From the Institut für Zellbiologie and
¶ Institut für Molekularbiologie, Departement Biologie,
ETH-Hönggerberg, CH-8093 Zürich, Switzerland and
Program in Molecular Medicine, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
Received for publication, January 23, 2003, and in revised form, March 6, 2003
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ABSTRACT |
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Nucleosomes inhibit DNA repair in
vitro, suggesting that chromatin remodeling activities might be
required for efficient repair in vivo. To investigate how
structural and dynamic properties of nucleosomes affect damage
recognition and processing, we investigated repair of UV lesions by
photolyase on a nucleosome positioned at one end of a 226-bp-long DNA
fragment. Repair was slow in the nucleosome but efficient outside. No
disruption or movement of the nucleosome was observed after UV
irradiation and during repair. However, incubation with the nucleosome
remodeling complex SWI/SNF and ATP altered the conformation of
nucleosomal DNA as judged by UV photo-footprinting and promoted more
homogeneous repair. Incubation with yISW2 and ATP moved the
nucleosome to a more central position, thereby altering the repair
pattern. This is the first demonstration that two different chromatin
remodeling complexes can act on UV-damaged nucleosomes and modulate
repair. Similar activities might relieve the inhibitory effect of
nucleosomes on DNA repair processes in living cells.
Folding of eukaryotic DNA into nucleosomes and higher order
structures restricts its accessibility to proteins, thereby repressing DNA-dependent processes like transcription and DNA repair.
Dynamic properties of nucleosomes and chromatin remodeling activities contribute to relieve the repressive role of chromatin. Although remodeling has been extensively studied in the context of
transcriptional regulation, its contribution to DNA repair remains
unclear (1-5). Here we show that two different nucleosome remodeling
activities can act on UV-damaged nucleosomes and modulate repair by photolyase.
The basic unit of chromatin is the nucleosome core particle. It
consists of 147 bp of DNA wrapped in 1.65 left-handed superhelical turns around an octamer of core histones. The octamer itself consists of a histone (H3-H4)2 tetramer, which binds the central
six turns of DNA, and two H2A-H2B dimers, which primarily bind distal
regions of the core DNA. The structure of DNA changes upon folding into nucleosomes (6).
In principle, all reactions that involve DNA can be regulated by
changing DNA packaging. A contribution to the regulation of DNA
accessibility comes from intrinsic properties of nucleosomes, such as
nucleosome mobility, unfolding, or partial disruption (7). Another
contribution is made by protein complexes that remodel chromatin
structures. One class consists of histone-modifying complexes that add
or remove covalent modifications from histone tails. Another class
utilizes the energy of ATP hydrolysis to modify chromatin structure in
a non-covalent manner (2, 3, 8, 9). The ATP-dependent
chromatin remodeling complexes contain an ATPase subunit that belongs
to the SNF2 superfamily of proteins. Based on the identity of this
subunit, they have been divided into the SWI2/SNF2 family, the ISWI
family, and the Mi-2 family (10). Although functional analysis of these
complexes has been focused mainly on transcription, there is increasing evidence that similar activities may assist recombination and repair
(1-3, 11-13).
Cyclobutane pyrimidine dimers
(CPDs)1 are the major class
of DNA lesions produced by UV light. The recent crystal structure of a
CPD in DNA showed that the overall helical axis bends ~30° toward
the major groove and unwinds ~9° (14). CPDs appear to have only
minimal effect on nucleosome stability but may affect nucleosome
positioning during chromatin assembly. CPD formation is modulated by
the structure of nucleosomes and by other protein-DNA interactions (4,
5). In most organisms, pyrimidine dimers are removed by the multistep
nucleotide excision repair (NER) pathway (15). As an alternative or
additional pathway, a wide variety of organisms can specifically revert
photoproducts to their native bases by DNA photolyase in the presence
of light (photoreactivation) (16).
Both DNA repair mechanisms are modulated by chromatin structure (4, 5).
Nucleosomes exert a repressive role on NER and photoreactivation
because repair is slow in regions of positioned nucleosomes and fast in
linker DNA of yeast (17-19). However, the repression is not tight,
since all lesions need to be repaired to prevent mutagenesis. In
vitro, reconstituted nucleosomes also exert an inhibitory effect
on repair by photolyase or T4-endonuclease V (20, 21) and by NER (13,
22-25). Repair of SV40 minichromosomes by NER was also reduced
compared with naked DNA (26). However, Xenopus extracts
proficient for NER can repair a lesion placed in a reconstituted
nucleosome (27), suggesting that factors responsible for modulating
nucleosome structure may be present in the extract.
Several proteins that are involved in DNA repair pathways belong to the
SNF2 superfamily of ATPases and thereby might provide chromatin
remodeling activities to facilitate DNA repair (1, 28). Nucleosome
remodeling activity was shown on undamaged nucleosomes for the
Cockayne's syndrome B (CSB) protein, which is involved in
transcription coupled repair (29), and for the INO80 chromatin remodeling complex of yeast. Mutations in INO80 cause sensitivity to
hydroxyurea, methyl methanesulfonate, and ultraviolet and ionizing radiation (30). On the other hand, the ACF remodeling complex was reported to facilitate NER of a lesion placed in linker DNA of a
dinucleosome but not of a lesion in the nucleosomes (25). In contrast
to ACF, the SWI/SNF remodeling complex was most recently reported to
stimulate excision nuclease activity on a nucleosome containing a bulky
acetylaminofluorene-guanine adduct (13). Thus, SWI/SNF and ACF might
act differently on damaged nucleosomes, or their remodeling activities
might be dependent on the nature and/or location of the damage within
the nucleosome. Here we show that two different chromatin remodeling
activities (SWI/SNF and ISW2) can act on UV-damaged nucleosomes and
facilitate repair by photolyase. In addition, the photoreactivation
pattern of remodeled nucleosomes reflected differences in the
activities of SWI/SNF and ISW2.
Preparation of DNA for Reconstitution--
The
HindIII/BamHI fragment of p8ATDED (31) was
subcloned in either orientation into the SacI site of pUC18
to generate p18ATDED and p18ATDED-c for top and bottom strand labeling,
respectively. The 226-bp fragments generated by cleavage with
SmaI and EcoRI were purified and the 3' ends
labeled by filling the recessed ends with
[ Nucleosome Reconstitution--
Reconstitution was done by
histone octamer transfer from chicken erythrocytes core particles (20).
200 ng of end-labeled DNA was incubated with a 40-fold excess of core
particles (8 µg) in 200 µl of 10 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 0.8 M NaCl at 37 °C for 30 min and at 4 °C for another 30 min. The samples were dialyzed at
4 °C in a microdialyzer (Pierce, membrane molecular weight cut-off
8000) overnight against dialysis buffer 1 (0.6 M NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 50 µM phenylmethylsulfonyl fluoride), 4-5 h against
dialysis buffer 2 (10 mM Tris (pH 7.5), 50 mM
NaCl, 1 mM EDTA (pH 7.5), 50 µM
phenylmethylsulfonyl fluoride), and finally 2-3 h against fresh
dialysis buffer 2.
Yeast SWI/SNF Remodeling--
Yeast SWI/SNF
was added to nucleosomes (160 ng for the DNase I footprint, 100 ng for
the UV footprint, and 800 ng for the photoreactivation experiments),
and the buffer was adjusted to final concentrations of 8 mM
Tris (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl2, 3% glycerol (in
final volumes of 30, 20, and 80 µl, respectively). Where indicated,
ATP was added to a final concentration of 0.5 or 1 mM. The
photoreactivation experiment contained in addition 46 µg/ml insulin
brought in by a new yeast SWI/SNF batch. The molar ratios of ySWI/SNF
to nucleosomes were 1 (10 ng of complex/ng of nucleosome) for the DNase
I footprinting (Fig. 3, A and B), 0.8 (8 ng of
complex/ng of nucleosome) for the UV photo-footprinting (Fig. 3,
B and C), and 0.6 (6 ng of complex/ng of
nucleosome) for the photoreactivation experiments (Fig. 4). The
reactions were incubated 30 min at 30 °C. For competition, 2-µl
aliquots were removed, and an excess of linear plasmid DNA (pBSFT99; 1 µg in 10 mM Tris (pH 7.5), 100 mM NaCl, 3%
glycerol) was added, resulting in a final volume of 5 µl. The
reaction was incubated for 45 min at room temperature.
Expression and Purification of the Yeast ISW2 Complex--
The
yISW2 complex was expressed using the Bac to Bac baculovirus expression
system (Invitrogen). SF21 cells were grown in suspension culture to a
density of 1 × 106 cells/ml in SF900 media and
infected with viral m.o.i. of 0.1 or 1.0 using a C-terminally
His6-tagged version of ISW2. Cells were harvested by
centrifugation 48 (for 1.0 m.o.i.) or 72 h (for 0.1 m.o.i.) post-infection. All subsequent steps were performed at 4 °C.
Cell lysis and binding to Talon metal affinity resin was carried out
according to the manufacturer's instructions
(Clontech) except that the binding buffer was
adjusted to pH 7.4. Elution was carried out with a linear gradient to
100 mM imidazole. Eluted material was loaded directly onto
a Porous HPQ column (Beckman Instruments) and eluted with a linear
gradient to 0.6 M KCl. Combined fractions were loaded on a
Sepharose G-400 gel filtration column run in 100 mM KCl, 20 mM Tris (pH 7.5). Peak fractions were concentrated using
Vivaspin concentrators (Sartorius).
yISW2 Remodeling--
His-tagged yISW2 was added to nucleosomes
(2 µg), and the buffer was adjusted to final concentrations of 10 mM Tris (pH 7.5), 90 mM NaCl, 5 mM
MgCl2, 1 mM dithiothreitol, 8% glycerol, 30 µg/ml bovine serum albumin (in a final volume of 100 µl). Where
indicated, ATP was added to a final concentration of 0.5 mM. The molar ratio of ISW2 to nucleosomes was 0.7 (1 ng of
complex/ng of nucleosome). The reactions were incubated for 30 min at
30 °C.
UV Irradiation--
Nucleosome (40 ng/µl), remodeled
nucleosome (5 ng/µl), and DNA (5-40 ng/µl) samples were split in
20-40-µl droplets and irradiated on parafilm strips placed on ice at
a fluence of 15 watts/m2 using germicidal lamps (G15WT8,
Sylvania) emitting predominantly at 254 nm.
Photoreactivation--
Irradiated nucleosomes or naked DNA
isolated from irradiated nucleosomes were mixed with Escherichia
coli photolyase (BD Biosciences) to yield a ratio of 70-75 ng
of photolyase/µg of DNA. Photoreactivation was performed by
irradiating the samples at 30 °C with six fluorescent lamps (15 watts, F15T8 BLB, Sylvania, peak emission at 375 nm) with a fluence of
17 watts/m2. In the SWI/SNF and ISW2 remodeling
experiments, samples were photoreactivated in remodeling buffer.
CPD Analysis--
SDS was added to the repair samples (0.5%
final concentration), followed by proteinase K digestion. DNA was
purified through QIAquick columns (Qiagen) and eluted in 50 mM Tris, 5 mM EDTA. DNA was incubated at
37 °C; T4-endonuclease V (Epicentre) was added (0.06 units/ng DNA),
and incubation was continued for 3 h. DNA was purified by
StrataClean resin (Stratagene) extraction, precipitated, and analyzed
on denaturing 8% acrylamide gels.
Native Nucleoprotein Gels--
Reconstitution products were
analyzed on 4% acrylamide gels run in 0.5× TBE. Where indicated, the
nucleoprotein gels also contained 10% glycerol (see figure legends).
Prior to loading, the samples were mixed with glycerol to yield a final
concentration of 6% glycerol and electrophoresed at 4 °C for 3-4 h
at 12 mA.
DNase I Footprinting--
DNase I digestion of nucleosomal and
naked DNA samples was performed in parallel. The samples were adjusted
to 5 mM MgCl2 and incubated with DNase I (Roche
Applied Science; 2 units of DNase I/µg of nucleosomal DNA and 0.2 units of DNase I/µg of naked DNA) at room temperature between 30 s and 6 min. The reactions were stopped by adjusting the samples to 10 mM EDTA. The DNA was purified and analyzed on 8%
denaturing acrylamide gels.
Restriction Enzyme Digestions--
1.8 µg of nucleosomes or
naked DNA was incubated with 50 units of restriction enzyme
(AflIII, HhaI, and XhoI) at 30 °C
for 3 h in a buffer adjusted to 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 mM Tris (pH 7.5), and 0.1 mg/ml bovine serum albumin for
the restriction enzymes AflIII and HhaI. Purified
DNA was analyzed on 8% denaturing acrylamide gels.
Quantification--
The gels were dried on Whatman DE81 and 3MM
papers and quantified using a PhosphorImager (Amersham
Biosciences).
The "ATDED-long Nucleosome," a Chromatin Substrate to Study
CPD Repair--
We reconstituted a nucleosome at the end of a 226-bp
DNA fragment. This "ATDED-long" fragment originates from the
natural yeast DED1 promoter and contains several
polypyrimidine tracts (31), which allowed us to monitor DNA structure
by UV footprinting and DNA damage accessibility by photolyase (Fig.
1A). This nucleosome has the
space to reassemble or slide to alternative positions as a consequence
of DNA damage formation and interactions with DNA repair enzymes and
remodeling complexes.
The DNA was end-labeled with 32P on either strand
separately, and nucleosomes were reconstituted by histone octamer
transfer from chicken erythrocytes core particles. Nucleoprotein gels
showed that reconstitution was efficient, with over 90% of the DNA
folded into nucleosomes. Only one preferential product was observed
(see examples below). DNase I digestion produced a 10-bp repeat pattern between map units 1360 and 1497, which is characteristic for
nucleosomes occupying a single rotational orientation (Fig. 1B,
lanes 2 and 3; Fig. 3B, lanes 5 and 6). No such patterns were generated in naked DNA
(Fig. 1B, lanes 4). The translational
position of the nucleosome was verified by digestion with restriction
enzymes. Reconstituted DNA was efficiently cleaved by AflIII
(84%) and HhaI (79%), whereas cleavage by XhoI
was strongly inhibited (2% cut; Fig. 1C). Thus, DNase I and
restriction analyses demonstrate that the nucleosome is positioned at
the right end of the DNA and adopts a preferential rotational setting.
For repair experiments, reconstituted nucleosomes were irradiated with
UV light at a dose of 750 J/m2 to generate about 1.5 CPDs
per fragment and exposed to E. coli DNA photolyase and
photoreactivating light for up to 120 min at 30 °C. Nucleoprotein
gels revealed that the nucleosomal fraction remained unchanged after UV
irradiation and during photoreactivation (Fig.
2A, lanes 3-9). Hence,
neither UV irradiation nor photolyase disrupted the nucleosome.
To assess the CPD distribution and their removal by photolyase, DNA
of UV-irradiated and photoreactivated nucleosomes was purified and cut
at CPDs with T4-endonuclease V, and the digestion products were
displayed by gel electrophoresis (Fig. 2B, lanes 2-9). To
test the activity of photolyase on naked DNA, DNA of UV-irradiated
nucleosomes was extracted and exposed to photolyase and light for up to
45 min (lanes 10-13). Decreasing band intensities with
increasing photoreactivation times showed site-specific repair. Damages
were quantified in pyrimidine clusters; the fraction of CPDs repaired
in 30 min is shown (Fig. 2C). CPD repair in naked DNA was
fast with 65-95% of the CPDs removed in 30 min. In reconstituted samples, repair was fast outside of the predicted nucleosome (clusters 1, 2, 3, 10, and 11) but slow and inefficient in the nucleosome (clusters 5-9 and 13-19). Clusters 12 and 4 are located toward the
left end of the nucleosome (on the "linker side") and were repaired more efficiently than those located in the central region and
toward the other end of the nucleosome. Interestingly, cluster 12 (map
units 1376-1378) appears to coincide with a DNase I-sensitive site
that is not part of the 10-bp ladder and therefore might indicate an
unusual structure at the end of the nucleosome (Fig. 1B, bottom
strand).
In conclusion, CPDs in nucleosomes are resistant to photoreactivation,
whereas CPDs outside are efficiently repaired. The results imply that
the nucleosome was not displaced either by UV damage formation or by
incubation with photolyase despite the length of the fragment. Thus, UV
lesions and E. coli photolyase are unable to displace the
histone octamer.
Remodeling by ySWI/SNF Alters the DNA Structure of
Nucleosomes--
In contrast to the results obtained in
vitro, DNA lesions are completely removed from nucleosomes
in vivo. Therefore, we tested whether nucleosome remodeling
activities can act on UV-damaged nucleosomes and facilitate CPD
accessibility and repair. We first tested ySWI/SNF. SWI/SNF is known to
generally enhance accessibility of nucleosomes to DNase I, restriction
endonucleases, and transcription factors in vitro, without
disruption of the histone octamer (32-35).
Reconstituted nucleosomes were incubated with ySWI/SNF either in the
absence or in the presence of ATP. In the nucleoprotein gel, all
labeled DNA was found in the well after addition of ySWI/SNF (Fig.
3A, lanes 3 and 4)
and ATP (lane 4), indicating that the nucleosomes were bound
to the remodeling complex. DNase I footprinting revealed that the
rotational setting of the nucleosome was maintained when complexed with
ySWI/SNF in the absence of ATP and lost after incubation with ySWI/SNF
and ATP (Fig. 3B, lanes 5-10). The resulting cutting
pattern was similar to naked DNA (lanes 2-4). Thus the complex apparently remodels the ATDED-long nucleosome as described for
other substrates (32, 34, 36).
CPD formation depends on the structure of DNA and can be altered by
folding of DNA in nucleosomes (37-39). ATDED-long nucleosomes were
analyzed by UV photo-footprinting during remodeling (Fig. 3,
C and D). Naked DNA, nucleosomes, and nucleosomes
treated with ySWI/SNF in the presence or absence of ATP were irradiated
with UV light at a dose of 500 J/m2 to generate about one
CPD per fragment. The nucleoprotein gel confirmed that prior to
irradiation, all nucleosomes were complexed with ySWI/SNF in the
presence and absence of ATP (Fig. 3C, lanes 3 and
4). In naked DNA, the CPD formation pattern was
heterogeneous, depending on the sequence and the unusual structure of
T-tracts which forms characteristic damage patterns (38, 40) (Fig. 3D, lane 4, clusters 13 and 16). The
damage pattern generated in reconstituted nucleosomes was clearly
different (Fig. 3D, lane 5) demonstrating that folding of
ATDED-DNA in a nucleosome alters its DNA structure. In particular,
folding of DNA into nucleosomes induced enhanced CPD formation in
cluster 16 and at the 5' end of cluster 13. The nucleosome pattern was
maintained when nucleosomes were irradiated in the presence of ySWI/SNF
(Fig. 3D, lane 6), but irradiation in the presence of
ySWI/SNF and ATP generated a pattern similar to that of naked DNA
(lane 7). These UV photo-footprinting results demonstrate a
change in the structure upon folding of DNA into nucleosomes and upon
remodeling by ySWI/SNF.
To test whether DNA might have been released from nucleosomes by
ySWI/SNF under our conditions, aliquots were incubated with an excess
of plasmid DNA to compete for ySWI/SNF and analyzed by nucleoprotein
gel electrophoresis (Fig. 3C, lanes 5-8). After competition
with plasmid DNA, only a small fraction of material was observed as
free DNA, whereas most of the material was found in nucleosomal
fractions (Fig. 3C, lanes 7 and 8). This is in agreement with previous work (34, 36, 41, 42), where no or very little
increase in naked DNA was observed in similar competition experiments.
Two major nucleosomal bands (Fig. 3C, N and N')
indicate that some nucleosomes might have joined to form dimers
following interactions with ySWI/SNF. In summary, the nucleoprotein gel demonstrates that ySWI/SNF did not promote a release of free DNA from
nucleosomes. Thus, the UV damage pattern observed by footprinting reflects the pattern of a nucleosome interacting with ySWI/SNF or being
remodeled by ySWI/SNF.
Nucleosome Remodeling by ySWI/SNF Facilitates Repair--
To address a possible contribution of nucleosome remodeling
activities to DNA repair, nucleosomes were irradiated with UV light at
a dose of 500 J/m2, incubated with ySWI/SNF either in the
absence or presence of ATP, and exposed to photolyase in the presence
of photoreactivating light. The nucleoprotein gel (Fig.
4A) showed that the
nucleosomes were stable over the course of the experiment (lanes
2-7). Addition of ySWI/SNF resulted in complexes that appeared as
a smear or remained stuck in the wells (Fig. 4A, lanes
8-15). Thus, ySWI/SNF can bind to damaged nucleosomes both in the
presence and absence of ATP. Photolyase does not disrupt the complexes
or may disrupt the complexes only transiently.
To address the state of the nucleosome during the repair experiment,
the ySWI/SNF complex was removed by competition with plasmid DNA (Fig.
4B). In all samples, addition of plasmid DNA resulted in the
recovery of the nucleosomal band (lanes 8-15) indicating
that most of the DNA remained incorporated into nucleosomes after
remodeling by ySWI/SNF and over the course of photoreactivation.
Repair analysis is shown in Fig. 4, C and D.
Repair of naked DNA was fast and complete after 30 min, whereas repair
of the nucleosomal substrate was fast outside and slow inside of the nucleosome as observed above. Incubation with ySWI/SNF in the absence
of ATP did not dramatically alter the repair pattern. Only clusters 12 and 19 were more slowly repaired than in nucleosomes. Thus, binding of
ySWI/SNF did not substantially inhibit the accessibility of CPDs,
neither outside nor inside the nucleosome. Substantial changes in
repair were observed in the presence of ySWI/SNF and ATP (Fig. 4,
C, lanes 10-12, and D). Repair of
clusters 10 and 11 was reduced, and repair of clusters 12-18 was
enhanced, resulting in a more homogeneous repair pattern over
the DNA fragment. Only cluster 19 remained poorly repaired. Since the
nucleosomes were not disrupted, we can conclude that, in the presence
of ATP, ySWI/SNF facilitates CPD accessibility and repair by remodeling
the chromatin substrate.
yISW2 Remodels ATDED-long Nucleosomes--
To investigate whether
other ATP-dependent remodeling complexes can promote CPD
repair in nucleosomes, His-tagged-yISW2 was tested in our model system.
The nucleosomes were irradiated with UV light at a dose of 500 J/m2, incubated with yISW2 either in the absence or in the
presence of ATP, and exposed to DNA photolyase and photoreactivating
light. The nucleoprotein gels (Fig.
5A) showed that incubation of
UV-irradiated nucleosomes with yISW2 in the absence of ATP did not
change the migration of the nucleosomes (lanes 7-10),
indicating that the majority of the nucleosome substrate was not firmly
bound to yISW2 in our conditions. In the presence of ATP, nucleosome
migration was retarded, and the altered mobility was maintained over
the course of photoreactivation (Fig. 5A, N2, lanes
11-14) indicating that yISW2 was actively remodeling the
substrate. Since nucleosomes located at a fragment end migrate faster
than those that are positioned more centrally in non-denaturing gels
(43), it is likely that yISW2 and ATP shifted the nucleosome to a more
central position. This behavior was not dependent on UV irradiation,
because similar results where obtained on non-damaged nucleosomes (data
not shown). This part of the experiment demonstrates that yISW2 can
remodel UV-damaged nucleosomes and that the remodeled nucleosomes
remain stable over the course of the experiment.
The DNA repair gels and the fraction of CPDs repaired in 30 min are
shown in Fig. 5, B and C. Repair of naked DNA was
complete after 30 min, whereas repair of nucleosomes was modulated as
described above. Although incubation with yISW2 had no dramatic effect
on CPD repair, yISW2 in the presence of ATP altered repair on both strands. Repair was inefficient in the central clusters 4-7 (top strand) and 12-17 (bottom strand), and enhanced in
clusters 8, 9, 18, and 19, which are located toward the end of the
fragment. Thus, the inhibition of repair observed on the right side of
the fragment in the nucleosome substrate was shifted toward the center of the fragment after incubation with yISW2 and ATP. These results are
consistent with a movement of the nucleosome toward more central positions by yISW2.
DNA accessibility in eukaryotes is affected by intrinsic
properties of nucleosomes, their higher order organization, and by chromatin modifying activities. Here we show that neither DNA damage
formation by UV light nor interaction with photolyase disrupted a
preformed nucleosome. However, two different chromatin remodeling activities were shown to remodel UV-damaged nucleosomes and modulate damage accessibility for a repair enzyme. Thus, our results suggest that remodeling activities could be engaged in cells to relieve the
inhibitory effect of nucleosomes in DNA repair processes.
UV Damage Formation and Nucleosome Stability--
UV-induced DNA
lesions represent distortions in the DNA structure. It is therefore
conceivable that DNA lesions interfere with the stability of
nucleosomes and may promote their disruption or promote sliding to
alternate positions. The currently available data are somewhat
controversial. Generally, UV-damaged nucleosomes appear to be quite
stable, since they can be purified from irradiated cells
(e.g. Ref. 44). On the other hand, irradiation was shown to
destabilize nucleosomes reconstituted on 5 S rDNA (45) and plasmids (46), whereas nucleosomes reconstituted on HISAT DNA and
5 S rDNA were not similarly affected (20, 38, 39). In the examples
shown in this study, UV lesions were not sufficient to disrupt the
nucleosomes. Thus, structural distortions introduced by UV lesions were
accommodated by both nucleosomes and nucleosome remodeling complexes.
This is consistent with the observation that crystallized nucleosomes
can accept the deficit of a base pair in one turn of the superhelix (6)
and therefore could as well accept a DNA lesion.
Inhibition of Photoreactivation by a Positioned
Nucleosome--
Previous work (20, 21) reported a strong inhibition of
CPD repair by photolyase and T4-endonuclease V in nucleosomes in vitro. Here we demonstrate that the inhibition of photolyase is restricted to nucleosomal DNA, whereas the nucleosome free "linker" DNA was rapidly repaired. Thus, it appears that binding and/or processing of CPDs is strongly inhibited on the nucleosome surface. Moreover, there is no obvious correlation of site-specific
photoreactivation with the rotational setting as determined by DNase I. In contrast to DNase I, which binds to the minor groove and generates
single strand cuts, photolyase bends DNA and flips out the pyrimidine dimer into its active site (47, 48). Despite the flexibility of
nucleosomal DNA with respect to damage accommodation, such a flip-out
mechanism appears to be severely inhibited by the structural constraints of nucleosomes. Consequently, efficient repair of nucleosomal DNA requires disruption or displacement of nucleosomes with
or without the help of remodeling activities.
Remodeling by ySWI/SNF Alters the Structure
of Nucleosomal DNA and Facilitates CPD Accessibility--
SWI/SNF
generally increases the accessibility of nucleosomal DNA to
transcription factors, DNase I, and restriction endonucleases (32-35)
and induces octamer sliding (49, 50). The UV photo-footprint experiments show that the DNA structure changes upon remodeling by
ySWI/SNF. In naked DNA, CPD formation is influenced by the DNA
structures, in particular by the unusual rigid structure of T-tracts.
The CPD pattern changed, when DNA was reconstituted in nucleosomes,
indicating that the histone octamer exerts a dominant constraint on the
structure of those sequences (38, 51). Binding of ySWI/SNF alone had no
effect on the CPD formation pattern, which suggests that the nucleosome
remained intact. However, addition of ySWI/SNF and ATP changed the CPD
pattern, which appeared similar to that of naked DNA, although the
complex remained bound. Thus, DNA appears to be relaxed or extended
enough in the nucleosome-SWI/SNF-ATP complex to allow formation of the
T-tract structure.
The repair data also support a generally better accessibility of
remodeled DNA. In contrast to the modulation of repair observed in
nucleosomes or nucleosomes with ySWI/SNF, ySWI/SNF and ATP allowed more
uniform repair along the reconstituted DNA fragment. Only cluster 19 at
the end was relatively poorly repaired. Therefore, ySWI/SNF activity
appears to generally facilitate the accessibility of photolyase to
nucleosomal DNA.
Remodeling apparently destabilizes the structure of the
histone-DNA complex to such an extent that the photo-footprint reveals a naked DNA-like structure, and the activity of a base flip-out enzyme
can be accommodated. The details of that structure are not known at
present. If nucleosome sliding were involved, the octamer must move to
and even overlap the DNA end (8) to allow repair of cluster 18 but
inhibit repair of cluster 19.
Nucleosome Mobilization by yISW2 Influences CPD Repair--
ISWI
containing complexes have been shown to induce octamer sliding
(52-55). Yeast ISW2 is a two-subunit complex belonging to the ISWI
group of ATP-dependent remodeling factors, which influences nucleosome positioning in vivo (56) and nucleosome spacing
in vitro (57). Here we provide two lines of evidence that
His-tagged yISW2 can act on UV-damaged nucleosomes and move the
nucleosome from the end to a more central position in an
ATP-dependent manner. First, an altered migration was
observed in nucleoprotein gels. Second, the photoreactivation revealed
an altered repair pattern, in particular enhanced repair at sites
toward the end of the fragment and inhibition in the center. It has to
be pointed that, in contrast to the ySWI/SNF, yISW2 was not firmly
bound to nucleosomes. Thus, the repair data do not reflect the
accessibility in the complex but rather the accessibility in the
remodeled products. Very recently, ISW2 has been shown to move
undamaged nucleosomes from an end position to a central position on a
DNA fragment (58), thus indicating that the ISW2 complex behaves
similarly on undamaged and damaged nucleosomes.
Several mechanisms are discussed how nucleosomes are mobilized by
ATP-dependent remodeling complexes: (i) disruption and
reformation of all histone-DNA contacts, (ii) formation of a DNA bulge,
or (iii) local twist of DNA that propagates on the histone surface (reviewed in Ref. 2). The results shown here demonstrate that ySWI/SNF
and yISW2 in the presence of ATP can remodel UV-damaged nucleosomes.
Thus, irrespective of the underlying mechanism, UV lesions do not
inhibit those structural transitions.
DNA Damage Accessibility in Vivo--
To defend the cells against
extensive mutagenesis of the genome, all DNA lesions need to be
repaired efficiently (15). Although there is a pronounced modulation by
nucleosomes and other protein-DNA interactions, both nucleotide
excision repair and photolyase almost completely remove UV-induced DNA
lesions (4, 5). High resolution repair analyses on positioned
nucleosomes of the URA3 gene in yeast showed similar repair
patterns for NER and photolyase: fast repair in the linker and a
decrease toward the center of the nucleosomes (17, 19, 59). In
combination with multiple positions found by nuclease footprinting,
those studies (59) suggest that intrinsic mobility of nucleosomes might
place a lesion in the linker DNA, thus providing a window of
accessibility for damage recognition.
Alternatively, there is an increasing number of repair-related proteins
with potential roles in chromatin remodeling (1). CSB and its yeast
homologue Rad26 belong to the SNF2 family and are involved in the
transcription-coupled repair rather than in the repair of
non-transcribed DNA (28, 60). Recombinant CSB was shown to remodel
undamaged nucleosomes and nucleosome arrays in vitro,
thereby being the first repair enzyme demonstrated to possess
remodeling activity (29). Rad7-Rad16 is a complex of the NER pathway of
yeast Saccharomyces cerevisiae that is essential for repair
of nontranscribed, nucleosomal chromatin (61) and recognizes UV lesions
in an ATP-dependent manner in vitro (62). Rad16
has homology to SNF2 (63) and therefore might play a role in nucleosome
remodeling to generate space for the other NER proteins (4). ACF, on
the other hand, is a chromatin assembly and remodeling factor
containing ISWI and Acf1, which was shown to facilitate NER of a
specific lesion located in linker DNA but not of a lesion in the
nucleosome (25). The observation made here that two remodeling activities, ySWI/SNF and yISW2, can act on damaged nucleosomes and
alter the accessibility for a base flip-out enzyme suggests that
similar activities could play a role in vivo. This
hypothesis is further supported by the recent findings that ySWI/SNF
stimulates the human excision reaction on a nucleosome containing a
bulky adduct (13) and that binding of transcriptional activators to their cognate sequences in the absence of transcription stimulates NER
by inducing a local chromatin remodeling mediated by ATP-driven chromatin remodelers and acetyltransferases (11).
For transcriptional regulation, nucleosome remodeling complexes are
recruited to the promoter regions of specific genes by transcription
factors. The situation is different for DNA repair, because DNA lesions
are generated almost randomly all over the genome. This implies that,
in principle, a DNA lesion needs to be recognized first, before
nucleosome remodeling activities can be recruited. Therefore, damage
accessibility depends on the structural properties of the region
containing the DNA lesion (e.g. nucleosome, linker, and
nucleosome-free region) and on the genetic activity, e.g.
whether it is transcribed or replicated. We might speculate about a
more general role of remodeling activities in chromatin organization.
Since all ATP-dependent remodeling complexes apparently can
change the structure or translational positions of nucleosomes, it
seems conceivable that these complexes might also act randomly on
the chromatin substrate in order to enhance the intrinsic dynamic properties of nucleosomes and keep chromatin in a "fluid" state. This would facilitate any DNA sequence recognition in
transcriptional regulation and repair and, in addition, adjust
packaging constraints imposed by chromosome metabolism. Thus,
remodeling complexes might perform a rather general role in the
maintenance of chromosome structure.
In this study, E. coli photolyase was used as a tool to
assess CPD accessibility and repair in nucleosomes. However, E. coli photolyase has many properties in common with the yeast
S. cerevisiae photolyases. Both photolyases share a high
sequence homology, require the same co-factors, and can partially
cross-complement each other in photoreactivation-deficient mutants
(reviewed in Ref. 16). Photolyase repair CDPs via a "flip-out"
mechanism (47, 48) as do many other DNA-processing enzymes, including methyltransferases and DNA glycosylases involved in base excision repair (64). It is conceivable that those flip-out enzymes have similar
structural requirements for DNA accessibility. Indeed, as observed for
photolyase and T4-endonuclease V (20), human uracil-DNA glycosylases,
UNG2 and SMUG1, were able to remove uracil from nucleosomes, but the
efficiency of uracil excision from nucleosomes was severely reduced
when compared with naked DNA (65). With our model system we have tested
a possible contribution of nucleosomes and remodeling activities toward
DNA repair of UV lesions. Thus, it will be important to investigate
whether similar observations can be made with other enzymes and DNA
lesions and whether a contribution of remodeling activities can be
detected as a requirement or help for repair processes in living cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dATP and dTTP.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (48K):
[in a new window]
Fig. 1.
The ATDED-long nucleosome, a model substrate
for DNA repair analysis. A, schematic drawing of the
ATDED-long nucleosome. Indicated are map units (MU),
position of the radioactive end label (asterisks),
pyrimidine clusters (bars and sequences numbered
from 1 to 19), cutting sites of restriction enzymes
(AflIII, HhaI, and XhoI), and location
of the histone octamer (ellipse). B, DNase I
footprints. Lane 1, sequence marker (A/G);
lanes 2 and 3, digestion products of nucleosomes
(Nucl.); lanes 4, digestion products of naked
DNA. Black dots and numbers mark
nucleosome-specific DNase I cutting sites. The ellipse marks
the position of the histone octamer and its putative center;
black bars mark the position of the clusters 1-19.
C, restriction enzymes accessibility experiment (top strand
labeled). Nucl., digestion products of nucleosomes;
DNA, digestion products of naked DNA; %cut, the
fraction of molecules cut in each lane.
View larger version (53K):
[in a new window]
Fig. 2.
Modulation of photoreactivation by a
positioned nucleosome. DNA was end-labeled either on the top or
bottom strand, reconstituted in nucleosomes, irradiated with 750 J/m2 UV (UV +), and exposed to photolyase and
photoreactivating light for 5-120 min. A, nucleoprotein
gels containing 10% glycerol. D, naked DNA; N,
nucleosomes; Phr., photolyase. B,
photoreactivation in nucleosomes and naked DNA. Nucl.,
UV-irradiated nucleosomes; DNA, DNA isolated from
irradiated nucleosomes; 5' to 120', minutes of
photoreactivation; T4endoV (+), DNA cleaved at
CPDs with T4-endonuclease V. Lanes 2, initial CPD
distribution; lanes 4-9, photoreactivation in nucleosomes;
lanes 10-13, photoreactivation in naked DNA; lanes
1 and 3, damaged DNA treated with no T4-endoV and an
excess of T4-endoV, respectively; ellipse, the position of
the histone octamer; black bars 1-19, pyrimidine clusters
(as in Fig. 1A). C, repair is shown as the
fraction of CPDs removed in 30 min in clusters 1-19. Nucl.,
nucleosomes (black bars); DNA, white
bars.
View larger version (67K):
[in a new window]
Fig. 3.
Remodeling of the nucleosome by
ySWI/SNF. Structural analysis by DNase I and UV
photo-footprinting. DNA was labeled at the bottom strand, reconstituted
in nucleosomes (Nucl.), and incubated for 30 min at 30 °C
with ySWI/SNF (one complex per nucleosome (A and
B) or 0.8 complex per nucleosome (B and
C)) in the presence or absence of 1 mM ATP.
A and C, nucleoprotein gels containing 10%
glycerol. B, naked DNA, nucleosomes and complexes were
treated with DNase I. The products are resolved in a sequencing gel.
D, naked DNA, nucleosomes and complexes were treated
with 500 J/m2 UV light. DNA was extracted and cut at CPDs
with T4-endoV. The products are resolved in a sequencing gel. Presence
of naked DNA, DNA, +; nucleosomes,
Nucl., +; remodeling complex,
SWI/SNF, +; ATP, ATP +;
competitor DNA, plasmid DNA, +; DNA cut at CPDs
with T4-endoV, T4-endoV +; well, W; migration of
free DNA, D; migration of nucleosomal complexes,
N, N'; DNase I pattern of nucleosomes,
dots and numbers 1383-1497;
pyrimidine clusters, black bars 12-19; the nucleosome
position, ellipse.
View larger version (40K):
[in a new window]
Fig. 4.
Enhanced CPD repair on nucleosomes remodeled
by ySWI/SNF. DNA was end-labeled on the bottom strand,
reconstituted in nucleosomes, irradiated with 500 J/m2 UV,
incubated with ySWI/SNF (0.6 complex per nucleosome), and exposed to
photolyase and photoreactivating light for up to 60 min. A,
nucleoprotein gel. D, naked DNA; N and
Nucl., nucleosomes; W, wells. B,
nucleoprotein gel analysis after competition of ySWI/SNF. The samples
described in A were incubated with an excess plasmid DNA for
45 min at room temperature. The fraction of material in the well
(W), the nucleosomal bands (N), and the naked DNA
bands (D) is indicated for each lane (bottom).
C, photoreactivation of naked DNA, nucleosomes, and
remodeled nucleosomes. Description is as in Fig. 2B.
Lane 2, initial CPD distribution; lanes 4-6,
photoreactivation in nucleosomes; lanes 7-9,
photoreactivation of nucleosomes and ySWI/SNF; lanes 10-12,
photoreactivation of nucleosomes, ySWI/SNF and ATP; lanes
13-15, photoreactivation of naked DNA isolated from irradiated
nucleosomes; lanes 1 and 3, damaged DNA treated
with no T4-endoV and an excess of T4-endoV, respectively;
ellipse, the position of the histone octamer; black
bars 10-19, pyrimidine clusters. D,
fraction of CPD repaired in 30 min in clusters 10-19. Nucleosomes
(open bars), nucleosomes incubated with ySWI/SNF (gray
bars), nucleosomes incubated with SWI/SNF and ATP (black
bars). (Data of Figs. 3 and 4 are from three independent
experiments reproducing ATP-dependent modulation of DNA
accessibility (Fig. 3B and Fig. 4, C and
D) and DNA structure (Fig. 3D and Fig.
4C).)
View larger version (58K):
[in a new window]
Fig. 5.
Nucleosome mobilization by yISW2 alters CPD
repair. DNA was end-labeled either at the top or bottom strand,
reconstituted in nucleosomes, irradiated with 500 J/m2 UV,
incubated with yISW2 (0.7 complex per nucleosome), and exposed to
photolyase and photoreactivating light for up to 60 min. A,
nucleoprotein gel. D, naked DNA; N1, nucleosomes;
N2, nucleosomes with reduced mobility. B,
photoreactivation in naked DNA, nucleosomes (Nucl.), and
remodeled nucleosomes. Description is as in Fig. 2B.
Lane 2, initial CPD distribution; lanes 4-6,
photoreactivation of nucleosomes; lanes 7-9,
photoreactivation of nucleosomes and yISW2; lanes 10-12,
photoreactivation of nucleosomes, yISW2, and ATP; lanes
13-15, photoreactivation of naked DNA isolated from irradiated
nucleosomes; lanes 1 and 3, damaged DNA treated
with no T4-endoV and an excess of T4-endoV, respectively;
ellipse, the position of the histone octamer; black
bars 1-19, pyrimidine clusters. C, fraction of CPD
repaired in 30 min in clusters 1-19. Nucleosomes (open
bars), nucleosomes incubated with yISW2 (gray bars),
nucleosomes incubated with yISW2 and ATP (black bars). (Data
are from two independent experiments (one each with the bottom and top
strand) reproducing yISW2 dependent modulation of DNA
accessibility.)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. R. E. Wellinger for discussions and Dr. U. Suter for continuous support.
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FOOTNOTES |
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* This work was supported by grants from the Swiss National Science Foundation, the ETH Zürich, and the Roche Research Foundation.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.
§ Present address: Dept. de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain.
** To whom correspondence should be addressed: Institut für Zellbiologie, ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Tel.: 41-1-633 33 23; Fax: 41-1-633 10 69; E-mail: thoma@cell.biol.ethz.ch.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M300770200
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ABBREVIATIONS |
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The abbreviations used are: CPDs, cyclobutane pyrimidine dimers; NER, nucleotide excision repair; T4endoV, T4-endonuclease V; CSB, Cockayne's syndrome B; m.o.i., multiplicity of infection; ACF, ATP-using chromatin assembly factor; y, yeast.
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