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J Biol Chem, Vol. 275, Issue 1, 657-668, January 7, 2000
From the Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
Several factors were evaluated to determine their
role in facilitating the presence of transcription-induced stresses in
a circular DNA. Transcription was done with T7 RNA polymerase in the
presence of E. coli topoisomerase I and closed circular
DNA. Positive stress was observed in hypotonic conditions or when one of the polyamines, spermidine or spermine, were present. Polycations such as polylysine, polyarginine, histone H1, histones H2A-H2B, and
protamine were observed to induce minimal positive stress. It is known
that polyamines influence DNA structure by causing both
self-association and sequence-specific structural alterations (polyamine-induced localized bending). Experimental evidence indicates that the likely cause of the positive stress is the induced bending. In
order to evaluate protein-mediated bending, transcription was done on
nucleosomes. A minimum of three nucleosomes on a DNA of 6055 bp was
sufficient to generate very high levels of positive stress. Histones
H3-H4 in the absence of H2A-H2B were responsible for this effect. Since
these histones by themselves are able to maintain negative coils on
DNA, it is concluded that protein-mediated bending is yet another
mechanism for placing rotational restriction on DNA. The bending of DNA
by either polyamines or histones is an effective mechanism for
promoting transcription-induced stresses at physiological ionic strength.
Extensive topological changes occur on DNA during the
transcription process. As RNA polymerase transcribes the DNA, it opens 10-15 bp1 of DNA and would
therefore be expected to rotate 360° for every 10.5 bp that is
transcribed. However, as the length of the transcript increases, the
rotational freedom of the polymerase decreases, and the DNA
preferentially rotates. This rotation causes an overwinding of the
helix in front of the polymerase (positive stress) and an underwinding
of the helix in the wake of the polymerase (negative stress). This twin
supercoiled domain model (1) has received a wealth of support from
in vivo (2-7) and in vitro (8-12)
experimentation in prokaryotic systems. These studies indicate that
Escherichia coli topoisomerase I functions to remove
negative stress and gyrase functions to remove positive stress by
introducing negative coils in an energy-dependent process.
E. coli topoisomerase I serves a critical role in
maintaining an overall negative state in the DNA. This enzyme senses
helical distortion (single-stranded character) in DNA that is caused by
the transcription-induced negative stress (13-15). The activity of
this enzyme decreases rapidly as the DNA becomes increasingly relaxed
and therefore provides a mechanism for maintenance of a basal level of
negative stress. In vitro transcription studies on closed
circular DNA have shown that when this enzyme is present, positive
stress is produced on the template (8). Various levels of positive
stress have been observed as a result of restricting rotational freedom
of both polymerase and DNA (9) or by immobilizing the nascent
transcript (4, 16, 17). Therefore, this topoisomerase has been shown to
be an effective reagent for the quantitation of helical distortions, as
observed by the accumulation of positive stress on DNA during transcription.
In contrast to the topoisomerases of the prokaryote, the eukaryotic
topoisomerases (I and II) relax both positively and negatively coiled
DNA equally well. A number of studies have been done in yeast with
deletion mutants of topoisomerase I and ts mutants of
topoisomerase II that indicate that these proteins function in a
similar way to remove topological stresses that are induced by
transcription (18-21). In this instance, the presence of nucleosomes does not appear to alter the formation of these induced stresses. Indeed, the induced positive stress in advance of the polymerase may
alter the structural state of the nucleosome (22, 23); an alteration
that may enhance transcription through them. It has also been suggested
that the negative stress in the wake of the polymerase has an important
role in facilitating activation of promoters, DNA replication, and
recombination (reviewed in Refs. 24-27). It is therefore of interest
to evaluate the parameters that facilitate the maintenance of
transcription-induced stress.
The in vitro experiments that have studied the induction of
transcription-induced stress have used variable conditions of ionic
strength or polyamine concentrations. We have evaluated these
components further in order to define the process whereby positive
stress is induced in template DNA when transcription is done in the
presence of E. coli topoisomerase I. We conclude that a
decrease in flexibility of DNA is a critical factor that is responsible
for this effect. This decreased flexibility enhances the maintenance of
transcription-induced stress in two ways as follows. 1) When reduced
flexibility is present, the induced topological stress has a more
profound effect on the helical state of the DNA. Any negative stress
that is generated causes a greater helical distortion (single-stranded
character), which can be rapidly removed by E. coli
topoisomerase I. This reduced flexibility is observed during
transcription in hypotonic conditions and in the presence of
polyamines. 2) The reduced flexibility as exhibited with the polyamines
also decreases the rotational freedom of DNA. Polyamines are known to
stabilize alternative DNA structures (i.e. Z DNA) and
facilitate sequence-specific bending (28-32). The stabilized bending
increases the effective diameter of DNA and restricts its rotation
during transcription. These two effects, which are a consequence of
reduced flexibility, enhance the maintenance of helical distortions and
are required for E. coli topoisomerase I to remove the
negative stress. We have also evaluated whether similar helical
distortions could be produced in the presence of nucleosomes. We
observe that as little as three nucleosomes in a plasmid capable of
holding 30 nucleosomes will facilitate the formation of very high
levels of positive stress. In this instance, a protein-mediated bending
of DNA has increased the effective diameter of DNA and reduced its
rotational freedom. Histones H3-H4 and not histones H2A-H2B were
responsible for this reduced rotational rate, which is an indication
that the presence of the stored negative coil is a primary determinate
for this rotational restriction.
Reagents--
Salmon protamine (grade X),
poly-L-lysine (4-15 kDa), poly-L-arginine
(5-15 kDa), poly(Asp-Glu) (7 kDa), spermine, spermidine, and protamine
were purchased from Sigma.
1,19-Bis(ethylamino)-5,10,15-tirazanonadecane (BE-4-4-4-4) was a gift
from Hirac Basu (University of Wisconsin, Madison).
Procedures for Purification of Proteins--
Histones were
purified from chicken erythrocytes as described previously (33).
Briefly, histones H2A-H2B were separated from H3-H4 by elution from a
hydroxylapatite column in which the chromatin had been immobilized.
Elution was done with a 0.1 M NaCl step gradient between
0.6 and 2.0 M NaCl. The histones were pooled, concentrated
on Amicon filters, and stored at
Eukaryotic topoisomerase I was isolated from MSB cells (chicken
leukemic cell line) using a modification (34) of the procedure of Liu
and Miller (35). One unit is defined as that quantity that achieves
100% relaxation of 0.5 µg of DNA in 30 min at 37 °C. This protein
is referred to here as MSB topoisomerase I.
Procaryotic topoisomerase I was isolated from a clone of E. coli topoisomerase I (pJW312; a gift of M. Gartenberg and J. C. Wang, Harvard). The procedure for isolation was as described
previously (36) except that after the final phosphocellulose column,
the enzyme was further purified from DNase and RNase activity by
collecting the flow-through from a Mono Q column and applying it to a
Mono S column. The enzyme eluted in a 0-0.5 M KCl
gradient. We have defined 1 unit of activity as the quantity that
relaxes 1 µg of DNA from
T7 RNA polymerase was prepared from E. coli strain BL 21, which contained plasmid pAR1219 (a gift of W. Studier and J. Dunn, Brookhaven National Laboratory). The enzyme was purified by the procedures of King et al. (37) except with modification as
described previously (38). One unit is defined as that amount of enzyme that will incorporate 1 nmol of CTP at 37 °C for 60 min.
Nuclear assembly protein 1 (NAP1) was prepared from E. coli
strain BL21, which contained plasmid pTN2 (gift of A. Kikuchi, Tokyo
Institute of Technology). The protein was purified by the procedure of
Fujii-Nakata et al. (39) except with the modification that
after the last purification on the Mono Q column, the protein was
reapplied to a Mono S column followed by concentration on a second Mono
Q column. This additional step provided a homogenous protein free of
RNase and DNase activity. This preparation provided maximum deposition
of histone when the ratio of NAP1 to histones was 1:1 by weight.
Preparation of DNA--
An additional 35-bp sequence containing
a T7 promoter was cloned into the XbaI-HindIII
site of the T7/T3-19 plasmid (2238 bp; Bethesda Research Laboratories)
to yield a plasmid with two T7 promoters 39 bp apart and a size of 2255 bp (2T7/T3-19). This DNA was used as the vector for the insertion of a
3800-bp SacI fragment excised from pT207-18 (gift of P. Yau
and M. Bradbury, University of California, Davis). This fragment
contains 18 repeats of the 207-bp 5 S gene of Lytechinus
variegatus (40). This insertion yields a DNA of 6055 bp with two
tandem promoters that directly transcribe into the insert
(p2T7/T3-207-18). Circular, covalently closed, negatively coiled DNA
(referred to here as "S") was purified on CsCl-ethidium bromide
density gradients followed by sedimentation on 5-20% sucrose
gradients. For the preparation to be used as a template for
transcription, the DNA was treated with MSB topoisomerase I at 0 °C
in buffer conditions of 10 mM MgCl2, 10 mM triethanolamine, pH 7.4. The angle between adjacent base
pairs in DNA decreases by about 0.011 rotational degree for each degree
centigrade decrease (41-43). By including 10 mM
MgCl2, the angle is reduced even further (44), so that
after the topoisomerase I activity is terminated and the temperature is
raised to 37 °C in isotonic conditions, the superhelical density is
+0.011 SD. The 6055-bp DNA will contain an average of +6.5 coils. After
treatment with the topoisomerase I, the DNA was
phenol/chloroform-extracted and ethanol-precipitated prior to use. This
DNA is referred to as "R". The R DNA was used for these studies in
order to discriminate by gel electrophoresis the template DNA that had
been nicked during post-transcriptional processing of samples from
template DNA that had remained covalently closed. In this way, it is
possible to determine more precisely the conversion of template DNA
from its original topological state (+0.011 SD) to a more positively
stressed condition as a result of transcription.
Conditions for Transcription--
The buffer conditions and
quantities of T7 RNA polymerase and E. coli topoisomerase I
that were used were varied as described. The remainder of the
transcription conditions were kept constant and included the following:
a final concentration of 50 µg/ml in template DNA and 0.8 mM each of ATP, GTP, CTP, UTP, and 5 µCi/ml [ Reconstitution of Histones with NAP1--
In the transcription
experiments in which histones were used, the buffer conditions were 100 mM NaCl, 40 mM Hepes, 6 mM
MgCl2, 10 mM DTT (pH 7.3 or 7.9), and 0.8 mM each ATP, GTP, and CTP. The histone-NAP1 complexes were
prepared in this same buffer at a final concentration of 100 µg/ml
histone. After a 5-min incubation at 37 °C of the histones in this
buffer, NAP1 (1:1 (w/w)) was added, and the incubation continued for 30 min. This solution was directly added to the mix containing the
template DNA, and the incubation continued for 10 min before
transcription was initiated with the addition of UTP. This 10-min
incubation is sufficient to complete the deposition of histones to the
template DNA by the NAP1 (see bottom panel of
Fig. 9).
The Level of Positive Stress Observed Is Highly Dependent on the
Ionic Environment in Which the Transcription Was Done--
These
transcription studies were done on a closed circular DNA (6055 bp) that
contains two tandemly arranged T7 promoters that are 39 bp apart. To
facilitate the analysis of changes in the topological state of DNA
during transcription, the DNA was preadjusted to a density of +0.011 SD
(see "Experimental Procedures"). As shown in Fig.
1A (lane
R), the DNA averages +6.5 coils. As shown in Fig.
1B, when this DNA was transcribed by 400 units/µg DNA of
T7 RNA polymerase in the buffer conditions of 10 mM Hepes, 6 mM MgCl2, 10 mM DTT, pH 7.9, and
in the presence of E. coli topoisomerase I (60 units/µg
DNA), the number of positive coils increased substantially as
transcription proceeded. That this change is due to an increase in
positive coils can be shown in the two-dimensional analysis of Fig.
1C. This pattern of distribution for DNA topoisomers is
characteristic of positive coils when the second electrophoretic
condition is in the presence of chloroquin (46). Fig. 1D
shows an analysis of the RNA that was produced during this
transcription and indicates that the rate of transcription was 95 bases/s. That this formation of high levels of positive stress is
dependent on the twin domain model of transcription-induced stress can
be seen in Fig. 1B (+ RNase lane). The
lack of increased positive stress in this lane is interpreted as
indicating that the RNase A is destroying the transcript as synthesis
occurs. The RNA polymerase is free to rotate on the DNA and therefore unable to induce stress.
We further characterized the conditions responsible for forming the
positive stress with the following experiments. As shown in Fig.
1E, when the quantity of polymerase was increased 4-fold, the number of positive coils increased so extensively that now one
major band of very highly positively coiled DNA was observed (Fig. 1,
region 1). An estimate of the superhelical
density can be determined by an analysis of the DNA on CsCl-ethidium
bromide gradients (46). Using this approach, we estimate the level of stress to average +0.15 SD (data not shown). These results are in
contrast to when the buffer condition was adjusted to include 100 mM NaCl. As shown in Fig. 1F, there was a
minimal change in the topological state of the DNA. This more
physiological condition has negated the ability of E. coli
topoisomerase I to remove negative coils that have been produced by
transcription. Graphically shown in Fig.
2A is an analysis of the
levels of positive stress that were generated as a function of
variations in ionic strength. Fig. 2 also shows the quantity of RNA
that was produced during transcription in order to correlate levels of
transcription with the quantity of positive stress that was produced.
When the NaCl concentration was raised from 0 to 120 mM,
there was a 2-fold increase in transcription. Yet even with this 2-fold
increase in transcription, there was a rapid and precipitous decrease
in the ability to form positive coils on the template. We interpret this observation as indicating that the monovalent ions are increasing the flexibility of the DNA through neutralization of the phosphates (47, 48). The induced negative and positive coils are rapidly neutralized by translational diffusion around the circular plasmid before sufficient stress is generated to cause the helical
perturbations (14, 15, 26).
Polyamines Greatly Increase the Formation of Transcriptionally
Induced Positive Stress--
The polyamines, putrescine (diamine) and
spermidine (triamine), are found in prokaryotic cells in millimolar
concentrations (49). In eukaryotic cells, spermine (tetramine) and
spermidine are found in submillimolar and millimolar concentrations,
respectively (50, 51). The polyamines are thought to function at
several levels of RNA and DNA processing (52). With regard to DNA,
polyamines interact with both the major and minor groove in a
sequence-dependent process (53-55). This selective binding
can result in an increased stabilization of unique DNA structures such
as Z DNA (28) as well as causing a general stiffness in the DNA by
interaction with the surface phosphates (29-31). Because of this
general neutralization of charge and the potential bridging of the
multivalent polyamines between DNAs, higher concentrations will
increase DNA-DNA affinity or self-association (56-59). As shown in
Fig. 3, when transcription was done for 5 min in 100 mM NaCl and in the presence of increasing spermidine (Fig. 3A) or spermine (Fig. 3C),
substantial positive stress was produced on the template. In both
cases, the DNA has a superhelical density estimated to be +0.10 SD
(Fig. 3C, region 2). The minimal polyamine
concentrations required to obtain this superhelical density were 4 and
0.8 mM, respectively. These data have been quantitated and
are graphically shown in Fig. 2B in order to compare with
the levels of transcription that produced this stress. These data
indicate that the transcription rate remained relatively unchanged
throughout the range in which positive stress continued to be amplified
for both polyamines. The increase in positive stress must be a result
of a change in the physical property of the DNA or RNA synthesized from
it. This altered character could be a change in the flexibility and/or
an increase in self-association (aggregation) between nucleic acid
molecules. The rate at which this positive stress is produced is shown
in Fig. 3F. In this experiment, the spermidine (6 mM) was added simultaneously with the initiation of
transcription. High levels of positive stress were observed after
30 s, which suggests that whatever physical property is changed by
the polyamines, it is changed very rapidly.
We evaluated the factors that contributed to the induced positive
stress by initially characterizing the aggregative state of the RNA and
DNA. Transcription was done in the presence of increasing quantities of
the polyamines, during which the samples were sedimented 27,000 × g for 5 min. Both the supernatant (S) and pellet
(P) were analyzed by gel electrophoresis for RNA and DNA
content. As shown in Fig. 4A,
when the spermidine concentration was increased, there was a
proportional increase in aggregation of both RNA and DNA. At 4 mM spermidine, approximately 10% of the DNA and RNA was in
the pellet. RNA was the major factor that caused this aggregation, for
in the absence of transcription no DNA was in the pellet (see Fig.
7A). We can differentiate between the role of DNA
flexibility and aggregation in inducing positive stress, providing we
can minimize this aggregation further. As shown in Fig. 4B,
transcription in the presence of 25 mg/ml BSA resulted in minimal
aggregation even at high spermidine concentrations. Fig. 3B
shows the positive stress that was produced in this nonaggregative condition. Minimal changes in the extent and level of positive stress
are observed (Fig. 3, compare A and B), which
suggests that a decrease in DNA flexibility is responsible for the
effect. To assess the factors that produced positive stress with
spermine, transcription was done in the presence (Fig. 4D)
and absence (Fig. 4C) of poly(Asp-Glu) followed by
centrifugation to remove the aggregated material. In the absence of
poly(Asp-Glu), complete aggregation of both DNA and RNA was observed at
1.6 mM spermine. When it was present, approximately 15% of
DNA and RNA aggregated. The poly(Asp-Glu) has substantially reduced the
aggregative behavior of spermine. A comparison of panels
C and D of Fig. 3 indicates that the positive
stress generated in the DNA for these two conditions remains unchanged.
The implication from this analysis is that the decrease in DNA
flexibility is again responsible for facilitating the presence of DNA
helical distortions during transcription. Does this indicate that
aggregation is unable to cause transcription-induced stress? To
evaluate this question, we repeated this analysis with the synthetic
polyamine BE-4-4-4-4. This polyamine is extremely effective at
aggregating nucleic acids at very low concentrations (60). As shown in
Fig. 4E, 0.4 mM BE-4-4-4-4 was sufficient to
cause complete aggregation of DNA and of the RNA synthesized in its
presence. This is a 4-fold lower concentration than was required for
spermine. If we now examine the level of positive stress generated in
BE-4-4-4-4 (Fig. 3E), we observed that positive stress was
generated, but at a much reduced level. We have increased the
polymerase and topoisomerase content in subsequent experiments and
observed that this pattern remains unchanged (data not shown). We
conclude that aggregation can cause positive stress, although it is
unlikely to be the major factor. The minimal effect of aggregation in
promoting the formation of positive stress will be further shown by our
analysis of the large polycations, polylysine and polyarginine,
described below.
An additional measure for the level of aggregation caused by the
polyamines is the observed catenation that occurs when circular single-stranded DNA is incubated with E. coli topoisomerase
I (56, 61). As shown in Fig.
5A, M13 single-stranded DNA
catenates into intramolecular knotted structures as well as
intermolecular larger molecular weights when in the presence of
topoisomerase and 6 mM spermidine, 6 mM
MgCl2, 10 mM Hepes, pH 8.0. At this low ionic
strength, spermidine causes aggregation (Ref. 58; data not shown), and,
as expected, catenates are formed. As shown in Fig. 5B, when
the NaCl concentration was increased, the extent of catenation
dramatically decreased such that by 80 mM NaCl, no
catenation of either form was observed in the 6 mM
spermidine. These observations are another indication that at the more
physiological salt concentrations, spermidine minimally promotes
intramolecular or intermolecular interactions of DNA.
It has been reported that the polyamines are effective at stabilizing
the double-stranded character of DNA (62, 63). As a result, E. coli topoisomerase I is substantially inhibited from relaxing
negatively coiled DNA when in the presence of spermidine (64, 65). This
point is illustrated in Fig. 6, in which
negatively coiled DNA was treated with the enzyme in the absence
(A) and presence (B) of 4 mM
spermidine. The presence of spermine extensively inhibited the enzyme.
These data are graphically shown in Fig. 7A, in which it is observed
that only 11% of the activity remained. Higher concentrations reduced
the activity even further so that at 10 mM spermidine, 4%
of the activity was observed (Fig. 7A). This inhibition was
not due to the aggregative state of the DNA, because, as shown in Fig.
7A, the DNA remained soluble even at 10 mM
spermidine, and indeed, as shown in this figure, the eukaryotic topoisomerase I (MSB) was extensively stimulated by the presence of
spermidine. This stimulation has been reported previously (65, 66) and
would not occur if aggregation were present. This point is illustrated
in Fig. 7B, in which a similar experiment was done using
spermine. As shown in this figure, spermine caused complete aggregation
at 1.2 mM, and no stimulation of MSB topoisomerase I
activity was observed (see also Ref. 60). However, as shown in Fig.
7C, aggregation by spermine was prevented when poly(Asp-Glu) was present, and in this condition the MSB topoisomerase I was now
extensively stimulated. This stimulation was to the same level as when
spermidine was used (Fig. 7A). Thus, when spermine and poly(Asp-Glu) are combined, the structural state of the DNA is very
similar to when spermidine alone is present. In both cases, the level
of positive stress that can be generated by transcription is the same
(Fig. 3, compare A and D) and is an indication
that the structural state of the DNA and not its aggregation is
primarily responsible for the effect.
With the highly inhibitory effect of spermidine on E. coli
topoisomerase I activity, the question arises as to how negative coils
are removed during transcription. Fig. 6C shows an
experiment in which negatively supercoiled DNA was treated with
topoisomerase I in the presence of 4 mM spermidine.
Transcription was initiated simultaneously with the addition of
topoisomerase I, and, as shown in this figure, the rate at which the
negative coils were removed and converted to positive coils far
exceeded the rate at which those coils were removed when transcription
did not occur (Fig. 6, compare B and C). The
transcription process generated negative coils, which were more
effectively relaxed by the topoisomerase. The actual mechanism that
facilitates this process is undefined. It probably involves both an
increase in the dynamic rather than static nature of the negative coils
and the induction of higher levels of negative stress by transcription.
Minimal Transcription-induced Stress Is Preserved on Template DNA
in the Presence of Polylysine or Polyarginine--
DNA was treated
with increasing quantities of either polylysine or polyarginine. Since
these polycations bind DNA, their presence was expected to also
decrease the flexibility of DNA and thus increase the likelihood of
helical distortions being induced during transcription. As shown in
Fig. 8A, the presence of
polylysine produced minimal positive stress on the DNA. Even at
concentrations in which the polylysine began to cause self-association
(aggregation) of the DNA (0.65:1; data not shown) (and, as shown in
Fig. 8A, significant transcription continued to occur), no
increase in positive stress was observed. For polyarginine, there was a
gradual increase in positive stress, but it still remained
significantly less than for the polyamines (compare Figs. 8A
and 2B). This lack of positive stress is not due to an
inability of E. coli topoisomerase I to access the DNA.
Control experiments have been done that show that negatively coiled DNA
is readily relaxed in the presence of these poly-amino acids (data not
shown). We interpret these observations as indicating that
peptide-induced changes in the physical state of the DNA as exhibited
by these poly-amino acids is substantially different from the
polyamines.
Extensive Positive Stress Is Generated by Transcription of DNA When
Histones Are Reconstituted under Specific Conditions--
Increasing
quantities of histone H1 or protamine were directly added to template
DNA, and, after an incubation of 10 min, the samples were transcribed
in the presence of E. coli topoisomerase I. As shown in Fig.
8B, very minimal positive stress was observed on the DNA for
either protein despite the presence of substantial transcription,
particularly at the lower protein:DNA ratios. E. coli
topoisomerase I was unable to detect helical distortion in the DNA
brought about by the transcription process. These observations are
consistent with the observations concerning the poly-amino acids, in
that the binding of protein to DNA does not necessarily provide
conditions in which transcription-induced stresses can be preserved.
We now considered whether the presence of core histones would preserve
transcription-induced stresses. Histones H2A-H2B and histones H3-H4
were reconstituted with DNA at increasingly greater ratios using the
same direct addition protocol as was used for the experiments with
histone H1 and protamine. Minimal positive stress was observed although
substantial transcription continued to occur (Fig. 8B).
However, it is known that the direct addition of core histones to DNA
at physiological ionic strength severely limits nucleosome formation.
Rapid binding is not conducive for proper interaction. When
reconstitution is done by salt gradient dialysis (67) or by mediation
with deposition factors (68), proper histone-histone and histone-DNA
contacts are established, and nucleosomes will form. To evaluate the
effect of nucleosomal structure in this process, core histones were
pretreated with the deposition factor, NAP1, and then added to DNA in
increasing quantities. The samples were split in half, and one aliquot
was incubated for 10 min in the presence of MSB topoisomerase I in order to determine the number of nucleosomes that were present. The
maintenance of negative coils by histones when in the presence of an
eukaryotic topoisomerase is an indication that nucleosomes are present
(67, 69). Each negative coil is equivalent to the presence of one
nucleosome. As shown in the bottom panel of Fig.
9A, an increase in the
histone:DNA ratio resulted in an expected increase in the number of
negative coils on the DNA. This 10-min incubation was sufficient to
complete this process, since a more prolonged 60-min incubation did not
alter these levels (data not shown). The second aliquot was incubated
for 10 min, during which the nucleosomes were allowed to form, except
this time E. coli topoisomerase I was present. After the
10-min incubation, transcription was initiated by the addition of the
UTP. Transcription was allowed to occur for 5 min, and the DNA was then
analyzed as shown in the upper panel of Fig.
9A. At a histone:DNA (H:D) ratio of 0.1:1, the very highly
positively coiled band (region 1) was observed. This very low ratio
equates to an average of three nucleosomes on a 6055-bp DNA (Fig.
9A, bottom panel). As the ratio was
increased and more nucleosomes were formed, the quantity of the highly
positively coiled band increased proportionately until a ratio of 0.5:1
H:D was reached, at which point no further increase was observed. These
data are graphically shown in Fig. 8C along with the level of transcription that was observed in this experiment. It is now useful
to compare these data with the data of Fig. 8B, in which histones were on the DNA in a nonnucleosomal form. There is a dramatic
difference in the level of positive stress, although the level of
transcription for both situations was very similar up to the
histone:DNA ratio of 0.6:1. We conclude that nucleosomes very
effectively promote the presence of transcription-induced stress.
To evaluate the role of the individual core histones in producing this
effect, this procedure was repeated except that only histones H3-H4
were preincubated with NAP1 prior to the addition to the DNA. As shown
in Fig. 9B (lower panel), the number
of negative coils increased as the ratio of H3-H4 to DNA was increased.
This observation has been reported previously (46, 70) and is an indication that histones H3-H4 can hold negative coils independent of
H2A-H2B. Increasing the length of incubation beyond 10 min did not
appreciably change the number of negative coils that were observed and
is again an indication that with these histones, deposition was
complete within the 10 min (data not shown). Transcription of these
reconstitutes in the presence of E. coli topoisomerase I
resulted in the formation of the highly positively coiled band (Fig.
9B, upper panel). This band was first
observed at the very low ratio of 0.05:1 H:D and is an indication that
H3-H4 are extremely effective in maintaining transcription-induced
stress. An additional point of interest regarding these data is that
when a ratio of 0.5:1 H:D was reached, all of the DNA was shifted to
the highly positively coiled state. This result is in contrast to when
core histones were used (Fig. 9A, upper
panel), in which a sizable percentage of DNA never became
highly positively coiled. We interpret this difference as being due to
the blockage of promoters by core histones in a subset of templates,
which does not occur in the absence of H2A-H2B. It is known that
nucleosomes will form on the T7 RNA polymerase promoter and repress
initiation (71).
As shown in the graphical analysis of Fig. 8C, the level of
positive stress that was produced in the presence of H3-H4 was 100%.
In other words, this level of positive stress is equivalent to the
level observed when transcription was done at low ionic strength
(Fig. 2A), a condition that up to this point produced the
maximum level of positive stress. The presence of H3-H4 has preserved
these induced stresses in physiological conditions of ionic strength
and MgCl2, a condition in which these stresses would
normally not be detected by E. coli topoisomerase I.
These experiments were repeated with histones H2A-H2B in order to
evaluate whether these proteins contributed to this process. In this
instance, NAP1 was not used as a deposition factor, since it is
ineffective in depositing H2A-H2B when H3-H4 are absent (72). Instead,
H2A-H2B were deposited by direct addition to the DNA (33). The
reconstitutes were then incubated with MSB topoisomerase I, and, as
shown in Fig. 9C (lower panel),
H2A-H2B were unable to hold negative coils. This observation has been reported previously by several investigators and is an indication that
H2A-H2B in the absence of H3-H4 are unable to hold negative coils on
DNA (70). The upper panel of Fig. 9 shows the
quantity of positive stress that was left on the template after
transcription in the presence of H2A-H2B. Even at a ratio of 1:1 H:D,
the level of positive stress that was observed was very low. These data along with the level of RNA produced during transcription are graphically shown in Fig. 8C. These data illustrate the
striking difference between these two sets of proteins. Histones
H2A-H2B produced a 10-fold lower level of positive stress compared with H3-H4. Additional information can be obtained from this graphical analysis. For the ratios between 0.025 and 0.40 H:D, the slopes of the
two lines describing the levels of positive stress that were observed
for all four histones compared with H3-H4 alone are nearly identical
(Fig. 8C). Similarly, if the number of stored negative coils
that the four histones maintain are compared with H3-H4 alone for this
same range of H:D ratios, we also observe that they are very similar
(Fig. 9, A and B; compare lower
panels). We therefore conclude that H2A-H2B substitute very
effectively for an equimolar amount of H3-H4, providing an equimolar
amount of H3-H4 is present at the same time. It is the storage of the left-handed supercoil by the complex of histones that provides the
bending of the DNA. Histones H2A-H2B can contribute to the bending,
provided that they are interacting with H3-H4 to form left-handed coils
as part of the nucleosomal complex. The bending of DNA into the coil
has increased the effective diameter of the DNA, which cannot be
mimicked by protamine, H1, or nonnucleosomal core histones (Fig.
8B).
We have also observed that the method of reconstitution, whether
protein-mediated (NAP1) or salt-mediated (NaCl dialysis), does not
alter these results. The experiments of Fig. 9 have been repeated using
NaCl dialysis (67), and the same results were observed in all three
cases (data not shown). In the subsequent studies of this report, we
focus on the use of histones H3-H4 rather than total core histones.
Histones H3-H4 do not block the T7 RNA polymerase from its promoter. We
can then more effectively define increases in positive stress during transcription.
A Comparison of the Positive Stress That Is Produced with
Spermidine, Spermine, and Histones H3-H4--
We have observed that
spermidine, spermine, and histones H3-H4 preserve transcription-induced
stress at physiological ionic strength. We now compared their relative
effectiveness in this process by transcribing under conditions in which
both the concentration of E. coli topoisomerase I (Fig.
10, A-C) and T7 RNA
polymerase (Fig. 10, D-F) were varied. The conditions that
were used were 2.0 mM spermidine (Fig. 10, A and
D), 0.8 mM spermine (Fig. 10, B and
E), and H3-H4 at 0.5:1 H:D (Fig. 10, C and
F). As shown in Fig. 10, A-C, increasing the
E. coli topoisomerase I content from 3.8 to 120 units/µg
DNA (lanes a-f) resulted in an increase in positive stress for all three conditions. However, any quantity of
topoisomerase I greater than 15 units/µg DNA (lane
c) did not succeed in significantly increasing this level of
stress. Each level appears to be unique for each agent. For spermidine,
the upper limit was +0.05 SD (region 3), for spermine it was +0.10 SD
(region 2), and for H3-H4 it was +0.15 SD (region 1). A similar result
was observed when the T7 RNA polymerase content was varied from 100 to
3200 units/µg DNA (Fig. 10, D-F). Therefore, regardless of the quantity of polymerase or topoisomerase that was used, these
concentrations of spermidine, spermine, and H3-H4 preserve different
levels of stress. We are observing well defined differences in rates at
which the negative and positive stresses are diffusing around the
circular DNA. The structural character of the DNA in these three
conditions is uniquely different.
We determined the number of polymerase molecules that were actively
transcribing in the experiments of Fig. 10, D-F, in order to correlate this number with the quantity of positive stress that was
generated. T7 RNA polymerase (100-3200 units/µg DNA) was added to
the template DNA in the presence of excess MSB topoisomerase I and
transcribed for 2 min. The principle behind this analysis is that each
polymerase opens approximately one turn of the DNA helix as it
transcribes (73). Since eukaryotic topoisomerases relax both positive
and negative stress equally well, closed circular DNA will have an
overall relaxed state until both transcription and topoisomerase I
activity is terminated by SDS treatment. The elongating polymerase is
then displaced, the helix is restored to the double-stranded state, and
a negative coil is now in the circular DNA. As shown in Fig.
10G, an average of six negative coils are present in
lane d, which indicates that on average six polymerases produced the quantity of positive stress that is shown in lane d of Fig. 10, D-F. Multiple
initiations are required to provide sufficient transcription-induced
stress so that this upper limit of positive stress can be obtained.
Reduced Rates of Transcription at 37 °C Decrease the Formation
of Positive Stress--
We have observed that it is possible to
decrease rates of transcription by T7 RNA polymerase at 37 °C by two
approaches, adjusting the pH from 7.9 to 7.3 and/or including GDP (2 or
4 mM) during transcription. Fig.
11A shows the size of RNA
that was produced at pH 7.9 (lanes a-c) and pH
7.3 (lanes d-f). When no GDP was present, the
transcription rates were 83 and 47 bases/s, respectively (lanes a and c); when 2 mM
GDP was present, the rates were 47 and 20 bases/s, respectively
(lanes b and e); and when 4 mM GDP was present, the rates were 25 and 10 bases/s,
respectively (lanes c and f). Using
these procedures, we analyzed the effect of transcription rate on the
ability to form positive stress. The H3-H4-NAP1 complex was
reconstituted with DNA at a ratio of 0.5:1 H:D and transcribed in the
presence of 4 mM GDP at pH 7.9 (Fig. 11A) and pH
7.3 (Fig. 11B). As shown in Fig. 11A, the 25 bases/s rate of transcription has generated a level of stress
considerably less and substantially varied as compared with the 83 bases/s rate of Fig. 9B (upper panel).
This 3.2-fold decrease in rate has substantially decreased the level of
stress and mimics the results of Fig. 10F, in which a
similar -fold decrease in quantity of T7 RNA polymerase caused a
similar reduction in positive stress. As shown in Fig. 11B,
transcription at 10 bases/s caused an even more dramatic decrease in
the formation of positive stress, which indicates that a decrease in
transcription rate is proportional to a decrease in number of
actively transcribing polymerases.
High Levels of Positive Stress Are Observed When Transcription
Rates Are Lowered by Decreasing the Temperature--
The high levels
of positive stress that were observed when transcription occurred in
the presence of histones H3-H4 were the result of a transcription rate
of 83 bases/s (see lane a of Fig. 11) and an
average of six elongating polymerases (Fig. 10G). This large
number of polymerases was presumably needed to counter the extensive
neutralization of negative and positive stress that occurs as these
stresses diffuse around the circular DNA. Theoretically, reducing the
rate of transcription by reducing temperature should result in a
proportional decrease in positive stress as was seen in Fig. 11.
However, there are also decreases in the thermal motion of DNA because
of the lowered temperature, which may counter the lowered transcription
rate. In order to evaluate this interrelationship, DNA was
reconstituted with the H3-H4-NAP1 complex at a ratio of 0.5:1 H:D and
then transcribed at four different temperatures. These temperatures
were 37, 23, 13, and 4 °C. The transcription rates were 83, 27, 8.2, and 2.6 bases/s, respectively (data not shown). As shown in Fig.
12, the highly positively coiled band (region 1) was present at all four temperatures. Except for the 4 °C
condition, the remaining temperatures produced equivalent quantities of
this highly positively coiled DNA. The rate at which this stress was
achieved was substantially slower at the lower temperatures. One
possible reason for the slower rate may be a change in the activity of
E. coli topoisomerase I. As noted in the data of Fig. 10,
A-C, the quantity of E. coli topoisomerase I is
important. We have assayed the topoisomerase at these temperatures and
determined its activity relative to the activity at 37 °C. The
activity was decreased 40-fold at 23 °C, 640-fold at 13 °C, and
4 × 104-fold at 4 °C (data not shown). These
rather sizable decreases in activity are probably a major cause for the
slower rate at which highly positively coiled DNA was generated at the
lower temperatures. Nevertheless, the transcription rates at 23 °C
(26 bases/s) and at 13 °C (8.2 bases/s) clearly produced the same high levels of stress that were observed at 37 °C (83 bases/s). It
is a level substantially greater than what was observed when we
produced comparable transcription rates at 37 °C in Fig. 11 (25 and
10 bases/s). The decrease in transcription rate because of the lowered
temperature must have closely matched a similar reduction in thermal
motion. Rates of diffusion for the transcription-induced stresses are
then also reduced. A major determinant for defining the availability of
negative coils that are to be neutralized by E. coli
topoisomerase I is this rate of diffusion.
These data indicate that the there are two potential means whereby
transcription-induced stresses can be preserved within DNA: 1) changes
in DNA flexibility inherent in the DNA and 2) changes in rotational
flexibility because of protein interaction. The observation that low
ionic strength conditions extensively increase positive stress in DNA
during transcription is an indication that stiffening of DNA is a
contributing factor to this process (Fig. 2A). An
alternative explanation for this effect would be that the increased
single-strandedness of the DNA at the lower ionic strength has
increased E. coli topoisomerase I activity. We have not
observed an increase in activity in this low ionic strength condition
(data not shown). We have also shown that increasing the topoisomerase
I activity beyond a critical level does not alter the final level of
positive stress (Fig. 10). Thus, a change in DNA flexibility must be
the critical factor. We surmise that the change is a lack of DNA
flexibility due to the lack of counterions on the DNA surface. The
decreased flexibility is predicted to increase the likelihood that a
lower level of the induced stresses will induce helical distortions as
these stresses diffuse around the circular DNA. The hypotonic
condition, however, is nonphysiological, and the question arises as to
whether a similar change in flexibility can be observed in a
physiological context. Spermidine and putrescine are the major
polyamines in E. coli and exist in average concentrations of
6 and 20 mM, respectively, although the effective free
concentration may be less (49). Polyamines are known to decrease the
flexibility of DNA (29-31), and at sufficiently high concentrations
they will promote self-association of DNA (56-59). We have attempted
to differentiate the relative contributions of these two effects in
facilitating the formation of positive stress. We have found that when
transcription is done in the presence of either BSA (Fig.
4B) or poly(Asp-Glu) (Fig. 4D), minimal
aggregation of either RNA or DNA is observed. In both cases, whether
with spermidine or spermine, the level of positive stress that was
generated remained the same (Fig. 3). Thus, the change in flexibility
of the DNA would appear to be the major factor facilitating the
formation of positive stress. The mode of action for BSA and
poly(Asp-Glu) in minimizing this aggregation is probably based on their
polyanionic nature. Any inter- and intrastrand bridging between nucleic
acid strands that is promoted by the polycationic polyamines is
competitively blocked by these agents. These observations may have
relevance to the environment of a cell that is known to be in a crowded
state, with macromolecular concentrations of total RNA and protein
approximating 340 mg/ml (74, 75). The observation that BSA at a 10-fold lower concentration is able to modulate this aggregative behavior may
be of importance in facilitating both molecular crowding and accessibility to the DNA. Therefore, the general stiffening of DNA
observed in the hypotonic conditions would appear to be at least
partially mimicked by the polyamines in physiological conditions.
In the data of Fig. 10 it was observed that a relatively defined level
of positive stress was induced that was uniquely different for
spermidine at 2 mM (+0.5 SD), spermine at 0.8 mM (+0.10), and histones H3-H4 at a H:D of 0.5 (+0.15 SD).
One possibility for these differences is that they result from a
variation in transcriptional activity. Transcription was increased by
nearly 2-fold in the presence of polyamines (Fig. 2B).
However, one would normally expect to see greater positive stress, not
less, when transcription is increased. Another possibility is that the
differences result from the inhibition of E. coli
topoisomerase I activity when in the presence of polyamines (Fig. 7).
In this instance, the reduction of activity would be expected to reduce
levels of positive stress. These two variables were evaluated in the
data of Fig. 10, and we observed that even when the topoisomerase I concentration was increased greater than 20-fold or the T7 RNA polymerase concentration was increased by 8-fold, no appreciable change
in these defined levels of positive stress was observed. Our
interpretation of this observation is that the rate-limiting step that
defines these levels of stress is the rate of flux in which the
positive and negative stresses are propagated around the plasmid. There
must be an upper level of steady state stress that is generated that is
unique to each of these three conditions.
We surmise that the decrease in flexibility of DNA is the critical
factor by which the polyamines facilitate stress-induced helical
distortion. It has been reported that polyamines facilitate the bending
of DNA in a sequence-specific manner (31, 32). This bending, which may
be minimal at any particular DNA sequence, would be expected to be
globally significant in the larger context of a circular DNA of
multiple sequences. The increase in effective diameter would be
expected to increase the viscous drag of the DNA and result in a
prolonged maintenance of transcription-induced stress (48). It is our
interpretation that the major factor that causes the variations in the
different levels of positive stress that were observed for spermidine
at 2 mM and spermine at 0.8 mM in Fig. 10 is
how effectively the viscous drag (bent state) was maintained in those
two conditions. Histones H3-H4 are particularly effective in bending
DNA and maintaining it in that state, hence very effective in promoting
this viscous drag. Thus, the level of induced positive stress with
H3-H4 is the highest of the three conditions. The common theme is the
bending of DNA whether by polyamines or by histones.
General stiffness, however, without the bending of DNA does not appear
to be effective in the maintenance of transcription-induced stress.
Minimal positive stress was obtained with polylysine, polyarginine,
histone H1, protamines, or the core histones when directly added to DNA
(Fig. 8). Even at higher quantities of these agents, at which point
self-association of the DNA was occurring, positive stress was
minimally formed. We conclude that neither the stiffness of the DNA as
promoted by these agents or the self-association into aggregative
complexes is sufficient to cause the rotational restriction necessary
to observe high levels of induced stress. This observation was a
surprise, since we had expected that such polycations would mimic the
hypotonic conditions and therefore enhance the presence of helical
distortions. It may be that such helical distortions are enhanced but
that these particular agents mask them by their interaction with DNA.
The direct interaction by the polyamines within the major and minor
grooves of the DNA must produce unique structural characteristics in
DNA (stiffness and bending) that cannot be mimicked by these other polycations.
Based on counterion theory, increasing the ionic environment by either
mono or divalent ions would be expected to increase the flexibility of
the DNA rather than decrease it (76). This increased flexibility was
observed when we raised the NaCl concentration and subsequently
produced a precipitous drop in levels of positive stress (Fig.
2A). The polyvalent polyamines do not appear in an additive
way to increase this flexibility. Rather, the reverse is observed,
which suggests that these ions maintain a more persistent interaction
with DNA, rather than as a highly mobile ion shield (48). We have also
studied the divalent ions Mg2+ and Ca2+ and
have observed that at elevated concentrations (20 mM) a
similar decrease in flexibility is observed based on the level of
positive stress that is produced during transcription
(11).2 This persistence of
interaction by these polycations is expected to be responsible for the
structural changes that decrease DNA flexibility. The maintenance of a
consistent stiffness in the DNA may be of importance in not only
facilitating stress-induced distortions of the DNA helix, which are
critically important in accessing and replicating DNA (24-27), but
also in ensuring that the flexibility of the DNA is not so great that
it "winds itself into a ball" before the transcription-induced
stresses are able to produce the helical distortions. E. coli topoisomerase I cannot relax DNA without the single-stranded
character that is produced by the helical distortions. In the
prokaryotic cell, it must do this in the presence of polyamines that
repress helical distortions except under the conditions for which they
are generated by transcription (Fig. 6).
When the core histones were deposited onto the DNA using NAP1 so that
proper nucleosomes could form, substantial positive stress was observed
(Fig. 9A). Even at the low ratio of 0.1:1 H:D, the region 1 band representing +0.15 SD was observed. This ratio equates to an
average of three nucleosomes on a circular DNA (6055 bp), which is
capable of holding 30 nucleosomes. This number of nucleosomes is not
sufficient to cause an overall structural change in DNA of this size as
was observed for the polyamines. It is also not likely that
nucleosome-nucleosome interactions between plasmids or within plasmids
can occur with this number of nucleosomes. It should be noted that the
protein-induced aggregation that was observed in the higher H:D ratios
of the nonnucleosomal DNA-histone complexes (Fig. 8B),
produced very minimal positive stress. It is therefore very likely that
the rotational restriction represents a property of the nucleosome
itself. We surmise that the bending of DNA into the left-handed coil is
a critical factor in this rotational restriction. Histones H3-H4 in the
absence of H2A-H2B were able to form left-handed coils in DNA (Fig.
9B, lower panel) and were very
effective in the maintenance of induced topological stress as seen by
the presence of very high levels of positive stress (Fig.
9B, upper panel). Histones H2A-H2B are unable to form left-handed coils independent of H3-H4 and were unable
to maintain induced topological stress (Fig. 9C). They were
able to substitute for an equimolar amount of H3-H4 as part of the
nucleosome (Fig. 8C), which again emphasizes the importance of the left-handed supercoil. It is unknown in these conditions whether
histone displacement occurs during transcription. We have observed that
when transcription was done in the presence of MSB topoisomerase I, a
displacement frequency of 1 in 4 nucleosomes was observed (77). Whether
this same frequency is maintained in the presence of a high level of
positive stress remains to be determined.
We have observed that the rates at which induced stresses diffuse
around the circular plasmid define the level of positive stress.
Reducing the transcription rate from 83 to 10 bases/s at 37 °C
resulted in a substantial reduction in positive stress (Fig. 11).
Insufficient transcription-induced stress was generated relative to the
rate of translational diffusion. Reducing the temperature to 13 °C,
which has a transcription rate of 8.2 bases/s, substantially decreased
this translational diffusion so that very high levels of positive
stress were now formed (Fig. 12). An implication from these
observations is that independent of whatever temperature an organism
lives at, the interrelationship between transcription rate and
translational diffusion ensures that these transcription-induced stresses are actively present to facilitate changes in the helical state of the DNA.
We thank Drs. M. Gartenberg, J. C. Wang,
W. Studier, J. Dunn, A. Kikuchi, P. Yau, and M. Bradbury for the gift
of plasmids that were used in these studies.
*
This work was supported by National Science Foundation Grant
MCB-94056718.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
H. F. Peng and V. Jackson, unpublished observations.
The abbreviations used are:
bp, base pair(s);
BE-4-4-4-4, 1,19-bis(ethylamino)-5,10,15-tirazanonadecane;
SD, superhelical density;
NAP1, nuclear assembly protein 1;
DTT, dithiothreitol;
H:D, histone:DNA ratio;
BSA, bovine serum
albumin.
In Vitro Studies on the Maintenance of
Transcription-induced Stress by Histones and Polyamines*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Histone Hl was purified by
a 0.6 M NaCl extraction of calf thymus chromatin that had
been immobilized on hydroxylapatite. This extract was then treated with
5% perchloric acid in which H1 is preferentially soluble. The H1 was
then extensively dialyzed against 10 mM Tris, pH 8.0, and
stored at 70 °C. Topoisomerase I frequently contaminates H1
preparations when eluted from chromatin by NaCl. It is important to
remove this activity when characterizing topological states. The
perchloric acid extraction destroys this activity.
0.05 superhelical density (SD) to 0.25
SD at 37 °C for 1 min.
-32P]GTP (NEN Life Science Products). The UTP was
excluded in the initial mix and was added later to initiate extended
transcription. The mixture was incubated for 3 min at 37 °C in the
presence of polymerase and topoisomerase. During this time, the
promoters are saturated with the polymerase and transcription proceeds
13 bases before a UTP is required. Incubation was then continued for an
additional 10 min in which the conditions were varied by including
polyamines, poly-amino acids, protamine, or histones at the
concentrations indicated. Synchronized transcription was then extended
by adding UTP, and at specific time points the reaction was terminated
by the addition of an equal volume of 0.4% SDS, 20% glycerol, 50 mM Tris, 25 mM EDTA, pH 8.0, and 0.25%
bromphenol blue. Samples were directly electrophoresed for RNA
analysis, or aliquots were taken for analysis of 32P
incorporation by trichloroacetic acid precipitation. The remainder was
treated with 20 µg/ml RNase A for 30 min followed by 200 µg/ml proteinase K for 5 h at 37 °C. The DNA was electrophoresed on 1.0% agarose gels using buffer conditions as described previously (45). DNA was visualized by staining with ethidium bromide, exposed to
uv, restained a second time, extensively destained in water,
photographed, and imaged on XAR-5 film for densitometric analysis. As
reported previously (46), highly positively coiled DNA intercalates
ethidium bromide very poorly. For accurate quantitation, the gel must
be stained and exposed to uv for 5 min in order to nick the DNA. The
gel is then restained a second time followed by extensive destaining in water.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of the positive stress in DNA after
transcription. A, the 6055-bp DNA in its negatively
coiled state (S) and the same DNA that has been changed to a
superhelical density of +0.011 SD (R). This R DNA was used
for the template in the subsequent transcription studies. The
negatively coiled DNA served as a marker for superhelical density. It
has a density of 0.05 SD and has the same relative mobility for a DNA
that is made positively coiled at +0.05 SD. B, the template
DNA after transcription in 10 mM Hepes, 6 mM
MgCl2, 10 mM DTT, pH 7.9, with 400 units/µg
DNA of T7 RNA polymerase and in the presence of 60 units/µg DNA of
E. coli topoisomerase I at 37 °C. The +RNase
lane is an incubation in which transcription was done in the
presence of 10 ng/µg DNA of RNase A for 10 min. C, a
second dimensional electrophoretic analysis of the 10-min time point of
B. The second dimension was done in 15 µM
chloroquin (46). D, an agarose gel showing the size of RNA
produced during transcription for the time points of B. The
RNA size markers (M) are 4241 (a), 2360 (b), and 580 bases (c). E, the
template DNA after transcription in the presence of 1600 units/µg DNA
of T7 RNA polymerase. All other conditions are the same as in
B. F, the template DNA after transcription in 100 mM NaCl, 40 mM Hepes, 6 mM
MgCl2, 10 mM DTT, and with 1600 units/µg DNA
of T7 RNA polymerase and 60 units/µg DNA of E. coli
topoisomerase I. The transcription rate in this buffer condition is 83 bases/s (see Fig. 11A). On the right
side of F are indicated regions of positive
superhelical density: region (reg.) 3 (+0.05), region 2 (+0.10), and region 1 (+0.15). These values are average numbers and are
based on comparison with the negatively coiled (S) marker
and analysis on CsCl-ethidium bromide gradients as described previously
(46). The left side of A indicates the
number of positive coils in the R DNA (average +6.5). N
marks the location for nicked DNA.
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Fig. 2.
Graphical analysis of levels of transcription
and positive stress that were produced in variable conditions of ionic
strength and polyamines. A, NaCl concentrations were
varied from 0 to 140 mM in a buffer background of 10 mM Hepes, 6 mM MgCl2, 10 mM DTT, pH 7.9. Results are shown in terms of the
percentage of production of RNA ( 
) and percentage of production of
positive stress (
). B, transcription in the presence of
spermidine (0-10 mM) or spermine (0-2.0 mM).
Shown are the percentage of production in the presence of spermidine of
RNA () and positive stress () and the percentage of production in
the presence of spermine of RNA (
) and positive stress (
). The
left side of the y axis indicates the
percentage of production of RNA. It was determined by measuring the
quantity of RNA that was produced (32P label) in that
condition relative to what was observed with transcription in 100 mM NaCl (100%). The right side of
the y axis is the percentage of positive stress. It was
calculated based on a quantitation of gel electrophoretic data similar
to that in Figs. 1 and 3. The quantity of positive stress for each lane
of the gel is equal to the sum of all of the superhelical densities
(topological state of the bands in that lane) after each has been
multiplied by the measured band intensities for the respective band.
These values are then plotted as the percentage of positive stress when
determined relative to the highest level of positive stress that was
observed (100%). This highest level is the 0 mM NaCl
condition shown in A.
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Fig. 3.
Transcription-induced positive stress that
was observed in the presence of polyamines. A,
spermidine (0-10 mM); B, spermidine (0-10
mM) and 25 mg/ml BSA; C, spermine (0-2.0
mM); D, spermine (0-2.0 mM) and 200 µg/ml poly(Asp-Glu); E, BE-4-4-4-4 (0-0.6
mM). Prior to the initiation of transcription with the
addition of UTP, the DNA was preincubated for 5 min with these agents.
After the addition of UTP, transcription was for 5 min; F,
time course of positive stress produced during transcription in 6 mM spermidine. The spermidine was added simultaneous with
the addition of UTP. The buffer condition was 100 mM NaCl,
40 mM Hepes, 6 mM MgCl2, 10 mM DTT, pH 7.9, 1600 units/µg DNA of T7 RNA polymerase,
and 60 units/µg DNA of E. coli topoisomerase I.
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Fig. 4.
Analysis of the aggregation of RNA and DNA
during transcription in the presence of the polyamines.
A, spermidine (0-10 mM); B,
spermidine (0-10 mM) and 25 mg/ml BSA; C,
spermine (0-2.0 mM); D, spermine (0-2.0
mM) and 200 µg/ml poly(Asp-Glu); E, BE-4-4-4-4
(0-0.6 mM). After the initiation of transcription, samples
were centrifuged at 27,000 × g for 5 min to separate
supernatant from pellet. Reactions were terminated by adding a
SDS-containing buffer to both. Total length of transcription during
this processing was 7 min, and the temperature was maintained between
32 and 37 °C during centrifugation. The RNA size markers are 4241 (a), 2360 (b), and 580 bases
(c).
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Fig. 5.
Analysis of the catenation of M13
single-stranded DNA by E. coli
topoisomerase I in the presence of spermidine. A,
rate of formation of catenates in the presence of 6 mM
spermidine, 6 mM MgCl2, 10 mM
Hepes, pH 8.0, and 22 units/µg DNA of E. coli
topoisomerase I; B, catenates that were formed as the NaCl
concentration was increased from 0 to 80 mM. The incubation
was for 5 min at 37 °C in this panel. The left
side of B indicates the size of the DNA.
k, knotted catenates (intramolecular); 2,
3, and m, multiple catenates
(intermolecular).
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Fig. 6.
An analysis of E. coli
topoisomerase I activity when in the presence of spermidine and
in the presence and absence of transcription. The rate of removal
of negative coils from negatively coiled (S) DNA in the
absence (A) and presence (B) of 4 mM
spermidine and 7.5 units/µg DNA of E. coli topoisomerase
I. C, the same as B except that transcription was
initiated simultaneously with the addition of the topoisomerase. T7 RNA
polymerase (1600 units/µg DNA) was present in all three
experiments.
View larger version (15K):
[in a new window]
Fig. 7.
Graphical analysis of the inhibition by
polyamines of E. coli topoisomerase I activity and the
activation of MSB topoisomerase I (eukaryotic) activity.
A, spermidine (0-10 mM); B, spermine
(0-2.0 mM); C, spermine (0-2.0 mM)
and 200 µg/ml poly(Asp-Glu). The percentage of solubility of the DNA
( ), percentage of activity of E. coli topoisomerase I
(), and percentage of activity of MSB topoisomerase I (
) are
shown.
View larger version (18K):
[in a new window]
Fig. 8.
Graphical analysis of levels of transcription
and positive stress that were generated in the presence of poly-amino
acids (A); protamine, H1, or core histone (direct
addition) (B); and H3-H4 core histone (deposited by
NAP1) or H2A-H2B (C). A, percentage of
production in the presence of polylysine of RNA ( ) and positive
stress () and in the presence of polyarginine of RNA () and
positive stress (). B, percentage of production in the
presence of protamine of RNA (
) and positive stress (), in the
presence of histone H1 of RNA () and positive stress (), and in the presence of
core histones (by direct addition) of RNA () and positive stress
(). C, percentage of production in the presence of
histones H3-H4 (deposited by NAP1) of RNA () and positive stress
(), in the presence of core histones (deposited by NAP1) of RNA
() and positive stress (), and in the presence of H2A-H2B
(deposited by direct addition) of RNA () and positive stress ().
The values of 100% for both RNA and positive stress are based on the
values described in the legend to Fig. 2. The buffer condition was 100 mM NaCl, 40 mM Hepes, 6 mM
MgCl2, 10 mM DTT, pH 7.9, which has a
transcription rate of 83 bases/s (see Fig. 11A).
View larger version (132K):
[in a new window]
Fig. 9.
Analysis of the positive stress in DNA that
was transcribed in the presence of core histones (A), histones
H3-H4 (B), and histones H2A-H2B (C). The bottom
panels of A-C indicate the number of negative
coils held by the histones at the different histone:DNA (H:D) ratios.
This information was obtained by incubation in the presence of MSB
topoisomerase I (200 units/µg DNA) for 10 min. This quantity of
topoisomerase I is sufficient to relax naked DNA within 10 s. The
upper panels of A 
C indicate the
level of positive stress in the template DNA after a 10-min incubation
followed by transcription for 5 min in the presence of E. coli topoisomerase I. The buffer condition was 100 mM
NaCl, 40 mM Hepes, 6 mM MgCl2, 10 mM DTT, pH 7.9, and included 1600 units/µg DNA of T7 RNA
polymerase and 60 units/µg DNA of E. coli topoisomerase I. The transcription rate was 83 bases/s (Fig. 11A). Except for
H2A-H2B, deposition of histones was done with NAP1 (see "Experimental
Procedures").
View larger version (98K):
[in a new window]
Fig. 10.
Analysis of the positive stress in DNA that
was transcribed in the presence of spermidine (A and
D), spermine (B and
E), and histones H3-H4 (C and
F) using variable quantities of E. coli
topoisomerase I (A-C) and T7 RNA polymerase
(D-F). For A-C, the quantities of
E. coli topoisomerase I used were 3.8 (a), 7.5 (b), 15 (c), 30 (d), 60 (e); and 120 units/µg DNA (f). The T7 RNA
polymerase content was kept at 1600 units/µg DNA. For
D-F, the quantities of T7 RNA polymerase used were 100 (a), 200 (b), 400 (c), 800 (d); 1600 (e), and 3200 units/µg DNA
(f). The E. coli topoisomerase I content was kept
at 60 units/µg DNA. Transcription was for 5 min in 2.0 mM
spermidine, 0.8 mM spermine, or H3-H4 at a ratio of 0.5:1
H:D. For G, transcription was done in the presence of MSB
topoisomerase I (200 units/µg DNA) for 2 min. Lanes a-f
used the same quantities of T7 RNA polymerase as were used for
panels D-F. Lane X is a
control showing the DNA in a fully relaxed state. The
numbers on the right of G are the
number of negative coils (number of polymerase molecules) on the
template.
View larger version (109K):
[in a new window]
Fig. 11.
Analysis of the positive stress that was
produced when the transcription rate was reduced at 37 °C.
A, RNA that was synthesized after transcription for 1 min at
pH 7.9 (lanes a-c) or at pH 7.3 (lanes d-f). Lanes a and
d show transcription in the absence of GDP; lanes
b and e show transcription in the presence of 2 mM GDP; lanes c and f show
transcription in the presence of 4 mM GDP. Transcription
rates as determined from these data are indicated in the text. The
molecular weight markers (M) for RNA are as described in the
legend to Fig. 1. B, rate at which positively coiled DNA was
formed when transcription was done at pH 7.9 with 4 mM GDP
(transcription rate of 25 bases/s). C, same as B
except that the pH was changed to pH 7.3 (transcription rate of 10 bases/s). For B and C, the DNA was reconstituted
with H3-H4 (0.5:1, H:D) and transcribed in 1600 units/µg DNA of T7
RNA polymerase and 60 units/µg DNA of E. coli
topoisomerase I.
View larger version (101K):
[in a new window]
Fig. 12.
Analysis of the positive stress that was
produced in DNA when transcription was at 37, 23, 13, and 4 °C.
Transcription is shown at different temperatures of template DNA
reconstituted with histones H3-H4 (0.5:1, H:D) using 1600 units/µg
DNA of T7 RNA polymerase and 60 units/µg DNA of E. coli
topoisomerase I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 414-456-8776;
Fax: 414-456-6510.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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