From the Interdisciplinary Graduate Program and
Program in Molecular Medicine, University of Massachusetts Medical
School, Worcester, Massachusetts 01605, and
Department of
Molecular Medicine and Institute of Biotechnology, University of Texas
Health Science Center at San Antonio, San Antonio, Texas 78245-3207
Received for publication, November 12, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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In eukaryotic cells, the repair of DNA
double-strand breaks by homologous recombination requires a
RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like
ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support
robust homologous pairing between single-stranded DNA and a chromatin
donor. In contrast, bacterial RecA is incapable of catalyzing
homologous pairing with a chromatin donor. We also show that Rad54p
possesses many of the biochemical properties of bona fide
ATP-dependent chromatin-remodeling enzymes, such as
ySWI/SNF. Rad54p can enhance the accessibility of DNA within
nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a
chromatin-remodeling enzyme that promotes homologous DNA pairing events
within the context of chromatin.
Chromosomal DNA double-strand breaks
(DSBs)1 arise through
exposure of cells to harmful environmental agents such as ionizing radiation or mutagenic chemicals (radiomimetics, alkylating agents, etc.). DSBs can also be caused by endogenously produced oxygen radicals, by errors in DNA replication, or as obligatory intermediates during programmed cellular processes, such as meiosis or V(D)J recombination (1-3). Cell survival and maintenance of genome integrity
depend on efficient repair of DSBs, because unrepaired or misrepaired
DSBs may lead to mutations, gene translocations, gross chromosomal
rearrangements, or cellular lethality.
Several pathways for repairing DSBs have evolved and are highly
conserved throughout eukaryotes. Homologous recombination (HR) is a
major pathway of DSB repair in all eukaryotes and has a distinct
advantage over other mechanisms in that it is mostly error-free. In
organisms ranging from yeast to human, HR is mediated by members of the
RAD52 epistasis group (RAD50, RAD51,
RAD52, RAD54, RAD55, RAD57,
RAD59, MRE11, and XRS2). Accordingly,
mutations in any one of these genes result in sensitivity to ionizing
radiation and other DSB-inducing agents (2). The importance of the HR pathway in maintaining genome integrity is underscored by the fact that
mutations in each one of its critical factors have been correlated with
chromosomal instability-related ailments, including ataxia
telangiectasia-like disease, Nijmegen breakage syndrome, Li Fraumeni
syndrome, as well as various forms of cancer (4).
In vivo and in vitro studies have suggested the
following sequence of molecular events that lead to the recombinational
repair of a DSB. First, the 5' ends of DNA that flank the break are
resected by an exonuclease to create ssDNA tails (5). Next, Rad51p
polymerizes onto these DNA tails to form a nucleoprotein filament that
has the capability to search for a homologous duplex DNA molecule. After DNA homology has been located, the Rad51-ssDNA nucleoprotein filament catalyzes the formation of a heteroduplex DNA joint with the
homolog. The process of DNA homology search and DNA joint molecule
formation is called "homologous DNA pairing and strand exchange."
Subsequent steps entail DNA synthesis to replace the missing
information followed by resolution of DNA intermediates to yield two
intact duplex DNA molecules (6).
The homologous DNA pairing activity of Rad51p is enhanced by Rad54p
(7). Rad54p is a member of the Swi2p/Snf2p protein family (8)
that has DNA-stimulated ATPase activity and physically interacts with
Rad51p (7, 9, 10). Because of its relatedness to the Swi2p/Snf2p
family of ATPases, Rad54p may have chromatin remodeling activities in
addition to its established role in facilitating Rad51p-mediated
homologous pairing reactions. In this study we show that Rad51p and
Rad54p mediate robust D-loop formation with a chromatin
donor, whereas the bacterial recombinase, RecA, can only function with
naked DNA. Furthermore, we find that the ATPase activity of Rad54p is
essential for D-loop formation on chromatin and that Rad54p
can use the free energy from ATP hydrolysis to enhance the
accessibility of nucleosomal DNA. Experiments are also presented to
suggest that chromatin remodeling by Rad54p and yeast SWI/SNF involves
DNA translocation.
DNA--
All DNA manipulations were carried out using standard
methods (11). Oligonucleotides were obtained from Operon Technologies (Alameda, CA). Plasmid pXG540 and T4 EndonucleaseVII used in the cruciform extrusion experiments were a kind gift of Dr. T. Owen-Hughes.
The oligonucleotide used for triplex formation was TFO
(triplex-forming oligonucleotide) (5'-TTCTTTTCTTTCTTCTTTCTTT-3'). To generate pMJ5, the annealed oligonucleotides TFOB5
(5'-TCGAGAAGAAAAGAAAGAAGAAAGAAAC-3') and TFOB3 (5'-TCGAGT
TTCTTTCTTCTTTCTTTTCTTC-3') were ligated to the product of a
XhoI digestion carried out on pCL7c (12). This yielded a
pBluescript SKII ( Reagent Preparation--
Recombinant yeast Rad51p, Rad54p,
rad54K341Ap, and rad54K341Rp were overexpressed in yeast and purified
as previously described (7). SWI/SNF purification was as described
(13). Histone octamers were purified from chicken erythrocytes as
described by Hansen et al. (14). Octamer concentrations were
determined by measurements of A230 (15). Nucleosomal array
DNA templates (pXG540, pMJ5, or 208-11) were labeled by the Klenow
polymerase fill-in reaction using [ D-loop Reactions--
Oligonucleotide D1 (90-mer)
used in the D-loop experiments has the sequence:
5'-AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT-3', being complementary to positions 1932-2022 of pBluescript SK( ATPase Assay--
Recombinant yeast Rad54p (1 nM)
was incubated at 30 °C or 37 °C with 5 nM of either
naked 208-11 dsDNA or reconstituted nucleosomal arrays in the presence
of 100 µM ATP, 2.5µCi [ Cruciform Formation Assay--
Cruciform formation assays were
performed as previously described (16). Briefly, 8 ng of
AvaI-linearized pXG540 (either naked, N, or nucleosomal, C)
were incubated with various concentrations of Rad54, Rad51, or rad54
K341A and 0.15 mg/ml EndoVII (except where noted), in the presence of
10 mM Hepes, pH 7.9, 50 mM NaCl, 3 mM MgCl2, 5% glycerol, 0.1 mM DTT,
1 mM ATP (except where noted), 3 mM
phosphoenolpyruvate, and 20 units/ml pyruvate kinase for 30 min at
30 °C. The products were resolved in 1.2% agarose gels and
visualized with Sybr Gold staining (Molecular Probes, Eugene, OR)
followed by analysis with ImageQuant software.
Chromatin-remodeling Reaction--
For the coupled SWI/SNF-or
Rad54-SalI reactions, reconstituted 208-11 nucleosomal
arrays (~1 nM final concentration) were preincubated at
37 °C for 20 min with 2.5units/µl SalI in a buffer containing (final concentrations) 50 mM NaCl, 5 mM MgCl2, 1 mM ATP, 3 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase, 1 mM DTT, 10 mM Tris-HCl, pH 8.0, 100 µg/ml
BSA, and 3% glycerol. Nucleosomal arrays were ~80% saturated with
nucleosomes. Buffer, 2 nM SWI/SNF complex, or various
concentrations of recombinant Rad51p and Rad54p were added and samples
were taken at the indicated time points, vigorously mixed for 10s with
25 µl TE and 50 µl 1:1 solution of phenol/chloroform. The purified
DNA fragments were resolved by electrophoresis in 1.2% agarose gels in
the presence of 50 µg/ml ethidium bromide. The gels were then dried
on 3MM Whatman paper. The fraction of cut and uncut DNA was determined by PhosphorImager analysis using a Molecular Dynamics PhosphorImager and ImageQuant software. Experiments were repeated independently at
least 3 times, which yielded very similar results.
Microccocal Nuclease Digestion--
15 nM
reconstituted 208-11 nucleosomal arrays were incubated at 37 °C
with 2 nM SWI/SNF, 100 nM Rad54p, or buffer, in
the presence of 2 mM ATP, 5 mM NaCl, 2.5 mM Tris-HCl, pH 8.0, 0.25 mM MgCl2,
0.3 mM CaCl2, 3 mM
phosphoenolpyruvate, 10 units/ml pyruvate kinase, 1 mM DTT,
10 µg/ml BSA, 0.5% glycerol. After 20 min, 0.0005 units of
Microccocal Nuclease (Worthington) was added to the reaction, and
aliquots were taken at the indicated time points and then treated for
20 min with 2 µg/µl proteinase K and extracted twice with a 1:1
solution of phenol:chloroform. The resulting digestion products were
resolved by electrophoresis in 2% agarose gels, run at 2.5 volts/cm
for 12 h. The gels were fixed, dried, and analyzed using a
Molecular Dynamics PhosphorImager and ImageQuant Software.
Triple-helix Displacement Assay--
Triple-helix formation was
performed as described (17). Briefly, equimolar concentrations (100 nM) of SspI-linearized pMJ5 and
32P-labeled TFO were mixed in buffer MM (25 mM
MES, pH 5.5, 10 mM MgCl2) at 57 °C for 15 min and left to cool to room temperature overnight. The resulting
triplex was either used directly or reconstituted into nucleosomal
arrays. To introduce nicks into the DNA, pMJ5 was exposed to various
concentrations of DNaseI (Promega, Madison, WI) for 2 min at 37 °C,
the reactions were stopped with 5 mM EDTA, vigorously mixed
for 10 s with a 1:1 solution of phenol/chloroform, ethanol-precipitated, and resuspended in water. The degree of nicking
introduced by DNaseI treatment was assessed by electrophoretic analysis
of native and heat-denatured samples (in the presence of 15%
formamide) on denaturing 1.3% agarose gels, followed by Sybr Gold
Stain (Molecular Probes, Eugene, OR).
The triplex-containing substrates (5 nM) were incubated at
30 °C with 5 nM recombinant Rad54 protein or SWI/SNF
complex, in a buffer containing 35 mM Tris-HCl, pH 7.2, 3 mM MgCl2, 100 µg/ml BSA, 50 mM
KCl, 1 mM DTT, 3 mM phosphocreatine, 28 µg/ml
creatine phosphokinase, and where noted, 3 mM ATP. Samples
were taken at the indicated time points, the reactions were quenched
with GSMB buffer (15% (w/v) glucose, 3% (w/v) SDS, 250 mM
4-morpholinepropanesulfonic acid, pH 5.5, 0.4 mg/ml bromphenol
blue), and analyzed in 1.2% agarose gels (40 mM Tris
acetate, 5 mM sodium acetate, 1 mM
MgCl2, pH 5.5) at 10 volts/cm for 1.5 h at 4 °C.
Gels were fixed in 5% acetic acid, 50% methanol for 1 h, and
dried. The proportion of bound and free TFO was determined using a
Molecular Dynamics PhosphorImager and ImageQuant Software.
Rad51p and Rad54p Promote DNA Pairing with a Chromatin
Donor--
Repair of a DSB by homologous recombination begins with the
invasion of a double-stranded, homologous donor by a Rad51-ssDNA nucleoprotein filament, also referred to as the presynaptic filament. This strand invasion reaction is typically monitored in
vitro by following the Rad51p-dependent formation of a
D-loop between a radiolabeled oligonucleotide and a
homologous double-stranded DNA donor (Fig.
1A). In this case, efficient
D-loop formation also requires the ATPase activity of
Rad54p. In vivo, however, the search for homology and strand
invasion involves a homologous donor that is assembled into chromatin.
Given that the Rad54p ATPase shows sequence relatedness to known
chromatin remodeling enzymes, it was of considerable interest to
examine the ability of Rad54p to promote Rad51p-dependent
D-loop formation with a nucleosomal donor.
Fig. 1 shows the results of D-loop assays that use
either a circular, naked DNA donor or this same circular DNA assembled into nucleosomes. Consistent with previous studies, the combination of
yeast Rad51p and Rad54p led to rapid and highly efficient
D-loop formation on the naked DNA donor (Fig.
1B). A similar level of D-loop formation was
also obtained when the bacterial recombinase RecA was used in these
assays with naked DNA (Fig. 1C). Surprisingly, assembly of
the circular donor into chromatin had no effect on the efficiency of
D-loop formation by Rad51p and Rad54p (Fig. 1B).
D-loop formation on the chromatin donor required ATP
hydrolysis by Rad54p, because nonhydrolyzable ATP analogs (ATP Nucleosomal DNA Protects Rad54p from Thermal Denaturation--
The
ATPase activity of Rad54p is required for many of its biological
functions in vivo and for enhancing Rad51p-mediated
homologous DNA pairing reactions in vitro, both on naked DNA
(18) and on chromatin (Fig. 1D). Given the latter finding,
we were interested in determining whether chromatin influences the
ATPase activity of Rad54p.
As shown in Fig. 2, both naked DNA
(solid squares) and chromatin (solid
triangles) stimulated the ATPase activity of Rad54p at 30 °C,
with naked DNA being somewhat more effective. At the low protein
concentrations at which these assays were performed (1 nM),
purified Rad54p is extremely temperature-labile and is rapidly
inactivated at 37 °C (Fig. 2; Ref. 36). Thus, as expected, the ATPase activity of Rad54p was not detectable in the presence of
naked DNA when the reactions were performed at 37 °C (Fig. 2,
open squares). Importantly, when the reaction was carried
out in the presence of chromatin (open triangles), the rate
of ATP hydrolysis at 37 °C was even greater than the rate obtained
in reactions conducted at 30 °C. Importantly, there was no
measurable ATPase activity associated with the nucleosomal arrays in
the absence of Rad54p, and BSA, free histones, and replication protein A were unable to stimulate the DNA-stimulated ATPase activity of
Rad54p at 37 °C. These results indicate that nucleosomal DNA is
uniquely able to protect Rad54p from thermal inactivation, and these
data suggest that Rad54p may physically interact with nucleosomes.
Rad54 Generates Unconstrained Superhelical Torsion in Nucleosomal
DNA--
A number of chromatin remodeling complexes that contain
Swi2/Snf2-related ATPases have been shown to alter chromatin
structure by generating superhelical torsion in DNA and nucleosomal
arrays (16). Indeed, the ability to introduce superhelical stress may represent a primary biomechanical activity of all Swi2/Snf2-like ATP-dependent DNA motors, and this activity is likely to be
crucial for catalyzing alterations in chromatin structure. Previous
studies have shown that Rad54p can also generate both negative and
positive supercoiled domains in dsDNA, and it has been suggested that
this activity reflects the tracking of Rad54p along DNA (19, 20).
We investigated whether Rad54p is able to introduce superhelical
torsion on nucleosomal substrates, using a cruciform extrusion test
that has been used for examining other chromatin remodeling enzymes
(Fig. 3A). In this assay,
superhelical torsion leads to extrusion of a cruciform that is then
recognized and cleaved by bacteriophage T4 endonuclease VII that has
high specificity for this DNA structure (16). Consistent with previous
studies (19), Rad54p action generates torsional stress on a linear,
dsDNA substrate (N) which leads to cruciform extrusion (Fig.
3B, lanes 4-6). Importantly, Rad54p was able to
generate torsional stress on the nucleosomal substrate (C)
with comparable efficiency (lanes 12-14). The addition of
100 nM Rad51p greatly stimulated the ability of Rad54p to
promote the formation of cruciform structures on both naked and
nucleosomal substrates (compare lanes 4 and 8,
and 12 and 16, respectively. Also note the
decreased levels of linear template in lanes 16-18). Importantly, Rad51p fails to support cruciform formation by itself (lanes 7 and 15). As expected, the generation of
torsional stress required ATP (lanes 19 and 20).
Furthermore, the ATP hydrolysis mutant variant rad54 K341A was inactive
in these assays (data not shown). These data indicate that Rad54p, like
other Swi2/Snf2 family members, uses the free energy from ATP
hydrolysis to alter DNA topology and that nucleosomal arrays constitute
excellent substrates for this activity.
Rad54p Can Disrupt a DNA Triple Helix--
How Rad54p introduces
topological stress in nucleosomal DNA is unclear. Previously, we
suggested that superhelical torsion might result from translocation of
Rad54p along the DNA double helix (19). Recently, chromatin remodeling
by the yeast RSC complex (which contains the
Swi2/Snf2-related ATPase, Sth1p) has been shown to involve
ATP-dependent DNA translocation (21). To further evaluate
the ability of Rad54p to translocate on DNA, we used a DNA
triple-helix-displacement assay that was originally developed to follow
the translocation of a type I restriction endonuclease along DNA (17).
The substrate used (see Fig.
4A) consists of a
radioactively labeled oligonucleotide (TFO*) bound via Hoogsteen
hydrogen bonds to the major groove of a 2.5-kb linear dsDNA.
Translocation of a protein along the DNA displaces the triplex, which
can be detected as dissociation of the radioactive TFO* from the DNA
triplex. Fig. 4B shows typical levels of triplex displacement in the absence or presence of Rad54p or yeast SWI/SNF. Both Rad54p and ySWI/SNF were able to efficiently displace a preformed triplex from both naked (squares) and nucleosomal
(triangles) substrates in an ATP-dependent
manner. Similar results were obtained when the TFO*-bound substrate
contained single-strand nicks (data not shown), strongly suggesting
that the displacement of the TFO* reflects translocation of Rad54p and
ySWI/SNF and that it is not due simply to the generation of torsional
stress. Thus, yeast RSC (21), ySWI/SNF, and Rad54p (Fig. 4) all share
the ability to use the free energy from ATP hydrolysis to disrupt
triplex DNA.
Rad54p Has ATPase Kinetics Diagnostic of a DNA-translocating
Enzyme--
The "inch-worm" model for DNA translocation,
originally envisioned (22) for DNA helicases and later modified by
Velankar et al. (23), proposes that the translocating enzyme
progresses along the contour of the DNA in steps of a single base, and
each step requires the hydrolysis of one ATP molecule. This model
predicts that the rate of ATP hydrolysis of a unidirectional DNA
translocating enzyme will depend on the length of the DNA (24).
To investigate whether Rad54p has ATPase properties characteristic of a
unidirectional DNA translocating enzyme, the rate of ATP hydrolysis was
measured in the presence of saturating amounts of single-stranded
oligonucleotides ranging from 10 to 100 nucleotides in length (Fig.
5). For comparison, we also monitored the
ATPase activity of yeast SWI/SNF (triangles). In the case of
Rad54p, oligonucleotides shorter than 40 bases failed to stimulate the ATPase activity of Rad54p (diamonds), whereas
oligonucleotides between 40 and 70 bases led to a stimulation of ATPase
activity that was proportional to DNA length. For oligonucleotides
longer than 70 bases, the ATPase activity no longer increased with the DNA length. When Rad51p was added to these reactions
(squares), shorter oligonucleotides became more effective in
promoting ATP hydrolysis, and the overall activity was enhanced.
Likewise, the ATPase activity of ySWI/SNF (triangles) was
also proportional to the DNA length, with a plateau reached at 60 bases.
These results are fully consistent with both Rad54p and ySWI/SNF
coupling ATP hydrolysis to unidirectional translocation, in which the
rate of DNA binding is slower than the rate of DNA translocation (21).
In this case, no ATP hydrolysis is observed with very short substrates,
presumably because a minimum DNA length is required for Rad54p or
ySWI/SNF to bind and to translocate before reaching an end and
releasing the DNA. When the substrate is ~30-40 nucleotides in
length, Rad54p and ySWI/SNF readily bind the substrate, and more
extended translocation events take place. The rate of ATP hydrolysis is
fairly constant with DNA substrates longer than 60-70 nucleotides,
reflecting the possibility that Rad54p and ySWI/SNF have little
processivity, and thus they release their substrate after ~60-70
bases regardless of the total length of the DNA molecule. Although the
triphasic kinetics of ATPase activity are consistent with a
DNA-translocation mechanism, it remains a possibility that the longer
single-stranded oligonucleotides exhibit more extended secondary
structures that are either more proficient at binding Rad54p (or
SWI/SNF) or stimulating its ATPase activity.
Rad54 Is an ATP-dependent Chromatin Remodeling
Enzyme--
Cairns and colleagues (21) proposed that short-range
translocation events may be the key feature of chromatin remodeling enzymes, leading to a "pumping" of DNA across the surface of the histone octamer, which then results in enhanced DNA accessibility and
nucleosome movements. To investigate whether Rad54p might also enhance
the accessibility of nucleosomal DNA, we used an assay in which
nucleosome remodeling activity is coupled to restriction enzyme
activity such that remodeling is revealed as an enhancement of
restriction-enzyme cleavage rates (12). This assay uses a nucleosomal
array substrate in which the central nucleosome of an 11-mer array
contains a unique SalI site located at the predicted dyad
axis of symmetry (see Fig.
6A). In the absence of a
remodeling enzyme, the rate of SalI cleavage is very slow
(Fig. 6A, solid diamonds), whereas
addition of a remodeling enzyme, such as yeast SWI/SNF, leads to
enhanced digestion (Fig. 6A, solid
squares). When Rad54p was added to the remodeling reactions,
SalI digestion was also dramatically enhanced
(solid circles, triangles), although a
higher concentration of this protein (50 nM) was required
to achieve a rate of digestion comparable with that of reactions that
contained yeast SWI/SNF (2 nM, squares).
However, when Rad51p (50 nM) and Rad54p (50 nM)
were both present in the reaction, much higher levels of remodeling
were attained (open circles). Note that Rad51p
has no intrinsic chromatin remodeling activity (open
diamonds). The stimulation of the Rad54p chromatin
remodeling activity by Rad51p is congruent with previous studies
showing that Rad51p enhances the rate of ATP hydrolysis and DNA
supercoiling by Rad54p (19, 25, see Fig. 3). Thus, the above data
indicate that Rad54p is sufficient for chromatin remodeling activity
but that the combination of Rad51p and Rad54p constitutes a more potent remodeling machine.
Rad54p Does Not Induce Significant Nucleosome Mobilization--
A
number of chromatin remodeling complexes that contain a
Swi2/Snf2-related ATPase (ySWI/SNF, dCHRAC, dNURF, and xMi-2)
can use the energy of ATP hydrolysis to move nucleosomes in cis
(26-30). To investigate whether Rad54p can also catalyze nucleosome
mobilization, 32P-end-labeled nucleosomal arrays were
incubated with buffer, ySWI/SNF, or Rad54p, and nucleosome positions
were mapped by micrococcal nuclease (Mnase) digestion (Fig.
6B). Mnase can only cleave DNA between nucleosomes, which
leads to a periodic ladder of digestion products indicative of a
positioned 11-mer nucleosomal array (Fig. 6B). Consistent
with our previous studies, incubation with ySWI/SNF (2 nM)
and ATP leads to a complete disruption of the Mnase digestion pattern,
indicative of nucleosome sliding (Fig. 6B, left
panel; also see Ref. 28). In contrast, addition of Rad54p (100 nM) and ATP had very little effect on the cleavage
periodicity (Fig. 6B, right panel). Likewise,
addition of both Rad51p and Rad54p (100 nM each) to these
assays did not change the Mnase digestion profile (data not shown).
Importantly, these experiments used concentrations of ySWI/SNF and
Rad54p that yielded similar levels of chromatin remodeling in the
restriction enzyme accessibility assay (Fig. 6A). Thus,
although Rad54p can enhance the accessibility of nucleosomal DNA to
restriction enzymes, this activity does not appear to reflect large
scale rearrangement of nucleosome positions.
In eukaryotes, chromatin presents an accessibility dilemma for all
DNA-mediated processes, including gene transcription and DNA repair.
Although much progress has been made on identifying the enzymes that
remodel chromatin structure to facilitate transcription, less is known
of how the DNA-repair machinery gains access to damaged DNA within
chromatin (reviewed in Ref. 31). In particular, it has not been clear
how the recombinational repair machinery can locate short regions of
DNA homology when those DNA donor sequences are assembled into
chromatin. Here we have shown that the yeast recombination proteins,
Rad51p and Rad54p, are sufficient to promote heteroduplex DNA joint
formation with chromatin. In contrast, the bacterial recombinase RecA
is completely inactive with a chromatin donor. The unique capacity of
the eukaryotic machinery to contend with chromatin likely reflects the
chromatin-remodeling activity of Rad54p, in which the free energy from
ATP hydrolysis enhances the accessibility of nucleosomal DNA. Strand
invasion with chromatin may also require a specific interaction between Rad51p and Rad54p because the chromatin remodeling activity of Rad54p
does not facilitate RecA-dependent D-loop
formation with chromatin (Fig. 1D). Recently, Alexiadis and
Kadonaga have reported that the Drosophila Rad51 and Rad54
proteins can also facilitate strand invasion with chromatin (35).
How Does Rad54p Remodel Chromatin Structure?--
Several studies
have shown that SWI/SNF-like chromatin remodeling enzymes can perform
two separable reactions: 1) they can use the free energy from ATP
hydrolysis to enhance the accessibility of nucleosomal DNA and 2) they
can use this free energy to mobilize nucleosomes in cis (reviewed in
Ref. 32). Recent work from Cairns and colleagues have suggested that
both of these activities may be caused by ATP-dependent
"pumping" of DNA into the nucleosome (21). In this model, small
amounts of DNA translocation might lead to transient exposure of small
"loops" of DNA on the surface of the histone octamer, whereas
larger quantities of DNA "pumped" into the nucleosome would lead to
changes in nucleosome positions. Our data support this model, as we
find that both yeast SWI/SNF and Rad54p, like yeast RSC (21), can
disrupt a DNA triplex in an ATP-dependent reaction,
presumably by translocation of DNA along the surface of the enzyme or
by translocation of the enzyme along the DNA. Furthermore, the ATPase
activities of ySWI/SNF and Rad54p are sensitive to DNA length, which is
diagnostic of DNA-translocating enzymes (21).
Although ySWI/SNF and Rad54p can both enhance the accessibility of
nucleosomal DNA, only ySWI/SNF appears to be proficient at changing
nucleosome positioning. This result suggests that the precise mechanism
of chromatin remodeling by Rad54p may be distinct from that of
ySWI/SNF. For instance, Rad54p may only be able to pump small amounts
of DNA across the histone octamer surface. Alternatively, Rad54p may
translocate along DNA, rather than pumping DNA into the nucleosome. In
this model, Rad54p may "pull" the Rad51-ssDNA nucleoprotein
filament along the chromatin fiber, leading to changes in nucleosomal
DNA topology and DNA accessibility. Such a DNA tracking mechanism might
play a key role in facilitating both the search for homology as well as
the strand invasion step.
Multiple Roles for Rad54p during Homologous Recombination--
Our
results suggest that Rad54p is an extremely versatile recombination
protein that plays key roles in several steps of homologous recombination. Recently, we found that Rad54p is required for optimal
recruitment of Rad51p to a double strand break in vivo, and
likewise Rad54p can promote formation of the presynaptic filament in vitro by helping Rad51p contend with the inhibitory
effects of the ssDNA-binding protein replication protein
A.2 Several studies over the
past few years have also shown that the ATPase activity of
Rad54p plays key roles subsequent to formation of the presynaptic
filament. For instance, Rad54p is required for the Rad51p-nucleoprotein
filament to form a heteroduplex joint DNA molecule, even when the
homologous donor is naked DNA (Fig. 1A; see also Refs. 7,
18, 33). In this case, it has been proposed that Rad54p might use the
free energy from ATP hydrolysis to translocate along DNA, which
facilitates the homology search process. This DNA-translocation model
is fully consistent with our findings that Rad54p can displace a DNA
triplex and that the ATPase activity of Rad54p is proportional to DNA
length. Rad54p also stimulates heteroduplex DNA extension of
established joint molecules (34). Finally, we have shown that Rad54p is
required for Rad51p-dependent heteroduplex joint molecule
formation with a chromatin donor. In this case, our results suggest
that the ATPase activity of Rad54p is used to translocate the enzyme
along the nucleosomal fiber, generating superhelical torsion, which leads to enhanced nucleosomal DNA accessibility. It seems likely that
this chromatin remodeling activity of Rad54p might also facilitate additional steps after heteroduplex joint formation. Future studies are
now poised to reconstitute the complete homologous recombinational repair reaction that fully mimics each step in the repair of
chromosomal DNA double strand breaks in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) plasmid containing 5 head-to-tail repeats of the
208-bp Lytechinus variegatus 5S rDNA nucleosome positioning element flanked by a TFO-binding site. The DNA template (208-11) for reconstituting nucleosomal arrays for the ATPase, remodeling, and Mnase assays consists a
NotI-HindIII fragment derived from pCL7b (12),
containing 11 head-to-tail repeats of a 5S rRNA gene from L. variegatus, each one possessing a nucleosome positioning sequence.
The sixth nucleosome is tagged by a unique SalI restriction site.
-32P]dCTP (3000 µCi/mmol, Amersham Biosciences). Nucleosomal arrays were
reconstituted by salt dialysis as previously described (13), and the
nucleosome saturation was determined to be 60-80% by digestion analysis.
) replicative form I DNA. Oligonucleotide D1 was 5' end-labeled with
32P using [
-32P]dATP and polynucleotide
kinase, as described (7). Buffer R (35 mM Tris-HCl, pH 7.4, 2.0 mM ATP, 2.5 mM MgCl2, 30 mM KCl, 1 mM DTT, and an ATP-regenerating
system consisting of 20 mM creatine phosphate and 30 µg/ml creatine kinase) was used for the reactions; all of the
incubation steps were carried out at 30 °C. Rad51 (0.8 µM) and Rad54 (120 nM) were incubated with
radiolabeled oligonucleotide D1 (2.4 µM nucleotides) for
5 min to assemble the presynaptic filament, which was then mixed with
naked pBluescript replicative form I DNA (38 µM base
pairs) or the same DNA assembled into chromatin (38 µM
base pairs). Chromatin assembly was monitored by following topological
changes as well as measuring the degree of occlusion of a unique
EcoRI restriction site close to the D1 sequence. Substrates were estimated to be ~80% saturated with nucleosomes. The reactions containing RecA protein (0.8 µM) were assembled in the
same manner, except that they were supplemented with an additional 12.5 mM MgCl2 at the time of incorporation of the
duplex substrates. At the indicated times, 4-µl portions of the
reactions were withdrawn and mixed with an equal volume of 1% SDS
containing 1 mg/ml proteinase K. After incubation at 37 °C for 5 min, the deproteinized samples were run in 1% agarose gels in TAE
buffer (40 mM Tris-HCl, pH 7.4, 0.5 mM EDTA) at
4 °C. The gels were dried, and the radiolabeled DNA species were
visualized and quantified by PhosphorImager analysis (Personal
Molecular Imager FX, Bio-Rad).
-32P]dATP (6000 µCi/mmol, Amersham Biosciences), 2.5% glycerol, 0.1% Tween 20, 20 mM Tris-HCl, pH 8.0, 200 µM DTT, 5 mM MgCl2, 100 µg/ml BSA. For the DNA
length-dependence assays, 5 nM Rad54p, 5 nM
Rad51p, or 10 nM SWI/SNF were used. Oligonucleotides
(random N-mers ranging from 10-100 nucleotides in length) were
PAGE-purified to ensure length homogeneity (Integrated DNA
Technologies, Inc., Coralville, IA). Samples were taken after 2, 5, 15, and 30 min and resolved by TLC. The proportion of liberated
32P-pyrophosphate was determined using the Molecular
Dynamics PhosphorImager and ImageQuant Software. ATPase assays were
independently repeated 3 times, yielding very similar results.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rad51p and Rad54p promote efficient DNA
strand invasion with chromatin. A, schematic of the
D-loop reaction. A radiolabeled oligonucleotide
(ss) pairs with a homologous duplex target
(dsDNA) to yield a D-loop, which, after
separation from the free oligonucleotide on an agarose gel, is
visualized and quantified by PhosphorImager analysis of the dried gel.
B, panel I shows D-loop reactions
mediated by Rad51p and Rad54p with the naked homologous duplex
(Naked DNA) and the homologous duplex assembled into
chromatin (Chromatin). The results from the experiments in
panel I are graphed in panel II. The
ordinate refers to the proportion of the homologous duplex
converted into D-loop. C, panel I
shows D-loop reactions mediated by RecA with the naked
homologous duplex (Naked DNA) and the homologous duplex
assembled into chromatin (Chromatin). The results from the
experiments in panel I are graphed in panel II.
D, panel I shows D-loop reactions in
which Rad51p and RecA were used either alone or in conjunction with
Rad54p or rad54 mutant variants with the naked homologous duplex
(Naked DNA) and the homologous duplex assembled
into chromatin (Chromatin), as indicated. ATP was omitted
from the reaction in lane 5, and ATP S (
S)
and AMP-PNP (PNP) replaced ATP in lanes 6 and
7, respectively. The reactions in lanes 8 and
9 contained ATP, but Rad54p was replaced with the
ATPase-defective variants rad54 K341A (KA) and rad54 K341R
(KR), respectively. The results from the experiments in
panel I are summarized in the bar graph in panel
II.
S and
AMP-PNP) were unable to substitute for ATP (Fig. 1C,
panel I, lanes 6 and 7), and two
ATPase-defective mutant variants of Rad54p, rad54K341Ap and rad54K341Rp
(18), were inactive (Fig. 1D). In contrast to reactions that
contained Rad51p/Rad54p, the activity of RecA was completely eliminated
when the donor was assembled into nucleosomes (Fig. 1C).
Furthermore, addition of Rad54p to the RecA reaction did not rescue
D-loop formation on chromatin (Fig. 1D,
panel I, lane 11). Thus, the eukaryotic
recombination proteins have the unique capability of performing the DNA
strand invasion reaction with a chromatin donor.
View larger version (12K):
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Fig. 2.
Nucleosomal DNA protects Rad54p from thermal
inactivation. ATPase assays. 1 nM Rad54p was incubated
at 30 °C with no DNA (circles), 5 nM naked
(closed squares) or nucleosomal dsDNA
(closed triangles), or at 37 °C with 5 nM naked (open squares) or
nucleosomal dsDNA (open triangles). Samples were
taken after 2, 5, 15, and 30 min.
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Fig. 3.
Rad54 generates superhelical torsion on
nucleosomal DNA. A, schematic illustration of the cruciform
extrusion assay. A linearized plasmid
(pXG540) containing an inverted repeat sequence is incubated
with T4 Endonuclease VII, a highly selective junction resolving enzyme,
and Rad54p, in the presence of ATP. Rad54p increases the local
unconstrained superhelical density, resulting in the extrusion of a
cruciform structure, which is recognized and cut by Endo VII. Adapted
from Havas et al. (16). B, results of a typical
cruciform formation assay. Supercoiled (lanes 1,
2), AvaI-linearized pXG540 DNA (lanes 3-10,
19), or nucleosomal pXG540 (lanes 11-18,
20) was incubated with 12.5, 25, or 50 nM Rad54p
as indicated, in the presence or absence of 100 nM Rad51p.
EndoVII was omitted in lane 1. ATP was omitted in
lanes 19 and 20. The numbers below each lane
(% cut) represent the percentage of pXG540 molecules
cleaved by EndoVII. s.c., supercoiled substrate;
C, chromatin substrate; N, naked linear DNA
substrate.
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Fig. 4.
Rad54p action displaces a preformed
triplex. A, typical results obtained with naked
triplex-containing substrate. The upper band corresponds to the
duplex-bound TFO*; the lower band corresponds to free TFO*. Reactions
contained 5 nM triplex substrate and 5 nM
Rad54p. B, percentage of free TFO* in four or more
experiments were averaged and plotted as a function of time. Note that
triplex displacement from the nucleosomal template occurs at equal
efficiency to that of naked DNA.
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Fig. 5.
Rad54 has ATPase kinetics typical of a
unidirectional DNA translocating enzyme. The rate of ATP
hydrolysis by 5 nM Rad54p (diamonds), 5 nM Rad54p + 5 nM Rad51p (squares),
or 10 nM SWI/SNF (triangles) was measured in the
presence of 50 µM ssDNA (n-mers) oligonucleotides of
different lengths. The average values from 3 independent experiments
were plotted. Rates were determined from experiments with at least four
time points.
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Fig. 6.
Rad54 is an ATP-dependent
chromatin remodeling enzyme. A, various concentrations
of recombinant Rad54p were tested for chromatin-remodeling activity in
a coupled remodeling-restriction enzyme cleavage assay. The nucleosomal
substrate was incubated with 50 nM (closed
circles) or 480 nM Rad54p
(triangles), 100 nM Rad51p (open
diamonds), 50 nM Rad54p + 50 nM
Rad51p (open circles), 2 nM ySWI/SNF
(squares), or buffer (closed
diamonds). B, 208-11 reconstituted nucleosomal
arrays were incubated at 37 °C with 2 nM SWI/SNF, 100 nM Rad54p, or buffer (control lanes). Aliquots were treated
with Mnase for the indicated times. The arrowheads in the
left panel indicate the alternate banding pattern as a
result of SWI/SNF-induced nucleosome movement.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Institutes of Health to C. L. P. (GM49650) and P. S. (GM57814).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.
§ Both authors contributed equally to this work.
¶ Supported by a U. S. Department of Defense Predoctoral Fellowship (DAMD17-02-1-0471).
** Supported by a U. S. Department of Defense Predoctoral Fellowship (DAMD17-01-1-0414).
Current Address: Dept. of Biological Sciences, University
of Alaska, 3211 Providence Dr., Anchorage, AK 99508.
§§ To whom correspondence should be addressed. Tel.: 508-856-5858; Fax: 508-856-5011; E-mail: Craig.Peterson@umassmed.edu.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211545200
2 B. Wolner, S. Van Komen, P. Sung, and C.L. Peterson, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
DSB, double-strand
break;
HR, homologous recombination;
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA;
TFO, triplex-forming oligonucleotide;
DTT, dithiothreitol;
BSA, bovine serum albumin;
ATPS, adenosine 5'-O-(thiotriphosphate);
AMP-PNP, adenosine
5'-(
,
-imino)triphosphate;
Mnase, micrococcal nuclease.
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