(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)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.
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) 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) .
Using the ZIBS assay, we examined Z-DNA formation in
d(CG) 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
.
Figure 1:
ZIBS assay using pUC12
and pJKCGn series containing d(CG) 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.
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) region.
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 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.
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) 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.
Figure 5:
Z-DNA
formation in pJKCG4 of different superhelical densities. Panel
A, plasmids isolated from E. coli topA 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.
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) 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) .
Figure 6:
Restriction inhibition analysis of
pJKCGn plasmids isolated from E. coli topA 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.
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 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.
Figure 8:
Comparison of Z-DNA formation in
d(CG)-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.
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) 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) .
Second, we determined that DNA supercoiling
is necessary for Z-DNA formation in short alternating d(CG) sequences.
Although d(CG) 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.
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 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.
The lack of a measurable effect of salt on Z-formation was
surprising given that d(CG) homopolymer (47) and d(CG) 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.
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) 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.
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 (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.
Z-DNA conformation in d(CG)-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.
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) 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.
In summary, we have carefully examined the relative
role of d(CG) 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.