used as a criterion for Z-DNA formation. Intensities
of unshifted restriction fragments were used as controls for
quantitation. stretches in plasmids isolated from E. coli DH5 (Fig. 1). No differences were observed for the
218-bp HinfI fragment containing the multiple cloning site of
pUC12 and the corresponding fragment of pJKCG3 (lanes 3 and 4, respectively), indicating no Z-DNA formation in the
d(CG)
sequence. For pJKCG4 (lane 5), about a 20%
decrease in intensity of the d(CG)-containing band relative to the
corresponding band of pJKCG3 was observed, indicating that about 20% of
the d(CG) sequence in pJKCG4 is in the Z-DNA conformation.
For pJKCG5, a decrease of about 90% band intensity was found (lane
6), indicating that the corresponding portion of the d(CG) sequence is nearly completely in Z-DNA conformation in the
supercoiled plasmid. Similarly, complete Z-DNA formation was observed
in plasmids containing alternating d(CG) sequences greater than 10 bp
in length, including d(CG)
and d(CG)
(lanes 7, 8, and data not shown). No band shift
was observed in linear pJKCG13 (lane 9), showing the
requirement of supercoiling for Z-DNA formation in these short d(CG)
stretches. This result indicated that the transition from B-DNA to
Z-DNA conformation occurs from d(CG) to d(CG) in supercoiled plasmids isolated from E. coli DH5.
inserts. Lanes 1 and 2 contain pUC12 and
pJKCG4 without antibody treatment. Lanes 3-8 contain
pUC12 and pJKCG3, 4, 5, 7, and 13, respectively, with Z22 antibody
treatment. Lane 9 contains pJKCG13 digested by HinfI
before antibody treatment. The arrow indicates the approximate
position of the 218 ± d(CG)-bp HinfI fragment containing d(CG) inserts.
Detection of B-Z Junctions Using S1 Nuclease
To
rule out the possibility that Z22 antibody is promoting Z-DNA formation (37, 38) in the ZIBS assay, we utilized an independent
method involving S1 nuclease. S1 nuclease is known to cleave at
single-stranded regions occurring at B-Z junctions(4) , and the
site of cleavage can be mapped at the nucleotide level by primer
extension analysis. S1 nuclease-treated pJKCGn plasmid DNAs
were denatured, annealed with P-labeled sequencing primer
(or reverse sequencing primer; data not shown), and then primers were
extended using the Klenow fragment of DNA polymerase I and dNTPs (Fig. 2). Synthesized cDNAs were resolved by denaturing
polyacrylamide gel electrophoresis together with sequencing reaction
mixtures generated using the same primers. S1 cleavage was detectable
at the ends of d(CG) sequences in pJKCG4 and to a greater
extent in pJKCG5 and pJKCG6 (lanes 3-5), reflecting
Z-DNA formation. The degree of Z-DNA formation in the S1
nuclease-primer extension experiment was consistent with the ZIBS assay
result, confirming that a d(CG) stretch is the minimum
length required for Z-DNA formation and that the Z-DNA formation
observed in the ZIBS assay is not a result of induction or
stabilization of left-handed conformation by antibody.
region.
Restriction Inhibition Assay for Z-DNA
Conformation
Activities of several restriction and modification
enzymes are known to be sensitive to DNA
conformation(39, 40) . It was shown that BssHII cleavage of a d(CG) sequence is inhibited
when present on a highly negatively supercoiled plasmid but not when
relaxed(39) , indicating that BssHII is sensitive to
DNA conformation. Z-DNA formation in the pJKCGn plasmid series
was examined by an inhibition assay using BssHII. For the
standard restriction inhibition assay, 1.5 µg of pJKCGn plasmids containing different lengths of d(CG) segments were
incubated with 6 units of BssHII. Aliquots were taken after
various time periods and electrophoresed in an agarose gel (data not
shown). The amount of supercoiled molecules remaining was quantitated
by densitometry and plotted as a function of time (Fig. 3).
Under these conditions, pJKCG4 and pJKCG5 were both completely cleaved,
but pJKCG4 was cleaved more slowly (30 min) than pJKCG5 (15 min).
Partial resistance to cleavage was observed for pJKCG6, and the
inhibition of BssHII cleavage increased for plasmids with
increasing d(CG) length. Plasmids containing d(CG) or longer stretches were cleaved only partially even when excess
enzyme was present (10 units/µg of DNA, 2 h; data not shown). For
50% cleavage of supercoiled plasmids, pJKCG5 required 5 min, pJKCG6
required 10 min, and pJKCG8 required about 2 h, indicating greater
Z-DNA conformation in longer d(CG) stretches.
were incubated with 6 units of BssHII at 37
°C for various times and analyzed by electrophoresis on an agarose
gel. The relative band intensity of supercoiled DNA was measured by
densitometry and plotted versus time.
Effect of NaCl Concentration on Z-DNA
Formation
Earlier studies showed that high salt concentration
induced B-Z transition in poly d(CG) nucleotides (1) and
plasmids containing long stretches of d(CG)
sequences(10, 41) . To determine the effect of NaCl on
shorter stretches of d(CG) repeats, we treated plasmids that in the
absence of salt were not in Z-DNA form (pJKCG3), or only partially
(20%) in Z-DNA form (pJKCG4), with various concentrations of NaCl (0.1,
1, 2, 3, and 4 M) and carried out the ZIBS assay. The result
in Fig. 4A showed that no detectable change in Z-DNA
formation could be observed with increasing NaCl concentrations in
either plasmid (lanes 1-10). The possibility of an
inhibitory effect of salt on Z22 antibody binding was ruled out by the
lack of reduction in antibody binding to pJKCG11 (Fig. 4B, lanes 1-5) (and also in pJKCG5
and pJKCG7; data not shown) at the higher NaCl concentrations. The
effect of NaCl on more negatively supercoiled DNA isolated from the E. coli topA mutant strain also showed no
distinguishable salt effect on Z-DNA formation (see below). These
results indicate that NaCl does not have a measurable effect on Z-DNA
formation in plasmids at the superhelical densities occurring in E.
coli.
sequences up to n = 17, including the
d(CG)
sequence in pJKCG11 (lanes 6-10),
showed Z-DNA formation in the 0.1-4 M NaCl concentration
range. These results showed that high salt concentrations cannot
stabilize Z-DNA in short d(CG) sequences flanked by large
regions of B-DNA segments in linear molecules.Effect of DNA Supercoiling on Z-DNA
Formation
Plasmid DNA of different superhelical densities were
obtained from an E. coli topA strain, JTT,
and an isogenic topA strain,
RS2(25, 42) . Differences in linking deficiency of
plasmids were observed in agarose gel electrophoresis containing 5
µM chloroquine, indicating that plasmids isolated from the topA RS2 strain were 25% more negatively supercoiled than from
the JTT strain (Fig. 5C). Plasmids pUC12 and pJKCG3
isolated from each strain showed no difference by the ZIBS assay (data
not shown); however, 40% more Z-DNA formation was observed in pJKCG4
isolated from RS2 (Fig. 5, A and B, lanes
5-8) than JTT (lanes 1-4). These results
confirmed the importance of DNA supercoiling on Z-DNA formation.
However, the addition of NaCl up to 4.5 M concentration (lanes 4 and 8) did not promote the formation of
Z-DNA in either pJKCG3 or pJKCG4, indicating that salt does not affect
the B-Z equilibrium.
strain JTT (lanes 1-4) and topA strain RS2 (lanes 5-8)
were analyzed by the ZIBS assay after incubation at NaCl concentrations
of 0.1 M (lanes 1 and 5), 1.5 M (lanes 2 and 6), 3 M (lanes 3 and 7), and 4.5 M (lanes 4 and 8). Panel B, densitometric analysis of the
d(CG)-containing band, with lanes corresponding to lanes in panel A. Panel C, measurement of
supercoiling density of pUC12 (lanes 1 and 2) and
pJKCG4 (lanes 3 and 4) isolated from E. coli JTT (lanes 1 and 3) and RS2 (lanes 2 and 4) was compared by electrophoresis in 1% agarose gels
containing 5 µM chloroquine at 50 mV for 15
h.
) and RS2 (topA) strains were treated with BssHII, and the intensity of supercoiled plasmid DNA was
plotted against time (Fig. 6). Plasmids isolated from the topA RS2 strain showed more resistance to
cleavage than the same plasmids isolated from the topA JTT strain. Plasmid pJKCG4 isolated from
the topA strain showed a similar level of
restriction inhibition as pJKCG6 isolated from the topA strain. Restriction inhibition of pJKCG5
from the topA strain was inhibited to an
extent similar to that of pJKCG8 isolated from the topA strain(29) .
JTT and topA RS2 strains. The
digestion reaction was carried out on pJKCG4, pJKCG5, and pJKCG6 as in Fig. 3, and the intensities of supercoiled DNA in samples were
plotted versus time of BssHII
digestion.
DNA Superhelical Density Is Highly Negative in H.
halobium
The linking deficiency of plasmids was previously shown
to be much more negative in H. halobium than in E. coli on average, but in several cases individual topoisomers were not
resolved due to the large sizes of these plasmids(24) .
Therefore, we used the smallest available plasmid with the ability to
replicate in H. halobium, pCY1, as a reporter of superhelical
density inside H. halobium. Topoisomers of pCY1 were generated
by treatment of the plasmid with topoisomerase I in the presence of
various concentrations of ethidium bromide, and they were compared with
natural pCY1 topoisomers isolated from NRC-1, by electrophoresis in
agarose gels containing 0-6 µM chloroquine (Fig. 7). The results showed that pCY1, which is 5.3 kb in size,
was composed of topoisomers with linking deficiency
(-
Lk) of 36-38, which would be manifested as
36-38 negative superhelical turns. Assuming that pCY1 is entirely
in B-form, the linking number (Lk
) is 5,300/10.4
= 510 when relaxed. Therefore, the total (constrained plus
unconstrained) superhelical density, 
, is as
follows.
values of -0.02 to -0.06
reported for E. coli (e.g. 43), our results indicate
that DNA superhelical density is significantly more negative in H.
halobium than in E. coli.Z-DNA Formation in H. halobium Plasmids
Linking
deficiency of plasmids are more negative in H. halobium than
in E. coli, indicating that DNA superhelical density is more
negative in the halophile (Fig. 7). To compare Z-DNA formation
in plasmids of H. halobium with those of E. coli,
plasmids capable of replication in both microorganisms and containing
d(CG) stretches were constructed. When Z-DNA formation of
plasmids containing d(CG), d(CG),
d(CG), and d(CG)
from both E. coli and H. halobium (Fig. 8, A and B, lanes 2-5 and 7-10) were compared by ZIBS
assays followed by densitometric analysis (Fig. 7C),
20-40% more Z-DNA formation was observed in d(CG),
d(CG), and d(CG) sequences from H.
halobium, a result consistent with the importance of DNA
supercoiling in promoting Z-DNA formation.
-containing E. coli-H. halobium shuttle plasmids isolated from E. coli (panel A)
and H. halobium (panel B) by the ZIBS assay. Lanes 1-10 contain pJKSUC (lanes 1 and 6), pJKSCG3 (lanes 2 and 7), pJKSCG4 (lanes 3 and 8), pJKSCG5 (lanes 4 and 9), and pJKSCG6 (lanes 5 and 10),
respectively. Panel C shows the relative intensity of the
d(CG)-containing band in corresponding lanes.
length required for Z-DNA
formation using the ZIBS and other assays (Fig. 1). Our results
showed that Z-DNA did not form in plasmids pUC12 and pJKCG3. About 20%
of the d(CG) insert in pJKCG4 and 90% of the pJKCG5 insert
were in Z-DNA conformation. Longer d(CG) stretches were
found to be essentially completely in Z-DNA form. Our systematic
analysis showed that the length of d(CG) sequences is
critical for Z-DNA formation and that d(CG) is the minimum
length required for Z-DNA formation at natural superhelical densities.
These findings are consistent with previously published results showing
that a 10-bp d(CG) sequence in a supercoiled plasmid from E.
coli(44) and an 8-bp alternating purine-pyrimidine
sequence in SV40 (45) can adopt Z-DNA conformation. They
provide a framework for predicting Z-DNA formation in natural
sequences(46) . sequences 10 bp or longer in supercoiled
plasmids were largely in the Z-form, no Z-DNA formation could be
detected when these plasmids were linearized, even in d(CG) sequences up to 34 bp long ( Fig. 1and data not shown).
The effect of DNA supercoiling on Z-DNA formation was also analyzed
using plasmids isolated from E. coli strains with wild-type
and defective topoisomerase I (topA) genes (Fig. 3). No
Z-DNA forming capacity was observed for pUC12 and pJKCG3 isolated from
either strain (data not shown). However, a significant increase in
Z-DNA formation was detected in more negatively supercoiled pJKCG4
isolated from the topA strain than in pJKCG4
isolated from the topA strain (Fig. 5). These results demonstrated and
confirmed(4, 10, 11) that DNA supercoiling
plays a critical role in Z-DNA formation. strain (data not shown). Similarly, no
enhancement of Z-DNA formation by salt was observed in linear plasmid
fragments containing alternating d(CG) sequences up to 34 bp long (Fig. 4B and data not shown). These results suggest
that high salt concentrations found in the cytoplasm of some halophilic
archaea such as H. halobium cannot directly enhance Z-DNA
formation in the genome. However, an indirect effect of high salt, via
increased supercoiling (as shown in Fig. 7, (24) ), is
likely. nucleotide (48) have long been known to adopt Z-DNA
conformation at high salt concentration. Moreover, salt is also known
to increase twist, which will contribute to increasing DNA supercoiling
and Z-DNA formation in covalently closed circular
molecules(49) . The lack of a salt effect on Z-DNA formation in
short d(CG) sequences in plasmids is at least partly due to
the presence of flanking B-DNA stretches, which increase the activation
energy necessary for Z-DNA formation(12) . An additional factor
may be that a long stretch of Z-DNA is stabilized by cooperative charge
shielding between phosphate groups in the DNA backbone by salt, but the
cooperativity is absent or insignificant in short sequences. sequences in supercoiled plasmids by
spermidine, spermine, and hexamine cobalt chloride. These results
indicate that the presence of monovalent or polyvalent cations was not
sufficient to convert short stretches of d(CG) sequences to
Z-DNA when they are flanked by B-DNA. (data not shown) were used to assay
for single-stranded DNA regions at predicted B-Z junctions flanking
d(CG) sequences. The finding of cleavage by S1 nuclease and
OsO at both ends of alternating d(CG) sequences in pJKCG4
and pJKCG5 was consistent with Z-DNA formation. Greater cleavage of
pJKCG5 than pJKCG4 was consistent with the results of ZIBS assays. -containing plasmids was also
analyzed by the BssHII restriction inhibition assay. BssHII cleaves at d(GC) sequences in the B-form
but not in the Z-form(39, 40) . Plasmids that are
partly in Z-DNA conformation such as pJKCG4 and pJKCG5 were completely
cleaved by BssHII, suggesting that the alternating d(CG)
sequences rapidly isomerize from Z- to B-form. Interestingly, faster
cleavage was observed for pJKCG5 than for pJKCG4, in spite of more
Z-DNA formation in the former, as revealed by by ZIBS and S1 nuclease.
This may be due to the presence of two overlapping BssHII
recognition sites in pJKCG5 which provides a larger target for the
restriction enzyme compared with pJKCG4. However, with longer stretches
of alternating d(CG) sequences, we observed greater inhibition of BssHII cleavage, confirming the occurrence of more Z-DNA
conformation. Using the restriction inhibition assay, we also compared
Z-DNA formation in pJKCG5 and pJKCG6 isolated from isogenic E. coli
topA and topA strains.
No difference in Z-DNA formation had been detectable by the ZIBS assay
as both were almost completely in Z-DNA conformation. However, a very
significant difference in cleavage rates, and therefore, in the rates
of Z- to B-DNA isomerization could be detected by the restriction
inhibition assay. In fact, more negatively supercoiled pJKCG5 plasmid
isolated from the topA strain showed greater
resistance to cleavage than less negatively supercoiled pJKCG6 plasmid
isolated from the topA strain. This result
suggested that the greater supercoiling in the topA strain, about 25% greater linking
deficiency compared with the topA strain,
stabilizes Z-DNA conformation more than one extra d(CG) dinucleotide. sequences indicated
that these sequences are rarely in B-form. Thus, it may be concluded
that DNA sequences in the range from d(CG) to d(CG) can exist in both B- and Z-forms at E. coli
topA superhelical density, and sequences in the
range from d(CG) to d(CG) can exist in both
forms at more negatively supercoiled condition present in the E.
coli topA strain and H. halobium (Fig. 7). d(CG) stretches longer than 14 bp are probably
frozen in Z-DNA conformation. We would predict that natural alternating
d(CG) sequences within the 8-14-bp range may have been recruited
for functions involving structural isomerization between B- and Z-DNA.
In this context, it is interesting to note that a large number of
natural sequences capable of forming Z-DNA-like structure have been
found in the H. halobium genome, ()including an
11-bp alternating purine-pyrimidine sequence centered 23 bp 5` to the bop gene transcription start site which may be required for
promoter activity(24) . A search of the bacterial portion of
GenBank showed the occurrence of more than 3,000 sequences of
d(CG) length, about 200 sequences of d(CG), 10
sequences of d(CG), and no sequences of d(CG) or longer. length, DNA supercoiling, and salt
concentration on Z-DNA formation. Among these factors, effects of
repeat length and DNA supercoiling on Z-DNA formation were much more
more pronounced than salt concentration. Therefore, high salt
concentration present in H. halobium may not contribute
directly to the stabilization of Z-DNA in natural sequences. However,
slightly more Z-DNA formation can be observed in plasmids isolated from H. halobium than in E. coli, most likely reflecting
the greater negative DNA supercoiling density in H. halobium.
Our findings provide a better framework for understanding Z-DNA
formation in a wide variety of organisms, especially those with
d(CG)-rich genomes.
))
We thank Dr. B. David Stollar for a gift of Z22
monoclonal antibody and for numerous helpful comments during the course
of this work and Caryn Evilia for experimental contributions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Morohashi, Y. Yamamoto, S. Kuwana, W. Morita, H. Shindo, A. P. Mitchell, and M. Shimizu Effect of Sequence-Directed Nucleosome Disruption on Cell-Type-Specific Repression by {alpha}2/Mcm1 in the Yeast Genome Eukaryot. Cell, November 1, 2006; 5(11): 1925 - 1933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-B. Oh, Y.-G. Kim, and A. Rich Z-DNA-binding proteins can act as potent effectors of gene expression invivo PNAS, December 24, 2002; 99(26): 16666 - 16671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-m. Kim and S. DasSarma Isolation and Chromosomal Distribution of Natural Z-DNA-forming Sequences in Halobacterium halobium J. Biol. Chem., August 16, 1996; 271(33): 19724 - 19731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Becker, H. A. O'Neill, J. M. Zimmerman, and L. J. Maher III In Vitro and in Vivo Ligation-mediated Polymerase Chain Reaction Analysis of a Polypurine/Polypyrimidine Sequence Upstream of the Mouse metallothionein-I Gene J. Biol. Chem., December 15, 2000; 275(51): 40218 - 40225. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |