From the Institute of Biochemistry and Biophysics,
Polish Academy of Sciences and ¶ Warsaw University, Laboratory
of Plant Molecular Biology, Pawinskiego 5A, 02-106 Warsaw, Poland
Received for publication, September 10, 2002, and in revised form, October 2, 2002
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ABSTRACT |
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Deficient in DNA Methylation 1 (DDM1) protein is
required to maintain the DNA methylation status of Arabidopsis
thaliana. DDM1 is a member of the broad SWI2/SNF2 protein family.
Because of its phylogenetic position, DDM1 has been speculated to act as a chromatin-remodeling factor. Here we used a purified recombinant DDM1 protein to investigate whether it can remodel chromatin in vitro. We show that DDM1 is an ATPase stimulated by both naked and nucleosomal DNA. DDM1 binds to the nucleosome and promotes chromatin remodeling in an ATP-dependent manner.
Specifically, it induces nucleosome repositioning on a short DNA
fragment. The enzymatic activity of DDM1 is not affected by DNA
methylation. The relevance of these findings to the in vivo
role of DDM1 is discussed.
The compaction of eukaryotic genomes into chromatin structures has
profound implications for nuclear processes such as replication (1, 2),
transcription (3, 4), DNA repair (5), and recombination (6, 7).
Evolution has created a diverse repertoire of regulatory mechanisms
affecting the dynamics of chromatin structure. One broad group of
mechanisms involves a battery of the enzymes that covalently modify
histones and DNA (8-10). Specific regulatory factors recognize the
modification status of chromatin fibers and render the structure active
or inactive (8). To ensure sufficient structural flexibility of the
chromatin, the cell employs a specialized class of multiprotein
complexes, which utilize the energy of ATP hydrolysis to change
chromatin folding in the poorly understood process of "chromatin
remodeling" (11, 12). Central to this activity are SWI2/SNF2-type
ATPases that form the catalytic core of these remodeling complexes. The
SWI2/SNF2 protein family falls within the large superfamily of
DEXD/H-ATPases (13). The distinctive signature of the SWI2/SNF2
family is the SNF2_N domain, a variant of the typical DEXD/H domain
that contains a well conserved C-terminal extension of ~100
amino acids.1
Phylogenetic analysis divides the SWI2/SNF2 family into a number of
subfamilies (13), which appear to be present in all major taxa. Outside
the SNF2_N and HelicC domains, which form the catalytic module, these
subfamilies are not well conserved. Members of three closely related
subfamilies (ISWI, Mi2/CHD, and SWI2/SNF2) have been shown to serve as
the catalytic subunits of chromatin-remodeling complexes (15-18).
Recently, remodeling activity has also been demonstrated for a complex
built around the more distantly related INO80 ATPase (19). All
catalytic subunits of the chromatin-remodeling complexes that have been
analyzed can act outside the complex context, although they require
associated proteins to achieve their full activity (16-18, 20).
CSB/ERCC6 defines a fifth group of remodelers, although it has been
analyzed only as the isolated, recombinant protein (21). During recent
years it has become clear that energy-dependent remodelers
act in concert with histone modifying enzymes (22-27).
So far only very few family members have been analyzed in biochemical
assays. At least one of them, MOT1, acts as a transcriptional repressor
that regulates TATA binding protein binding to promoters (28-30). Therefore, it is likely that only a subset of SWI2/SNF2-like proteins serve as true chromatin-remodeling factors.
SWI2/SNF2-like proteins participate in various nuclear activities
including transcriptional control (24, 31), DNA repair (Ref. 13 and
references therein), chromosome segregation (32), and chromosome
folding (32, 33). The members of two SWI2/SNF2-type subfamilies, ATRX
and DDM1, are involved in the control of DNA methylation status (34,
35). In the plant Arabidopsis thaliana, the ddm1
genetic background results in a 70% decrease in the DNA methylation
level (36). In consequence, this leads to deregulation of gene
expression and the reactivation of silent transposons (37-42). DDM1
belongs to a small protein family conserved in plants (A. thaliana DDM1, NCBI accession number AAD28303; Zea mays, NCBI AAL73042), fungi (Saccharomyces cerevisiae
Yfr038wp, NCBI NP_116696; Aspergillus fumigatus, NCBI
CAD28443), and mammals (Mus musculus
lymphocyte-specific helicase, NCBI NP_032260 and Homo
sapiens proliferation-associated SNF2-like gene, NCBI AAF82262).
No genes encoding DDM1-like proteins have been found in the two fully
sequenced invertebrate genomes. Evolutionary analysis places the DDM1
subfamily close to ISWI and SNF2/SWI2 groups
(43).2 It is presently
unclear how DDM1 acts to maintain DNA methylation status, although it
has been speculated that it could remodel chromatin structure to
facilitate the access of DNA methylases to the substrate (35). However,
the remodeling activity of DDM1 has not been analyzed, and this
hypothesis is based solely on the phylogenetic position of the DDM1 subfamily.
To address the above issue we have expressed recombinant DDM1 and
characterized its biochemical activities. We find that DDM1 is an
ATPase stimulated by both naked and nucleosomal DNA. It binds to
nucleosomes and promotes nucleosome repositioning in an
ATP-dependent manner. Our data indicate that DDM1 defines a novel class of chromatin-remodeling factors. Remarkably, considering its apparent function in the nucleus, we find that DDM1 activity is not
affected by DNA methylation status.
Production of Recombinant DDM1 Protein--
The DDM1 cDNA
was amplified by PCR and cloned into the pFAST BAC 1 vector
(Invitrogen) cleaved with BamHI and SalI sites. The 3' primer contained a DNA sequence encoding a hexahistidine tag.
The PCR product encoded a DDM1 fusion protein carrying a C-terminal His
tag. The sequence of this fragment was verified by DNA sequencing.
After transfection and amplification, the recombinant virus was used to
infect SF21 insect cells. Three days after infection, the cells were
collected, washed with phosphate-buffered saline, and resuspended in
extraction buffer (20 mM HEPES-KOH, pH 7.8, 300 mM KCl, 5 mM imidazole,10% glycerol, 0.1%
Nonidet P-40). After homogenization cellular debris were removed by
centrifugation, and the extract was applied to a nickel-agarose column.
The resin was washed extensively with homogenization buffer containing
25 mM imidazole. Bound proteins were then eluted with the
buffer containing 500 mM imidazole. Further purification
and imidazole removal were achieved by gel filtration chromatography.
Purity was estimated to be about 90% by SDS-PAGE. The protein identity was confirmed by mass fingerprinting using a QTof (Micromass) spectrometer. The preparation was tested for contaminating nuclease activity. To exclude any effect of contaminants, a "mock"
preparation was made using uninfected cells. This preparation was
tested in all experiments and did not show any activity in either the
enzymatic or band-shift assays.
DNA Fragments and Chromatin Reconstitution--
For the initial
ATPase assays, pBR322 plasmid DNA was used. Chromatin was reconstituted
on the pBR322 DNA by the salt dialysis method using purified chicken
erythrocyte histones (44). Assembly quality was verified by micrococcal
nuclease digestion. For nucleosome mobility assay, mononucleosomes were
assembled on a 248-bp fragment of mouse rDNA (45). The DNA was labeled
with 32P by PCR. After gel purification, the PCR products
were converted into nucleosomes by the addition of core histones and
salt dialysis. Different ratios of DNA to histones were used, and
reconstitution products were analyzed by electromobility gel-shift
assay to reveal the optimal conditions for nucleosome assembly. For
nucleosome mobility assays, nucleosomes positioned on specific DNA
sequences were resolved by electrophoresis on a 4.5% polyacrylamide
gel and purified (45). For assays involving DNA methylation, the 210-bp
ClaI fragment of the A. thaliana FWA promoter was
used (positions 1287-1496 of NCBI accession number AF178688). DNA was
methylated with SssI DNA methylase (New England
Biolabs), and methylation was confirmed by digestion with
Fnu4HI. Labeling of DNA and the optimization of nucleosome
assembly conditions were performed as described above.
Band-shift Assays--
Free or nucleosomal DNA (15 fmol) was
incubated with purified DDM1 (1-50 fmol) in buffer consisting of 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1.5 mM MgCl2, 1 mM ATPase Assay--
The reactions were performed in buffer
consisting of 20 mM Tris-HCl, pH 8.0, 50 mM
NaCl, 1.5 mM MgCl2, 1 mM
Nucleosome Mobility Assay--
The nucleosome mobility assay was
performed essentially as described by Laengst et al.
(45). Gel-purified nucleosomes (60 fmol) were incubated with
recombinant DDM1 (1-50 fmol) in the ATPase buffer containing 1 mM ATP for 1 h at 25 °C in a final volume of 10 µl. The reaction was stopped by the addition of 500 ng of competing
plasmid DNA and further incubation for 10 min. Nucleosomes were then
separated by native gel electrophoresis on a 4.5% polyacrylamide gel
in 1× Tris acetate EDTA buffer. Gel-resolved bands were
visualized with the PhosphorImager.
DDM1 Interacts with Nucleosomal DNA--
Sequence analysis of
proteins of the DDM1 family did not reveal any known domain that might
be implicated in DNA or nucleosome recognition. Therefore, it was
necessary to determine whether DDM1 itself can bind to its potential
substrates, naked DNA, or nucleosomes. To this end purified,
recombinant DDM1 was used in band-shift assays with short fragments of
DNA and with in vitro assembled mononucleosomes.
To produce sufficient quantities of DDM1 protein, a recombinant
baculovirus expressing A. thaliana DDM1 was constructed and used to infect SF21 cells. The expressed DDM1 protein was isolated and
found to be ~90% pure (Fig.
1A). A 248-bp fragment of
mouse rDNA was amplified by PCR and used as free DNA or in a form of mononucleosomes assembled with purified chicken core histones. A
band-shift assay with DDM1 and free DNA demonstrated the formation of
nonspecific DNA-protein complexes (Fig. 1B). Increasing the protein concentration resulted in a smearing pattern, indicating the binding at the multiple sites. In contrast, the interaction of DDM1 with the mononucleosomes resulted in the formation of one distinct shifted band (Fig. 1C), indicating the
production of one bound species. Despite these differences in
the nature of the complexes formed, band-shift assay showed that DDM1
binds to both targets with similar affinity.
ATPase Activity of DDM1 Is Stimulated by Both Free and Nucleosomal
DNA--
Although SWI2/SNF2-type chromatin-remodeling ATPases occur
in vivo in multi-protein complexes, all of the enzymes
analyzed thus far display ATPase activity in the absence of other
subunits. To examine the activity of purified DDM1 protein, the ATPase
assays were performed in the presence of core histones, plasmid DNA, or
plasmid-assembled chromatin (Fig.
2A). Buffer was used as
reference. To exclude any effect of potential contaminants, the mock
preparation from uninfected cells was assayed in parallel reactions. To
optimize the assay, DDM1 activity was analyzed under a broad spectrum
of different conditions. The optimal reaction conditions were a buffer of pH 8.0 containing 50 mM NaCl, 100 µg/ml bovine serum
albumin, 1 mM
To understand how DNA and nucleosomes stimulate the ATPase of DDM1, a
kinetic analysis of the ATP hydrolysis reaction was performed. A time
course of the ATP hydrolysis showed that the reaction is linear for 90 min (data not shown). To ensure linearity of the reaction, the assays
were stopped after 30 min. The velocities of the ATP hydrolysis by
recombinant DDM1 in the absence or presence of DNA or nucleosomes were
determined by titrating with ATP (Fig. 2B). The maximal
velocity (Vmax) and the Michaelis-Menten
constant (Km) were then determined by curve fitting
and double-reciprocal plot (Fig. 2C). Both
Vmax and Km of ATP hydrolysis
by recombinant DDM1 were higher than the corresponding values
determined for the isolated Mi2 DDM1 Is a Chromatin-remodeling Factor--
The interaction
with mononucleosomes and the stimulation of ATPase activity
by nucleosomal DNA suggested that DDM1 could be a chromatin-remodeling
factor. To address this issue directly a well established sliding
assay, which measures the movement of a histone octamer along a short
DNA fragment, was used (45). The histone octamers were deposited on a
248-bp rDNA fragment. Gel electrophoresis of the assembled products
revealed two discrete species (Fig.
3A) that correspond to
nucleosomes positioned at the center or at the end of the DNA fragment
(45). Positioned nucleosomes were gel-purified and incubated with
increasing amounts of DDM1 protein. DDM1 was able to induce, in an
ATP-dependent manner, the redistribution of the histone
octamer (Fig. 3B). DDM1 was found to move nucleosomes from
the end to the center of the DNA fragment with much greater efficiency
than in the opposite direction.
DDM1 ATPase Activity Is Not Stimulated by DNA
Methylation--
DDM1 is involved in the maintenance of CpG DNA
methylation in vivo (36). Therefore, we wished to see
whether DNA methylation would affect the activity of recombinant DDM1
in in vitro assays. To this end DDM1 activity was compared
in the ATPase assay performed with nucleosomes assembled on methylated
and on non-methylated DNA. We decided to use an ATPase assay since it
gives the most quantitative and directly comparable results. A fragment
of A. thaliana FWA gene promoter was used because it
contains a high number of CpG sequences (18 methylcytosines in a
210-bp-long DNA fragment) and has been shown to undergo demethylation
in ddm1 mutant plants (40). The DNA was amplified by PCR and
methylated with SssI methylase. The efficiency of DNA
methylation was checked by cleavage with the restriction enzyme
Fnu4HI, which is sensitive to methylation of its recognition
sequence (Fig. 4A). Methylated and non-methylated FWA fragments were then used in ATPase
assays as both free and nucleosomal DNA. This experiment showed that there was no influence of DNA methylation on the ATPase activity of
DDM1 (Fig. 4B).
The SWI2/SNF2 protein family falls within the DEXD/H superfamily
of ATPases. It is defined by the SNF2_N domain, a variant of
DEXD/H-ATPase domain that contains an additional conserved region of
about 100 amino acids extending C-terminally to the classical DEXD/H
domain.1 Based on phylogenetic analysis the SWI2/SNF2
family can be divided into several subfamilies that share the SNF2_N
and HelicC domains but are not well conserved outside these regions
(13).2 Chromatin-remodeling activity has been demonstrated
for proteins of the closely related SWI2/SNF2, ISWI, and Mi2
subfamilies (15-17, 18). Recently the proteins of two other more
distant subfamilies, INO80 and ERCC6/CSB, have been shown to act in a
similar manner (19, 21). Here we present experimental evidences of
chromatin-remodeling activity by a member of the DDM1 subfamily.
DDM1 Interacts with the Nucleosome--
Our results show that DDM1
binds to both free DNA and nucleosomes with similar affinity (Fig. 1).
However, the binding to the nucleosomal target resulted in a different
pattern of gel-resolved complexes compared with the binding to free
DNA. The diffused pattern formed by free DNA-DDM1 complexes indicated
nonspecific interactions, whereas binding to the nucleosome resulted in
the formation of a single distinct complex. These results indicate that
in contrast to free DNA, the nucleosomal structure has a preferred
binding site for DDM1. We did not observe any stable interaction of
DDM1 with core histones in a pull-down assay (data not shown). These
data suggest that although DNA is a major target for DDM1 binding, it
is the nucleosomal structure that provides a specific structural
framework for complex formation. This phenomenon is reminiscent of
substrate binding by ISWI, another member of SWI2/SNF2
family.3
Enzymatic Activity of DDM1--
Purified recombinant DDM1
has weak intrinsic ATPase activity in vitro. ATP
hydrolysis was greatly stimulated by naked DNA. A slight additional
stimulation by nucleosomal DNA was seen (Fig. 2). The catalytic
subunits of all chromatin-remodeling complexes characterized so far
display ATPase activity when assayed as isolated polypeptides. However,
they differ in their response to DNA and chromatin. SWI2/SNF2-like
proteins are fully active in the presence of DNA, and no additional
stimulation by chromatin is observed (43). In contrast, other
remodelers such as Miz
The determination of the kinetic parameters of ATP hydrolysis in the
absence or presence of DNA or nucleosomes showed that both DNA and
nucleosomes increased primarily the Vmax of the
reaction (2.9 and 3.1 times, respectively) while only marginally
affecting the Km (1.2-fold increase in both cases).
Because Vmax measures the turnover number of an
enzyme, the above kinetic data indicate that both free DNA and
nucleosomes stimulate the ATPase of DDM1 mainly by increasing its
turnover number. Similar dependence was found earlier for
Mi2
Purified DDM1 showed nucleosome-remodeling activity in
vitro. It is able to catalyze the redistribution of histone
octamers on short DNA fragments in a process that requires the energy
provided by ATP hydrolysis. Similar "nucleosome-sliding" activity
has been observed for ISWI (15) and dMi2 (16).
As reported previously (46), ISWI repositioned only nucleosomes located
at the center of a 248-bp rDNA fragment but not those located at the
end. Interestingly, the same group has reported that the ISWI
containing complex ACF and purified dMi2 behave in exactly the opposite
way (20, 16). Most likely, these observations reflect mechanistic
differences between the remodelers. We found that DDM1, like ACF and
dMi2, was considerably more efficient at mobilizing nucleosomes
occupying the fragment termini than located at the center of the 248-bp
rDNA. The mechanistic reasons for this directionality of nucleosome
sliding are not clear.
The nucleosome sliding activity of DDM1 appears to be relatively weak.
To move ~50% of the nucleosomes positioned at the end of
a 248-bp rDNA fragment (~30 fmol) to the center of the DNA molecule,
50 fmol of recombinant DDM1 were required. To achieve comparable
efficiency it is sufficient to use about 7 times less of isolated,
recombinant ISWI or 150 times less of ACF complex (20). However, it is
not clear if different recombinant proteins can be directly compared.
This observation relates DDM1 to other remodelers that require the
context of the native complex for full activity. As demonstrated for
other SWI2/SNF2-type proteins such as hBrm, BRG1 (47), and Mi2
The low efficiency of DDM1 in remodeling assays may also
reflect the intrinsic limitations of our experimental system. To demonstrate the remodeling activity of DDM1, standard
chromatin-remodeling assay has been used. However, it is possible that
this assay, developed for other remodelers, is not perfectly suited for
DDM1. Chromatin-remodeling complexes appear to have evolved to fulfill various nuclear functions and to induce different changes in the structure of chromatin. It is likely that some novel assay system is
required to fully characterize the mechanisms of DDM1-induced chromatin remodeling.
DDM1 Activity and DNA Methylation Status--
DDM1 is involved in
the maintenance of DNA methylation (36). Similarly to ddm1,
mutations in Lsh, a gene encoding a mouse homolog of DDM1,
also cause alterations in the DNA methylation pattern (46). Therefore,
we wished to see whether DNA methylation can affect DDM1 activity. It
has been suggested that DDM1 may alter the chromatin
structure to facilitate methyltransferase access to DNA (35). According
to this scenario, fully CpG-methylated DNA would represent the final
product of the DDM1-assisted reaction. Therefore, it might be
anticipated that methylated DNA would inhibit or at least not activate
DDM1. Alternatively, DDM1 could act to compact the chromatin structure
of methylated DNA to render these regions of the genome inaccessible to
de-methylation and, therefore, help to preserve the methylation
pattern. If this were the case, preferential activation of DDM1 by
methylated DNA might be predicted. Surprisingly, we did not observe any
effect of DNA methylation on the ATPase activity of DDM1 (Fig. 4).
Band-shift assays also failed to show any effect of DNA methylation on
substrate binding affinity (data not shown). One possible explanation
for these findings is that DDM1 requires other associated proteins to
sense the DNA methylation status. Alternatively, DNA methylation may have some subtle qualitative influence on the DDM1-catalyzed reaction that was not detected in the experimental system used.
Recently the CpXpG DNA methylation catalyzed by the chromomethylase 3 was found to depend on the activity of kryptonite (47), the product of
one of 15 genes in Arabidopsis that potentially encode
histone H3 Lys-9 methyltransferases (48). However,
ddm1 mutations also affect CpG DNA methylation (37), which
is catalyzed by another DNA methylase MET1 (49, 50). It has been
demonstrated that in Neurospora crassa the CpG DNA
methylation acts genetically downstream of dim5 (51), which
encodes a homolog of Drosophila melanogaster histone H3
Lys-9 methyltransferase Su(var)3-9. Interestingly, Gendrel et
al. (52) recently demonstrated the alteration of the chromosomal
distribution of Lys-9-methylated histone H3 in ddm1 mutants.
Therefore, we speculate that DDM1 could be involved in histone H3 Lys-9
methylation. According to this scenario the effect on DNA methylation,
which is seen in ddm1 plants, would be indirect. We are
actively investigating this possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol,
0.05% Nonidet P-40, 100 µg/ml bovine serum albumin, and 10%
glycerol at 15 °C for 20 min. The reaction products were then
separated by electrophoresis on a 1.3% agarose gel and visualized with PhosphorImager.
-mercaptoethanol, and 100 µg/ml bovine serum albumin. Ten fmol of
recombinant DDM1 were incubated with 100 ng of free or nucleosomal DNA
in the presence of 66 µM ATP and a trace amount of
[
-32P]ATP. After a 60-min incubation at 25 °C,
1-µl samples were spotted onto polyethyleneimine-cellulose plates,
and the reaction products were separated from nonhydrolyzed ATP by thin
layer chromatography. For kinetic analysis, the samples were incubated
with ATP at concentrations ranging from 10 to 500 µM for
30 min. Spots were quantified with a Storm PhosphorImager using
ImageQuant software (Molecular Dynamics).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Recombinant DDM1 binds to free DNA and
nucleosomes. A, recombinant DDM1 (400 ng) was resolved
by SDS-PAGE and stained with Coomassie Blue. B, recombinant
DDM1 (1-50 fmol) was incubated with 248 of naked DNA (15 fmol) and
analyzed by electrophoresis on a 1.3% agarose gel. C,
recombinant DDM1 (1-50 fmol) was incubated with 248 mononucleosomes
(15 fmol) and then analyzed by electrophoresis on a 1.3% agarose
gel.
-mercaptoethanol, and 1 mM
MgCl2 and a temperature of 25 °C. In such a reaction
ATPase activity of DDM1 was clearly stimulated by free DNA. Very little
additional stimulation was seen in the presence of an equivalent amount
of DNA assembled into chromatin. The addition of core histones had no
effect on the DDM1 activity. These findings are consistent with the
results of band-shift assays and imply that interaction with both DNA
and nucleosomes stimulates the ATPase activity to similar levels.
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Fig. 2.
Recombinant DDM1 is an ATPase stimulated by
both naked and nucleosomal DNA. A, recombinant DDM1 (10 fmol) was used in an ATPase assay in the absence of any cofactor
(buffer) or in the presence of 100 ng of core histones
(Histones) or 100 ng of naked DNA (DNA) or 100 ng
of nucleosomal arrays (Chromatin). DDM1-containing reactions
are shown as gray bars. A mock protein preparation from
uninfected cells was used as a negative control (black
bars). B, recombinant DDM1 (10 fmol) was used in an
ATPase assay in the absence of any cofactor (squares) or in
the presence of 100 ng of free DNA (triangles) or
nucleosomal arrays (diamonds). The assays were performed
with the increasing concentrations of ATP ranging from 10 to 500 mM. C, double-reciprocal plots for kinetic
analysis. The regression equations are shown in the figure.
-remodeling ATPase (17), although
they were of the same order of magnitude (Table
I). The analysis revealed that DNA and
nucleosomes increased the Vmax 2.9- and
3.1-fold, respectively, and the Km only 1.2-fold.
Because Vmax measures the turnover number, our
data indicate that both cofactors stimulate the ATPase of DDM1 mainly
by increasing its turnover number while changing the affinity of DDM1
to ATP only slightly.
Kinetic parameters of ATP hydrolysis by different chromatin remodeling
factors
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Fig. 3.
Recombinant DDM1 is capable of remodeling
nucleosomes in vitro. A, mononucleosomes
were assembled on a 248-bp rDNA fragment and resolved by native
polyacrylamide gel electrophoresis to reveal two positioned nucleosomal
species. B, upper panel, nucleosomes positioned
at the center of an rDNA fragment (60 fmol) were gel-purified and
incubated with recombinant DDM1 (lane 1, 1 fmol; lane
2, 5 fmol; lane 3, 50 fmol) in the presence of ATP. As
a negative control DDM1 (50 fmol) was used in the absence of ATP
(lane 4, no ATP). Lower panel, 60 fmol of
isolated end-positioned nucleosomes were incubated with recombinant
DDM1 (lane 1, 1 fmol; lane 2, 5 fmol; lane
3, 50 fmol) in the presence or absence (no ATP) of ATP. All
samples were analyzed by electrophoresis in a native polyacrylamide
gel. The positions of free DNA (double line) and two
nucleosomal species (gray ovals) are indicated to the
left of the figure.
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Fig. 4.
DNA methylation status does not influence the
ATPase activity of DDM1. A, in vitro
methylation of the FWA 210 bp fragment with SssI
methylase demonstrated by Fnu4HI digestion and gel
electrophoresis (lane 1, nonmethylated DNA; lane
2, methylated DNA incubated with Fnu4HI; lane
3, nonmethylated DNA incubated with Fnu4HI).
B, recombinant DDM1 (10 fmol) was used in an ATPase assay in
the absence (buffer) or presence of 100 ng of nonmethylated
210-bp FWA naked DNA (DNA, gray
bar) or 100 ng of methylated DNA (Met-DNA, black
bar) or 100 ng of mononucleosomes assembled on nonmethylated
(Chromatin, gray bar) or methylated
(Chromatin, black bar) DNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ISWI require nucleosomal DNA to
achieve their full activity (15-17). This observation suggests that
functionally, DDM1 may be more closely related to the SWI2/SNF2
subfamily than to ISWI or CHD/Mi2.
-remodeling ATPase (17).
(17),
the associated proteins are essential to increase the rate of ATP
hydrolysis. ATPase-associated subunits have also been shown to act as
coupling factors that allow more efficient energy usage (20). However,
we can only speculate on the role of putative DDM1-associated proteins,
as DDM1 has not yet been shown to form a larger complex.
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ACKNOWLEDGEMENTS |
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We are indebted to Gernot Langst and Anton Eberharter for the gift of reagents, technical advice, and sharing of unpublished data. We thank Wim Soppe and Maarten Koornneef for the FWA plasmid clone. We also thank John Gittins for reagents and critical reading of the manuscript. The laboratory is supported by the Center of Excellence in Molecular Biotechnology program.
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FOOTNOTES |
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* This work was supported by Howard Hughes Medical Institute Grant 55000312, Polish Committee for Scientific Research Grants 6PO4A 00320 and PBZ-039/PO4/2001, and Foundation for Polish Science Grant 2/2000.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.
§ To whom correspondence should be addressed. Tel.: 48-22-659-6072; Fax: 48-22-658-4626; E-mail: jbrzeski@ibb.waw.pl.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M209260200
1 See http://pfam.wustl.edu/cgi-bin/getdesc?name=SNF2_N.
2 J. Brzeski, manuscript in preparation.
3 G. Langst, personal communication.
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