(Received for publication, September 14, 1994; and in revised form, November 21, 1994)
From the
We have asked whether a DNA helicase can unwind DNA contained within both isolated native chromatin and reconstituted chromatin containing regularly spaced arrays of nucleosome cores on a linear tandem repeat sequence. We find that Escherichia coli recBCD enzyme is capable of unwinding these DNA substrates and displacing the nucleosomes, although both the rate and the processivity of enzymatic unwinding are inhibited (a maximum of 3- and >25-fold, respectively) as the nucleosome density on the template is increased. The observed rate of unwinding is not affected if the histone octamer is chemically cross-linked; thus, dissociation, or splitting, of the histone octamer is not required for unwinding to occur. The unwinding of native chromatin isolated from HeLa cell nuclei occurs both in the absence and in the presence of linker histone H1. These results suggest that as helicases unwind DNA, they facilitate nuclear processes by acting to clear DNA of histones or DNA-binding proteins in general.
In the eukaryotic nucleus, processes such as DNA replication, transcription, and recombination are highly regulated. One factor which affects these processes is the structure of the DNA, which is maintained in a highly condensed state by its assembly into chromatin. The mechanistic interplay between these nuclear processes and chromatin structure is complex and has proven difficult to elucidate. Recent investigations using defined chromatin templates have demonstrated that chromatin structure plays essential roles in the mechanism of transcription activation (reviewed in Refs. 1, 2). The effect of chromatin structure on DNA replication and recombination has not been extensively studied in vitro, although genetic studies in yeast have indicated that alteration of chromatin structure influences the recombinogenicity of DNA sites (see, for example, (3, 4, 5, 6) ).
Direct
measurement of the effect of nucleosomes on DNA replication was
conducted in vitro by Bonne-Andrea et
al.(7) . Using the entire complement of purified T4
bacteriophage replication proteins, the replication of plasmid
DNA-containing nucleosomes was examined. This hybrid system was used
because such a defined, well-characterized system offered a means by
which modest changes in activity could be detected. On a DNA template
which was moderately reconstituted with nucleosomes (3/4.7 kb (
)molecule), the replication fork was able to pass through
the nucleosome structure after a certain amount of pausing, resulting
in an overall slower rate of synthesis compared to that on protein-free
DNA. Interestingly, this reaction absolutely required a DNA helicase,
the T4 dda protein, presumably to assist in the displacement of protein
blockades on the duplex DNA ahead of the replication fork. The data
were interpreted to show that the core particle remained intact
throughout the replication process and that the histone octamer
interacted, at least transiently, with ssDNA. Other studies have
indicated that histone octamers do not bind ssDNA(8) , although
the possibility that the histones formed a suboctameric association
with ssDNA was not excluded. Alternatively, these observations may be
explained by either binding of the histone octamer to secondary
structure within the replicating DNA, intramolecular transfer of the
octamer(9, 10) , or transient displacement of the
octamer followed by diffusion back onto the DNA(11) .
To address how the packaging of DNA into chromatin affects recombination, DNA strand exchange promoted by the Escherichia coli recA protein was examined in vitro using substrates which were reconstituted with either E. coli HU protein or rat liver core histones(12, 13) . The presence of HU protein on the dsDNA recipient molecule did not affect the overall ability of recA protein to form paranemic (non-intertwined) joint molecules, but it did inhibit the formation of topologically linked plectonemic molecules. If HU protein was bound to the ssDNA donor molecule to which recA protein binds initially, neither paranemic nor plectonemic joint molecule formation was affected. On templates containing eukaryotic chromatin structure, homologous pairing occurred on templates reconstituted at histone/DNA weight ratios up to 1.6:1 (nearly twice the in vivo ratio), although DNA strand exchange was prevented. The addition of histone H1 to the chromatin DNA inhibited even the initial homologous pairing of the DNA molecules. These results suggest that another factor may be required in vivo to disrupt this type of nucleoprotein structure so that the exchange of homologous DNA strands can occur.
One candidate for such a factor is a DNA helicase. Both prokaryotic and eukaryotic cells contain many DNA helicases, which possess different substrate specificities and which function in various biological processes. In principle, any DNA helicase must contend with proteins which organize the chromosome as well as with other DNA-binding proteins. We have utilized a heterologous system assembled from purified components to characterize how DNA helicase activity is affected by the presence of nucleosomes.
In E. coli, the primary helicase involved in homologous recombination is recBCD enzyme, a heterotrimeric, 330-kDa protein (for reviews, see (14, 15, 16) ). We chose to use this enzyme for these studies because its helicase activity is well defined with regard to reaction requirements and enzymatic parameters. Although initially identified as a potent, ATP-dependent nuclease capable of degrading DNA exo- and endonucleolytically(17, 18, 19) , recBCD enzyme is also a highly processive DNA helicase (20, 21) capable of unwinding an average of 30 kb/binding event in vitro(22) . Unwinding occurs at a rate of approximately 1000-1500 bp/s at 37 °C (23, 24) and requires the hydrolysis of 2-3 ATP molecules/bp unwound(25) . Unlike most helicases involved in replication or repair, recBCD enzyme catalyzes DNA unwinding without the requirement for an accessory factor or a specialized substrate, such as a tailed molecule having a defined polarity. Thus, this enzyme is an ideal candidate for the study of how helicase activity is influenced by the presence of chromatin structure.
We assessed the DNA unwinding activity of recBCD enzyme on two types of linear chromatin template. The first was a completely defined array of nucleosome cores assembled onto a template containing tandem repeats of the 207-bp nucleosome positioning sequence derived from the 5 S rRNA gene of Lytechinus variegatus(26) . The reconstitution of regularly spaced arrays of nucleosome cores onto this sequence from donor histone octamers has been thoroughly characterized(26, 27, 28, 29) . The second chromatin template was isolated from HeLa cell nuclei. The ability of recBCD enzyme to unwind both of these substrates was primarily monitored using a fluorometric assay, and the results were confirmed by analyzing the products of an unwinding reaction on neutral sucrose gradients. The experiments described below demonstrate that recBCD enzyme can generate ssDNA products from both types of chromatin template via displacement of histone proteins from the DNA substrate. They also suggest a general role of helicases in displacing DNA-binding proteins.
Histone octamers were prepared from HeLa cell nuclei as described(35) . Cross-linking of the histone octamers with dimethyl suberimidate was performed as described(36) .
Restriction enzymes and DNA modification enzymes were obtained from Pharmacia, New England Biolabs, and Boehringer Mannheim.
Since reconstitution of the nucleosomal templates involved dialysis, the concentration of the reconstituted DNAs was not known precisely. Any resultant variation, however, is accounted for by the following control reaction. To obtain a value for the total amount of fluorescence quenching possible, an equivalent nominal concentration of the protein-free or chromatin DNA was heat-denatured at 95 °C for 7 min and was immediately quenched in ice water. This DNA was then added to a cuvette containing all of the remaining components of the helicase assay except recBCD enzyme, and the fluorescence change after the addition of the heat-denatured DNA was measured. This value indicates directly the maximal amount of fluorescence quenching expected if the DNA is fully unwound and was independently determined for each DNA sample.
At the
end of the run, the gradients were fractionated into 0.5 ml
aliquots. To portions of each, a 0.1 volume of 2% SDS, 50 mM EDTA was added. The sample was then extracted once with
phenol/chloroform and ethanol-precipitated. The pellet was resuspended
in 0.3 M NaOH and heated at 65 °C for 1 h. 20
SSC (30) was added to a final concentration of 6
, and the
samples were applied to a nylon membrane. The membrane was probed with
5` end-labeled DNA made from the same native chromatin preparation
which had been extracted with phenol/chloroform. Radioactivity was
quantitated using a Fuji BA1000 PhosphorImaging system. Some signal
loss was observed when chromatin DNA was unwound by recBCD enzyme. We
are not certain as to the cause of this, but it is likely that since
the enzyme's translocation rate is reduced in the presence of
bound histone octamers, chromatin DNA is nicked more
frequently(31) , and these smaller ssDNA fragments may be less
efficiently precipitated during the sample preparation.
Figure 1: Model for unwinding of nucleosomal templates by recBCD enzyme. Initially, the linear duplex DNA template is either protein-free (not shown) or contains bound histone octamers (cylinders, A). A fully reconstituted template is shown, but the density of nucleosomes is manipulated experimentally. RecBCD enzyme (square/circle/triangle) binds to the end of a DNA molecule and initiates unwinding when ATP is added. This unwinding disrupts the association of the histone octamers and DNA and facilitates the binding of SSB protein (pentagons) to the newly formed ssDNA (B). Although unwinding cannot reinitiate from an end which has been unwound by greater than 25 nucleotides, both ends of the molecule can be utilized, as illustrated by the binding of a second enzyme molecule to the end which was not unwound initially (C). This process is detected using a fluorometric helicase assay, which measures the quenching of the intrinsic fluorescence of SSB protein when it binds to ssDNA (indicated by the change in shading of the pentagons).
Fig. 2A shows raw fluorescence data obtained using a subsaturating
concentration of recBCD enzyme and in vitro reconstituted
chromatin templates. In these experiments, the histone/DNA weight ratio (n) during the reconstitution procedure ranged from 0
(protein-free DNA) to 1 (highly reconstituted, with essentially every
positioning sequence on all DNA molecules occupied). We interpret the
quenching of SSB protein fluorescence as direct evidence that the
histones are being displaced from the DNA as it is unwound, although
other models are possible (see ``Discussion''). From such
data, the apparent rates of unwinding were calculated as described
under ``Experimental Procedures'' and are plotted in Fig. 2B. As the nucleosome density on the template
increases, the rate of unwinding decreases (). The inhibitory
effect of the presence of nucleosomes at the highest levels of
reconstitution lowers the rate of unwinding to
30% of that
obtained with protein-free DNA (10 ± 2.6 versus 33
± 6.6 nm bp/s). The maximum inhibition of unwinding occurs at
physiologically relevant nucleosome densities (n
0.8-1.0). We conclude that since even highly
reconstituted DNA is acted upon ( Fig. 2and data not shown),
chromatin DNA can be unwound and nucleosomes are displaced by the
helicase activity of recBCD enzyme.
Figure 2:
Unwinding of reconstituted templates as a
function of nucleosome density. The reaction contained 20 mM Tris acetate, pH 7.5, 1 mM Mg(OAc), 0.1
mM dithiothreitol, 20 mM NaCl, 2 µM SSB
protein, 10 µM nucleotide (2.6 nM DNA ends)
linear 207
DNA substrate, and 0.5 nM (0.17 nM functional) recBCD enzyme. This concentration of enzyme
corresponds to <0.1 functional enzyme molecule/DNA end. After
equilibrating the reaction to 25 °C, ATP was added to a final
concentration of 3 mM to allow recBCD enzyme to initiate
unwinding of the DNA. When indicated, 10 µM nucleotide
supercoiled pBR322 DNA was also included. A, shows the raw
data obtained using the fluorometric helicase assay, with the following
C
values for the reconstituted substrate: 0, filled circles; 0.2, open circles; 0.4, filled
triangles; 0.6, open triangles; 0.8, filled
diamonds; 1.0, open diamonds. In B, the initial
rates of unwinding were calculated as described under
``Experimental Procedures.'' Reactions without supercoiled
DNA (
); reactions with supercoiled DNA
(
).
One trivial explanation for the
reduction in helicase activity might be that the displaced histone
octamers were directly affecting the enzyme by binding to it. To test
this hypothesis, reactions were also performed in the presence of an
equimolar amount of supercoiled pBR322 DNA (Fig. 2B,
). This DNA can act as a trap to bind displaced histones but will
not interfere with the unwinding reaction because supercoiled DNA,
lacking an end, is not detectably bound by recBCD
enzyme(23, 39) . As expected, the addition of this DNA
does not affect the rate of unwinding on protein-free DNA (33 ±
6.6 versus 31 ± 7.9 nM bp/s), nor is there an
effect even with highly reconstituted chromatin substrates (Fig. 2B). In addition, supplementation of reactions
containing either protein-free DNA or moderately reconstituted DNA
(C
) with purified histone octamers does not affect the
rate of unwinding (26 versus 24 and 20 versus 18
nM bp/s, respectively; data not shown). Alternatively, the
displaced histones may interfere with the binding of SSB protein to
ssDNA. Direct titrations of SSB protein and free histone octamers with
ssDNA were performed to determine whether histones compete with SSB
protein for ssDNA-binding sites. When SSB protein is bound to ssDNA and
histones are subsequently added, a gradual decrease in the amount of
fluorescence quenching is observed with increasing molar concentrations
of free histone octamers; however, even at a 0.7 molar ratio
(histone/DNA), 85% of the expected fluorescence decrease is obtained
(data not shown). Similarly, the addition of both proteins
simultaneously results in the maximal fluorescence quenching expected
(data not shown). Thus, displaced histones do not inhibit unwinding to
a significant extent by either binding to recBCD enzyme, preventing the
binding of SSB protein, or renaturing the unwound ssDNA.
To confirm that the enzyme was acting catalytically on these substrates, the reactions were repeated using a 4-fold higher, but still subsaturating, concentration of enzyme (2 nM total enzyme; 0.25 functional enzyme molecule/DNA end). As expected, this increase in enzyme concentration results in a proportionate increase (4-fold) in the apparent rate of the reaction at all reconstitution ratios (data not shown).
To obtain the
unwinding processivity, the percentage of the total DNA unwound during
the reaction (i.e. the extent of unwinding) was calculated (Fig. 3A). All of the 207 DNA substrate
(
3.8 kb in length) should be readily unwound unless the
processivity of the enzyme is reduced >15-fold (30 kb/end/1.9
kb/end) by the presence of nucleosomes. This expectation is true for
reconstitution ratios of less than C
but not for higher
reconstitution ratios (Fig. 3A). Thus, the average
distance that recBCD enzyme can unwind is reduced by the presence of
nucleosomes, with higher degrees of reconstitution demonstrating a
greater effect. Even near saturating nucleosome density, however, a
majority (60%) of the total DNA is unwound.
Figure 3:
Processivity of unwinding on reconstituted
DNA templates. In A, the extents of the unwinding reactions (i.e. the percentage of DNA unwound) shown in Fig. 2are plotted. The values of N, the average
distance unwound by the helicase per binding event, were calculated as
described previously (22) and are plotted in B. For
extents which are approximately 100% (), it is impossible to
calculate an accurate processivity value (indicated by the arrows). At the higher reconstitution ratios, however, the
extents of unwinding are consistently less than 100% (
); these
data provide quantitative information regarding the processivity of the
enzyme under these conditions.
To illustrate the
dramatic effect nucleosome structure has on processivity, the values
for the extent of unwinding were converted to values of N (the
average number of base pairs unwound per DNA end; Fig. 3B). Because extent values near 100% are
uninformative for processivity determinations, we cannot estimate the
processivity at low reconstitution ratios (<C). At the
highest reconstitution ratios, when approximately 60% of the DNA is
unwound, N is
1.2 kb/end, which corresponds to a 25-fold
decrease in processivity compared to the value obtained on protein-free
DNA(22) . Additionally, the use of a 4-fold higher enzyme
concentration (which is still subsaturating with respect to the
concentration of DNA ends in the reaction) yields results for the
processivity parameter N which are within the experimental
error of these values (data not shown).
Because the fluorescent
helicase assay measures the average properties of a DNA population, it
cannot distinguish between differences in the ability of subpopulations
of molecules to be unwound. The susceptibility of the template
molecules to unwinding was examined on an agarose gel. If a population
of molecules is resistant to recBCD enzyme helicase activity, it will
migrate in the position of the starting material; otherwise, all of the
substrate band will disappear as the molecules are unwound. Consistent
with the latter proposal, the protein-free DNA substrate is rapidly
converted into a heterogeneous smear of unwound ssDNA fragments (data
not shown; see (31) , for an example using M13 DNA). The
disappearance of the highly reconstituted (C) DNA is less
rapid, but 90 ± 10% of the substrate DNA disappears after 10 min
of incubation. Since a subsaturating concentration of recBCD enzyme
(0.1 functional enzyme molecule/DNA end) was used, this result confirms
that the enzyme is acting catalytically. If a higher concentration of
enzyme is used, all of the DNA substrate is also unwound (data not
shown).
To determine
whether the enzyme was being sequestered, an order of addition
experiment was performed. DNA, either protein-free (C) or
highly reconstituted (C
), was unwound using a
substoichiometric amount of enzyme (<0.1 functional enzyme
molecule/DNA end). After the reaction was complete, a second aliquot of
DNA, either C
or C
, was added. The extent and
rate data are summarized in Table 1. When the DNA in both
aliquots is free of nucleosomes, all of the DNA is unwound (i.e. the extent is
100%), and the rate of unwinding of the second
aliquot is slightly slower than that of the first aliquot due to the
time-dependent loss of helicase activity when recBCD enzyme is
incubated in dilute solution(23) . If C
DNA is
unwound to completion and then C
DNA is added, both DNAs
are unwound to the extent and at the rate observed when either DNA is
unwound alone. These results show that the activity of the enzyme is
not significantly affected as a result of repeated cycles of unwinding.
The situation is strikingly different when a reconstituted chromatin
template (C) is unwound initially, however. If the second
aliquot of DNA is also C
, the rate of unwinding of the
second aliquot of DNA is reduced
5-fold, and the extent of
unwinding is apparently reduced by
60% (although this reduction
may be an overestimate because this reaction does not reach a distinct
end point). A more dramatic effect is observed when the second aliquot
contains protein-free DNA. If all of the recBCD enzyme molecules have
completed unwinding of the first DNA substrate, have been unaffected in
the process, and are free in solution, then the aliquot of C
DNA should be unwound at the rate and to the extent expected if
the enzyme had not been previously exposed to the reconstituted DNA.
Instead, the rate of unwinding is reduced 5-fold, although the reaction
still goes to completion. This result indicates that the concentration
of enzyme available to unwind this DNA is less than that present at the
start of the reaction, suggesting that most (
80%) of the enzyme
molecules have been sequestered onto the DNA containing nucleosomes or
have been otherwise affected with regard to their ability to initiate
unwinding on subsequent molecules (see ``Discussion''). The
enzyme molecules which are free in solution at the time the second DNA
is added are able to act catalytically on that DNA; hence, all of the
C
DNA is unwound.
To examine the role of
histone octamer dissociation on the helicase activity of recBCD enzyme,
nucleosome cores were reconstituted onto linear 207 DNA at
a ratio of 0.8:1 using donor histone octamers which had been
extensively cross-linked with dimethyl suberimidate(36) . If
octamer dissociation is required for recBCD enzyme to gain access to
and separate the DNA strands, unwinding would be inhibited. Using 2
nM recBCD enzyme, the initial rate of unwinding of the
chromatin substrate containing the chemically cross-linked histone
octamers is not significantly different than that which is observed
with the non-cross-linked substrate (40 ± 5 versus 49
± 6 nM bp/s, respectively; data not shown). The extent
of unwinding is also unaffected (49 ± 2 versus 46
± 2%, respectively; data not shown). This result demonstrates
that dissociation of the histone octamer is not necessary for helicase
activity on nucleosomal templates.
Using the fluorometric assay, we found that
recBCD enzyme was able to unwind the native chromatin substrate (Table 2). The rate of unwinding of the phenol-extracted native
DNA is lower than that observed with the protein-free 207 template. This result suggests that the DNA pool is intrinsically
less suitable for unwinding by recBCD enzyme, perhaps due to fraying or
degradation of the DNA ends or to internal nicking by micrococcal
nuclease during the preparation and isolation of this DNA. The rate of
unwinding of the H1-depleted native chromatin DNA at 0 mM NaCl
is also less than that obtained with the reconstituted chromatin (at 20
mM NaCl); nevertheless, this chromatin, which should be
roughly equivalent in nucleosome density to the highly reconstituted
templates used previously, is unwound at a rate which is 50% that of
the protein-free DNA (5.9 ± 0.3 versus 10.8 ±
0.9 nM bp/s, respectively), demonstrating good agreement
between the two types of substrates. In addition, the extent of
unwinding of the H1-depleted chromatin is similar to the previous
results using reconstituted substrates (Table 2). For the native
chromatin preparation, the protein-free DNA is unwound to an extent of
only 75%, rather than 100%. Assuming that this value represents the
maximum amount of unwinding achievable with this DNA, the observed
extent of 42% for the H1-depleted chromatin corresponds to
60% of
the maximum (the ``corrected'' extent), in agreement with the
highly reconstituted chromatin substrates. One unexpected finding was
that H1-containing chromatin could be unwound, albeit at a lesser rate
than H1-depleted chromatin (Table 2).
These reactions were
conducted at varying NaCl concentrations to assess the effect of
chromatin condensation on the rate of unwinding. At low salt
concentration, the DNA will be at its most extended state, whereas
increasing salt concentrations will cause the nucleosomes to fold into
higher order structures. When the salt concentration was increased to
100 mM, the rate of unwinding of the chromatin DNAs increased (Table 2). This result is not entirely unexpected since the rate
of recBCD enzyme helicase activity on protein-free DNA increases 2-fold
from 4 to 80-100 mM NaCl (Table 2(23, 24) ). At even higher salt
concentrations (200 mM NaCl), the rate of unwinding decreases
to a value 30% of that at 100 mM NaCl. Because the effect
of salt in these reactions appears to reflect the salt sensitivity of
unwinding by recBCD enzyme on protein-free DNA, there appears to be
little effect of chromatin condensation on unwinding.
Figure 4:
Characterization of the DNA products of a
native chromatin unwinding reaction by sucrose gradient fractionation.
An unwinding reaction containing protein-free DNA (A) or
H1-containing chromatin (B) was conducted as described under
``Experimental Procedures.'' At a time sufficient for
complete unwinding, the reaction was stopped, and a portion of each
reaction was centrifuged through neutral sucrose gradients. The
gradients were fractionated and the DNA content was identified as
described under ``Experimental Procedures.'' RecBCD enzyme
omitted (); recBCD enzyme included
(
]).
Reconstituted chromatin DNA containing arrays of regularly spaced, positioned nucleosomes can be unwound by recBCD enzyme, a well-characterized helicase. The rate of unwinding is reduced when nucleosomes are present (Fig. 2), with a maximum inhibition of approximately 60-70% when the template is reconstituted at a histone/DNA weight ratio of 1:1, a ratio which produces templates which are completely bound by histone octamers(27, 29, 42, 43) . Experiments using native chromatin isolated from HeLa cell nuclei (Table 2; Fig. 4) demonstrate that this physiological template is also capable of being unwound. While the results with the H1-depleted DNA are quantitatively similar to those obtained with highly reconstituted chromatin templates, chromatin substrates containing the linker histone H1 are unwound to a lesser, although measurable, extent.
There are two possible fates for the displaced
histone octamers. The dissociated octamers may remain free in solution
in equilibrium with H3/H4 tetramers and H2A/H2B dimers. Alternatively,
there may be a nonspecific association of various histone assemblies
with the unwound ssDNA. Since we observe 60% quenching at the
highest reconstitution ratios, it could be postulated that all of the
DNA is unwound and that the histones associate with one strand, while
SSB binds only to the other strand. Direct competition experiments
between SSB protein and free histone octamers, however, yield 85% of
the expected quenching of SSB protein fluorescence, indicating that no
more than 15% of the ssDNA produced by unwinding highly reconstituted
chromatin (C
= 0.7) is bound by (or renatured by)
histones (data not shown). Silver staining of polyacrylamide gels of
sucrose gradient fractions of native chromatin unwinding reactions
confirms that, in the presence of recBCD enzyme, free histones are
present at the top of the gradient, as expected if they are displaced
from the DNA during unwinding. (
)In addition, in vivo experiments detect at least partial dissociation of nucleosomes
during transcription and replication(44) ; it is likely that a
similar process occurs during unwinding by a helicase. Our results do
not necessarily conflict with previous studies which indicated that
nucleosomes transfer directly from the region ahead of a transcribing
RNA polymerase molecule to the DNA behind the enzyme (10) because in the helicase assay, renaturation of the DNA
behind the enzyme is precluded by the binding of SSB protein.
Consequently, no duplex DNA acceptor is available for direct transfer
of the histone octamer. In vivo, however, replication and
recombination processes utilize SSB proteins to maintain the transient
single-stranded character of the unwound DNA; thus, our system models
those physiological processes.
More significant than the reduction
in the rate of unwinding is the decrease in the processivity of
helicase activity (Fig. 3). On templates with a moderate to high
density of nucleosomes, the processivity of recBCD enzyme is reduced to
1.2 kb/end, a value 25-fold less than that observed on protein-free
DNA (30 kb/end(22) ). Although it might be argued that the
observed unwinding occurs only on DNA molecules which are
nucleosome-free, this explanation for the reduction in extent is
unlikely for two reasons. First, under the conditions used to assemble
the chromatin DNA, at a weight ratio of 1:1, the DNA is fully
reconstituted with positioned
nucleosomes(27, 29, 42, 43) .
Second, nucleosome cores do not assemble cooperatively onto these
repeat sequences(43) . Thus, at even low extents of
reconstitution, the majority of nucleosome cores should be randomly
distributed, and not clustered, on the DNA substrate. One explanation
which might be proposed to account for this observation is that the
enzyme is incapable of displacing histone octamers, and instead pushes
them along the DNA until there is no linker DNA separating neighboring
nucleosomes. Assuming this model to be valid, only
1100 bp (18
positioning sequences
60 bp of linker DNA/sequence) could be
unwound on a fully reconstituted molecule. We detect twice as much
unwound DNA, however, suggesting that the histones are instead
displaced.
Our finding that unwinding of chromatin DNA does not require dissociation of the histone octamer is consistent with previous data that transcription by T7 RNA polymerase is not affected by extensive cross-linking of the histone octamer(36) . Thus, models which propose transient association of a tetramer with ssDNA during transcription (see, for example Ref.41), and presumably during other processes which denature DNA, do not need to be invoked.
While
recBCD enzyme is less efficient at unwinding nucleosomal DNA, the
precise mechanism by which the rate and processivity of the
enzyme's helicase activity are reduced has not been determined.
It was possible that the observed unwinding rate on the nucleosomal
templates is a composite of two rates: one from the linker DNA between
core particles which is similar to that measured on protein-free DNA,
and a slower rate of unwinding from the DNA within the core particle.
We attempted to confirm this model by separating the products of an
unwinding reaction on either agarose or polyacrylamide gels. Time
points of an unwinding reaction were treated with S nuclease to degrade the unwound ssDNA tails (under the acidic
conditions of the digestion, SSB protein dissociates from the ssDNA and
does not interfere with quantitation)(22) . If unwinding of the
linker DNA is fast relative to that within the core particle and
remained synchronous(40) , a ladder of bands with a spacing of
207 bp should be observed. No discrete bands were detected;
instead, a disperse population of partially unwound DNA molecules was
observed (data not shown), arguing against such a scheme.
Based upon
the reduction in rate of unwinding observed on an aliquot of DNA added
after the enzyme has been exposed to chromatin DNA (Table 1), it
appears that some proportion of the enzyme is effectively inactivated.
Because inhibition of unwinding is not observed when free histone
octamers are added to a reaction, it is unlikely that the displaced
histones directly interfere with the helicase activity of the enzyme. A
more reasonable explanation for this behavior is that some fraction of
the enzyme population becomes either sequestered (in a paused but
otherwise active form) or inactivated on the nucleosomal template, or
is unable to reinitiate unwinding on a subsequent DNA molecule.
Although we have no data to suggest why enzyme dissociation or
reinitiation appears to be impaired, recent studies concerning the
effect of the recombination hotspot on the activities of recBCD
enzyme may provide insight into this behavior(45) . When recBCD
enzyme encounters a
site (5`-GCTGGTGG-3`), its dsDNA exonuclease
activity is attenuated, although it is still able to unwind
DNA(40, 46) . If the reaction contains ATP
concentration in excess of the Mg
concentration, the
altered enzyme is unable to reinitiate unwinding on a subsequent DNA
molecule(45) . Because this phenomenon is also observed with in vitro reconstituted recBC enzyme lacking the recD subunit,
it has been proposed that the productive interaction of recBCD enzyme
with
results in functional inactivation or loss of the recD
subunit. This modified enzyme is capable of unwinding the DNA molecule
with which it is associated but cannot reinitiate unwinding on other
DNA molecules(45) . All of the experiments reported herein
utilized conditions (3 mM ATP, 1 mM Mg(OAc)
) which would maintain the inactivated state if
it were to form. Although the 207
DNA fragment lacks a
sequence, it is possible that the presence of nucleosomes
infrequently results in a similar inactivation or dissociation of the
recD subunit, thus producing enzyme which is unable to reinitiate
unwinding.
Despite the heterologous nature of this system,
inferences can be made concerning the in vivo applicability of
such studies. Within the E. coli cell, the chromosomal DNA is
assembled into higher order structure by small, basic proteins such as
HU and IHF. In addition, sequence-specific DNA-binding proteins such as
repressors and other regulatory proteins will be bound to the DNA.
Although this problem is typically ignored when in vitro studies are performed, it is clear that any global process such as
replication, recombination, or repair must deal with these types of
nucleoprotein structures. From these results, we propose that one
method by which this is accomplished is through the action of a DNA
helicase. In E. coli, helicases play critical roles in each of
these processes, and perhaps one reason for this is that such an
activity is required to clear the DNA of other proteinDNA
complexes. The in vitro biochemistry of these processes as
derived from eukaryotic organisms is not as developed, but it is not
unreasonable to propose a similar, and perhaps even more critical, role
for DNA helicases in these organisms, which contain the additional
barrier of highly condensed chromatin.
In some respects, these studies parallel what has been observed in studies of transcription on chromatin templates. Although it was previously believed that once transcription was initiated, subsequent elongation of the transcript was unaffected by the presence of nucleosomes on the gene being transcribed, recent studies indicate an effect on both initiation and elongation by T7 RNA polymerase and RNA polymerase II(35, 47, 48) . Likewise, in these unwinding studies, an effect on both the rate of unwinding and its elongation, or processivity, is observed. Unlike results observed with transcription studies, however, we detect unwinding in the presence of condensed chromatin and histone H1. The presence of H1 linker histone has been shown to significantly inhibit transcription, replication, and recombination reactions on chromatin DNA in vitro (see, for example, Refs. 13, 49-51). This finding therefore indicates perhaps one means by which this barrier is overcome in vivo through the action of a DNA helicase. These results may also explain the difference between in vivo and in vitro measurements of the processivity of recBCD enzyme(22, 52, 53) . Roman et al.(22) proposed that this discrepancy (i.e. the lower apparent processivity in vivo) might be due to the presence of DNA binding proteins on the physiological substrate which would inhibit translocation of the helicase.