Download Chromatin Remodeling Facilitates DNA Incision in UV

Mol. Cells, Vol. 18, No. 1, pp. 100-106
M olecules
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Cells
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KSMCB 2004
Chromatin Remodeling Facilitates DNA Incision in UV-damaged
Nucleosomes
Kyungeun Lee, Deok Ryong Kim1, and Byungchan Ahn*
Department of Life Sciences, College of Natural Sciences, University of Ulsan, Ulsan 680-749, Korea;
1
Department of Biochemistry, College of Medicine, Gyeongsang National University, Jinju 660-751, Korea.
(Received April 17, 2004; Accepted May 26, 2004)
The DNA repair machinery must locate and repair
DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that
chromatin remodeling might be required for efficient
repair in vivo. To investigate a possible contribution of
nucleosome dynamics and chromatin remodeling to the
repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on
the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4endoV) in reconstituted mononucleosomes positioned at
one end of a 175-bp long DNA fragment. Repair by
ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the
human nucleosome remodeling complex, hSWI/SNF,
promoted more homogeneous repair by ML-UV endo
and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be
facilitated by a fluid state of the chromatin resulting
from chromatin remodeling activities.
Keywords: Chromatin Remodeling; DNA Repair; Reconstituted Nucleosome.
Introduction
The DNA of eukaryotic cells is packaged into chromatin
by association with histone proteins (Khorasanizadeh,
2004; Kornberg and Lorch, 1999). The basic unit of
chromatin is the nucleosome core particle. The nucleosomal unit, 1.65 left-handed superhelical turns of
DNA wrapped around an octamer of four core histones, is
* To whom correspondence should be addressed.
Tel: 82-52-259-2359; Fax:82-52-259-1694
E-mail: [email protected]
the first level of chromatin compaction (Luger et al.,
1997; Richmond and Davey, 2003). The folding of DNA
into nucleosomes creates persistent, tight curvatures of
the DNA in which DNA segments are bent into the minor
groove, generating structural constraints on the nucleosomes (Richmond and Davey, 2003). Thus, the higher
order structure of eukaryotic DNA within nucleosomes
restricts its accessibility to DNA-binding proteins, thereby
inhibiting transcription and V(D)J recombination; however the effect on DNA repair remains unclear (Fyodorov
and Kadonaga, 2001; Green and Almouzni, 2002; Kwon
et al., 2000; Narlikar et al., 2001; Smerdon and Conconi,
1999; Thoma, 1999).
DNA repair in chromatin has been examined in vivo as
well in vitro. The initial biochemical studies, using crude
cell extracts to perform repair, showed that repair synthesis is strongly inhibited in reconstituted nucleosomes containing UV damage and in UV-irradiated simian virus 40
(SV40) minichromosomes (Araki et al., 2000). Furthermore, a detailed study of repair at specific sites in a
mononucleosome demonstrated that removal of cyclobutane pyrimidine dimers (CPDs) is inhibited at most nucleosomal positions (Kosmoski and Smerdon, 1999;
Schieferstein and Thoma, 1998). The efficiency of repair
of UV damage by photolyase or T4-endonuclase V is severely reduced in reconstituted nucleosomes. Recently,
purified human nucleotide excision repair (NER) factors
have been used to investigate DNA repair in nucleosomal
contexts (Hara and Sancar, 2002; Hara et al., 2000; Liu
and Smerdon, 2000; Wang et al., 1991). Damage excision
by purified NER factors is also inhibited in both UVirradiated SV40 minichromosomes and reconstituted nu-
Abbreviations: CPD, cyclobutane pyrimidine dimer; ML-UV
endo, Micrococcus luteus UV endonuclease; NER, nucleotide
excision repair; T4-endo V, T4-endonuclease V.
Kyungeun Lee et al.
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cleosomes. Repair in dinucleosomes is also strongly inhibited by this chromatin structure even when the lesion is
located within the linker DNA (Ura et al., 2001). Thus,
these studies show that DNA repair in cells is affected by
chromatin structure.
In principle all DNA processing must require access to
specific regions of eukaryotic genomes, and efficient repair of UV damage throughout the genome is needed to
prevent mutagenesis. The accessibility of DNA is modulated not only by the dynamic nature of nucleosomes,
such as nucleosome mobility, unfolding, and partial disruption, but also by protein complexes that remodel
chromatin structures (Widom, 1998). One class of chromatin remodeling machine consists of histone-modifying
complexes that covalently alter histone tails. Another
class, energy-dependent chromatin remodeling complexes,
alters chromatin structures using the energy of ATP hydrolysis (Peterson, 2002). These activities facilitate access
to buried sites in chromatin.
Thus, reactions are required that permit access of the
repair machinery to DNA damage in chromatin in initial
repair steps. What processes are involved? Extracts of
Xenopus eggs proficient in NER repaired a single UV
radiation photoproduct, and photoproducts at separate
sites, with variable efficiency (Kosmoski et al., 2001),
indicating that activities responsible for stimulating nucleosome repair might be present in the extracts. In support of this notion, chromatin remodeling factors have
recently been employed in repair reactions in vitro. The
yeast chromatin remodeling factors, ySWI/SNF and
yISW2, facilitate NER of acetylaminofluorene-guanine
adducts (AAF-G) and (6-4) photoproducts, and photolyase
action on UV damage in mononucleosome, respectively
(Gaillard et al., 2003; Hara and Sancar, 2002). On the
other hand, addition of recombinant ACF (ATP-utilizing
chromatin assembly and remodeling factor) to a purified
in vitro NER system largely relieved inhibition of repair
of dinucleosomal structures specifically at DNA lesions
placed in linker DNA (Ura et al., 2001). Thus, ATPdependent chromatin remodeling activities are likely to be
required for repair in nucleosomes.
Since DNA lesions are generated almost randomly over
the genome, a DNA lesion needs to be recognized before
chromatin remodeling factors can be recruited. Therefore,
damage accessibility depends on the structural properties
of the region within nucleosomes containing the DNA
lesion. This leads to the prediction that chromatin remodeling complexes may act randomly on chromatin in order
to enhance the dynamic properties of nucleosomes and
keep the chromatin in a fluid state. To test this we used a
eukaryotic chromatin remodeling factor together with
bacterial and viral repair enzymes. We show here that the
human SWI/SNF complex acts on UV-damaged nucleosomes to facilitate repair by T4-endoV and ML-UV
endo.
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Materials and Methods
DNA substrate and proteins Xenopus 5S rRNA gene fragments were isolated from a plasmid (pKS-5S, a kind gift from
Dr. Smerdon, WSU, Pullman, USA). The plasmid was linearized with SexAI and the 5′ end of the DNA was labeled with
T4 polynucleotide kinase in the presence of [γ-32P]ATP (Amersham Biosciences). The labeled DNA was digested with SalI,
and a 175-bp DNA fragment was purified from an agarose gel
using a gel extraction kit (Qiagen). T4 endonuclease V was purchased from Trevigene (Gaitherburg, USA). Micrococcus luteus
UV endonuclease was a generous gift from Dr. L. Grossman,
USA. Human SWI/SNF was a gift from Dr. J. Kwon (Ewha
Womans University, Korea).
Reconstitution of nucleosome core particles Radiolabeled
DNA samples were reconstituted into nucleosomes by salt gradient-mediated histone octamer exchange from chicken erythrocyte core particles as described (Liu et al., 2000). Briefly, endlabeled 5S rDNA (50 ng) was mixed with 42 µg of chicken
erythrocyte core particles in 1 M NaCl, 10 mM Tris-HCl, pH 7.5,
1 mM EDTA, 0.2 mM PMSF for 30 min at 4°C. The samples
were dialyzed in a microdialyzer (PIERCE, membrane MWCO
3500) against, sequentially, buffers containing 0.6 M NaCl and
50 mM NaCl, to complete reconstitution. The fraction of 5S
rDNA-reconstituted nucleosomes in the reconstitution reaction
was monitored by a DNA mobility shift assay on a 6% nondenaturing polyacrylamide gel in TBE buffer run for 1.2 h at
120 V.
To make UV-damaged nucleosomes, naked DNA was UVirradiated and then subjected to in vitro nucleosome reconstitution.
UV-irradiation DNA was irradiated on parafilm strips placed
on ice at a dose 500 J/m2 using a germicidal lamp (G15WT8,
Sylvania) emitting predominantly at 254 nm. UV dose was
measured using a UVX radiometer (UVP, USA) equipped with
a 254 nm photocell (model UVX-25, UVP, USA).
Assay of DNA repair To measure DNA repair activity, naked
DNA or reconstituted nucleosomes (1−2 ng) were incubated
with ML-UV endo in reaction buffer (10 mM Tris-HCl, pH 7.9,
10 mM MgCl2, 50 mM NaCl, 1 mM DTT. T4 endo V activity
was assayed in a different reaction buffer (25 mM NaPO4, pH
6.8, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA)
for the indicated times at 37°C.
After repair, each sample was combined with stop buffer (5
mM Tris-HCl, pH 7.6, 10 mM EDTA Proteinase K, 40 µg/µl final
concentration) and heated to 42°C for 3 min. Proteins were extracted with phenol/chroloform/isoamylalcohol, and the DNA was
precipitated. The precipitated DNA was resuspended in 95% formamide loading buffer before separation on an 8% sequencing
polyacrylamide gel (7 M urea in 1× TBE). The gels were dried
and exposed to X-ray films, and the densities of the incision bands
were quantified with Scion software (NIH image, USA).
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Repairing DNA Damage in Mononucleosomes
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Results
Reconstitution of mononucleosomes Nucleosomes were
reconstituted onto the 5′-end labeled 5S rDNA by stepwise dialyses from chicken erythrocyte core particles. A
schematic diagram of the 175-bp 5S rDNA fragment used
in this study is shown Fig. 1A. The ovals represent the
two major positions of the 5S nucleosome found previously in larger fragments (Panetta et al., 1998). Gel mobility shift analyses were carried out to assess nucleosome
reconstitution of both UV-irradiated and non-irradiated
5S rDNA fragments. When the mixture was analyzed on a
nondenaturing polyacrylamide gel, at least 90% of the
DNA was folded into mononucleosomes (Fig. 1B). During the assembly of nucleosomes, multiple nucleosome
positions would result in a heterogeneous nucleosome
population. However, since it has been reported that the
Xenopus 5S rRNA gene contains a specific nucleosomepositioning sequence (Liu and Smerdon, 2000; Panetta et
al., 1998), the homogeneous nucleosome population obtained in this study points to unique translational setting
of mononucleosomes at one end of the 175-bp long DNA
fragment (Fig. 1B). In addition, this reconstituted mononucleosome has a 28-bp space at one end which may
permit reassembly or sliding of nucleosomes as a consequence of interactions with DNA repair enzymes or
chromatin remodeling complexes.
Effect of the nucleosome on repair endonucleases Incision of CPDs in nucleosomes by E. coli photolyase and
T4-endo V is severely reduced when compared with naked DNA (Kosmoski and Smerdon, 1999; Schieferstein
and Thoma, 1998), suggesting a repressive role of nucleosomes on repair processes. However, the dynamic
properties of nucleosomes and different mechanisms for
damage recognition could contribute to the damage accessibility. Thus, we asked how DNA repair on nucleosomes
is affected by repair enzymes and nucleosome positioning.
We used ML-UV endo that contains both endonuclease
and AP lyase activities but whose damage recognition
mechanism is unclear (Grafstrom et al., 1982; Shiota and
Nakayama, 1997). In addition, since the nucleosome reconstituted with 175-bp 5S rDNA is known to contain a
limited number of molecules with different translational
settings, this reconstituted nucleosome has minimal nucleosome dynamics. For repair experiments, ML-UV
endo or T4-endoV was incubated with the reconstituted
175-bp 5S rDNA nucleosomes, and the products of digestion by the repair enzymes were displayed by sequencing
gel electrophoresis. The bands in lanes 2 and 4 in Fig. 2
show DNA damage-specific incisions at CPD sites on
naked UV-damaged DNA. In contrast, the CPDs of the
reconstituted nucleosomes in lanes 3 and 6 were resistant
to T4-endo V and ML-UV endo and the overall repair
efficiency of the two repair enzymes was greatly reduced.
A
B
Fig. 1. Reconstitution of nucleosome core particles. A. Schematic diagram of the reconstituted mononucleosome. The arrow
line is the 175-bp fragment and the ovals are the predominant
sites of nucleosome formation. Restriction sites used to generate
this fragment are shown. B. The 175-bp fragment of the
Xenopus 5S rRNA gene containing CPDs was reconstituted into
nucleosome core particles by salt-gradient mediated exchange.
A characteristic band shift on a native 6% polyacrylamide gel
was produced. Lane 1, undamaged naked DNA; lane 2, undamaged nucleosome; lane 3, UV-damaged naked DNA; lane 4,
UV-damaged nucleosome. D, naked DNA; RN, reconstituted
nucleosome.
These results imply that the reconstituted nucleosomes are
not displaced either by UV damage or by incubation with
the repair enzymes. The extent of incision was not
changed when incubation time was increased up to 2 h
(data not shown).
Effect of SWI/SNF on incision of CPDs in nucleosomes
In contrast to the inhibition of incision in nucleosomes in
vitro, DNA lesions in nucleosomes are repaired in vivo.
Therefore, we asked whether nucleosome remodeling
activities can promote CPD repair in UV-damaged nucleosomes. We tested the human SWI/SNF (hSWI/SNF)
complex in nucleosome repair with ML-UV endo and T4endo V. Human SWI/SNF is known to enhance the accessibility of nucleosomes to restriction enzymes and transcription factors in vitro without disrupting the histone
octamer (Cote et al., 1998; Logie and Peterson, 1997;
Owen-Hughes and Workman, 1996; Utley et al., 1997).
Nucleosomes composed of UV-damaged DNA were
incubated with hSWI/SNF and either ML-UV endo or T4
endo V. The results are shown in Fig. 3. A substantial
increase in repair was observed when the nucleosomes
were incubated with either ML-UV endo or T4 endo V in
Kyungeun Lee et al.
103
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A
B
Fig. 2. Incision of naked DNA and reconstituted nucleosomes
by ML-UV endo (ML) and T4-endoV (T4). The 175-bp 5S
rDNA fragments was end-labeled at its SexA 1 site, irradiated
with 500 J/m2, reconstituted into nucleosomes, and incubated
with the repair enzymes for 120 min. The DNA samples were
separated on a DNA sequencing gel. A major translational position of the 5S nucleosome is indicated by the oval on the right
side of the gel.
the presence of hSWI/SNF (lanes 3 and 6). Clearly, incision of CPDs in the nucleosomes was stimulated by
hSWI/SNF (lanes 3 and 6 in Fig. 2). Interestingly, when
ML-UV endo was incubated with nucleosomes in the
presence of hSWI/SNF, the incision of the nucleosomal
DNA was somewhat more efficient than with T4 endo V
(Fig. 3B). Although ML-UV endo acts on CPDs as does
T4-endoV, the residues for damage recognition are not the
same (Shiota and Nakayama, 1997). Thus, this result implies that ML-UV endo may act on CPDs by a different
mechanism and that the different extents of incision may
be due to different degrees of accessibility of the repair
enzymes resulting from chromatin remodeling.
It has been shown that yeast SWI/SNF and ISW2
stimulate both repair by human NER (Hara and Sancar,
2002) and by E. coli photolyase (Gaillard et al., 2003).
Therefore, the results of the present study support these
previous findings. Since the chromatin remodeling factors
and repair proteins are from different organisms, specific
protein-protein interaction between the remodeling factors
and the repair proteins cannot account for the facilitation
of repair. Rather, it is likely that remodeling activities
alter the dynamic properties of the chromatin structures in
a random manner throughout the chromatin.
Fig. 3. Effect of SWI/SNF on the kinetics of incision of CPDs in
nucleosomes by ML-UV endo and T4-endoV. A. UV-damaged
nucleosomes were incubated with the repair enzymes in the
presence of 0.48 nM SWI/SNF for 2 h at 30°C. The reaction
products were separated on a denaturing sequencing gel (8% in
1× TBE). B. Proportions of CPDs repaired in 120 min. a-e. Nucleosomes incubated with hSWI/SNF and ML-endo (black bars),
nucleosomes incubated with hSWI/SNF and T4 (open bars).
(Data in Fig. 3B are a mean of two independent experiments.)
Discussion
We have investigated in vitro repair incision in nucleosomes. The activities of ML-UV endo and T4 endo V
were strongly impaired on nucleosomes, suggesting that
the accessibility of these enzymes to DNA damage in
chromatin is restricted. However, the use of chromatin
remodeling factors facilitated repair of the DNA damage
in nucleosomes. Thus, our results suggest that chromatin
remodeling is necessary in cells to overcome the inhibition of repair in nucleosomes.
The initiation of DNA transactions in chromatin im-
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Repairing DNA Damage in Mononucleosomes
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plies that nucleosome positioning is biased to facilitate
access by initiation factors. Thus, preferential positioning
of nucleosomes could place factor-binding sequences in
favorable positions for transcription (Flaus and Richmond,
1998). Furthermore, nucleosomes are intrinsically mobile
and permit access to their DNA in vitro (Polach and
Widom, 1995; Studitsky et al., 1997). On the other hand,
different translational settings of the DNA on nucleosomes
can alter the affinity for transcription factors and expose it
to restriction enzymes (Anderson et al., 2002).
When reconstituted nucleosomes are used in repair, the
variability in translational settings of the DNA on the histone octamer surface could permit transient exposure of
DNA damage. Indeed, it has been demonstrated that different settings of DNA on a histone octamer permit transient exposure of a uracil residue (Beard et al., 2003),
permitting some excision of the uracil by DNA glycosylase. However, when the translational setting of DNA was
locked at specific sequences, access of a restriction enzyme and repair proteins was completely prevented
(Beard et al., 2003).
The 175-bp DNA used in this study has been shown to
have a unique preferential translational setting for exonuclease III (Liu et al., 2000). As shown Fig. 1B, only one
nucleosome structure was reconstituted in our experiments. If there were multiple translational settings,
smeared bands or multiple shifted bands would have been
observed. When subjected to UV light, the major UV
photoproducts, cyclobutane pyrimidine dimers (CPD), are
formed. The overall helical axis of DNA containing a
CPD bends ~30° toward the major groove and unwinds
~9° (Park et al., 2002). However, an extensive study of
the effect of UV irradiation on nucleosome positioning
showed no significant change in either translational setting or rotational positioning (Liu et al., 2000).
Previous studies have shown that in vitro repair by
photolyase and T4 endo V is severely inhibited in nucleosomes (Kosmoski and Smerdon, 1999; Schieferstein
and Thoma, 1998). Since both enzymes bend DNA and
flip out a pyrimidine dimer into their active sites (Park et
al., 1995; Vassylyev et al., 1995), it might be expected
that the ability of the repair enzymes to alter DNA structure would permit access to the DNA damage as a result
of the dynamics of the nucleosomal DNA. However, such
a flip-out mechanism appears to be severely inhibited by
the structural constraints of nucleosomes (Kosmoski,
1999; Schieferstein, 1998). Thus, it is of interest to know
whether different types of repair enzymes behave differently with regard to recognizing damage in nucleosomes.
In this study, we tested Micrococcus luteus UV endonuclease (ML-UV endo) to see whether it could recognize
and incise DNA damage on nucleosomes. ML-UV endo is
known to have glycosylase and AP lyase activities
(Grafstrom et al., 1982; Shiota and Nakayama, 1997) and
to incise UV-photoproducts specifically, as do photolyase
and T4 endo V. However, the DNA damage recognition
mechanism of ML-UV endo is unclear because the conserved structural domain found in proteins that act by a
flip-out mechanism has not been found in ML-UV endo.
As shown in Fig. 2, incision of DNA damage by ML-UV
endo was greatly inhibited on nucleosomes. This result
suggests that ML-UV endo is not able to destabilize the
nucleosome, and thus to allow it to recognize DNA damage, or to process along the core particle until it encounters a CPD. Furthermore, the E. coli NER protein, the
UvrABC endonuclease that uses ATP-hydrolysis to recognize DNA damage, also cannot repair CPDs in nucleosomes (unpublished data), suggesting that the unwinding activity of the UvrAB complex cannot relieve the
structural constraints of nucleosomes. Consequently, efficient repair of nucleosomal DNA must require alteration
of the nucleosomes with or without the help of remodeling activities.
Some chromatin remodeling factors that generally increase the accessibility of nucleosomal DNA to transcription factors, DNase I, and restriction endonucleases
(Logie and Peterson, 1997; Utley et al., 1997) and induce
octamer sliding (Jaskelioff et al., 2000; Whitehouse et al.,
1999) have been found to facilitate DNA repair in nucleosomes. Indeed, the yeast SWI/SNF and ISW2 complexes promote repair of CPDs by E. coli photolyase and
human NER proteins (Gaillard et al., 2003). In addition,
another chromatin remodeling factor, ACF, is reported to
enhance human NER of a lesion in the linker region of
dinucleosomes (Ura et al., 2001).
Our repair data show that hSWI/SNF stimulates repair
along the reconstituted DNA fragment. Therefore, hSWI/
SNF appears to generally alter the accessibility of nucleosomal DNA to UV-DNA glycosylases. Surprisingly,
human chromatin remodeling factor can act on chicken
nucleosomes, and facilitates repair of CPDs by bacteriophage and bacterial repair enzymes. This implies that
chromatin remodeling factors act randomly on the chromatin substrate rather than specifically by interacting with
repair proteins. Therefore, the DNA in the nucleosome
appears to be sufficiently relaxed, or extended, by the
SWI/SNF complex to allow the repair proteins to recognize DNA damage randomly. Two other remodeling factors, ISW1 and ISW2, that are capable of moving nucleosomes to a more central region of DNA, had no effect
on repair by T4-endo V (unpublished data). As the DNA
fragment has a space of only 28-bp from the edge of the
nucleosome, repositioning of the nucleosome in the central region by ISW1 and ISW2 may not be enough to alter
the repair pattern, even if they cause nucleosome movement.
To defend cells against extensive mutagenesis of the
genome, all DNA lesions need to be repaired efficiently.
Since DNA lesions are formed randomly over the genome
they need first to be recognized before nucleosome re-
Kyungeun Lee et al.
/
modeling activities can be recruited. Therefore, damage
accessibility depends on the structural properties of the
region containing the DNA lesions (such as nucleosomes,
linkers, and nucleosome-free regions) and on DNArelated processes such as whether the DNA is being transcribed or replicated. Since all ATP-dependent chromatin
remodeling factors can apparently modulate the structure
and settings of nucleosomes, it is possible that they play a
more general role in chromatin organization, acting randomly on the chromatin to enhance the intrinsic dynamic
properties of the nucleosomes and to keep the chromatin
in a ‘fluid’ state. Such general structural alterations of
nucleosomes by remodeling activities appear to be required for DNA damage recognition and repair. However,
it remains to be seen how chromatin remodeling activities
contribute to repair within the living cell.
Acknowledgments This work was supported by the Korean
Research Foundation Grant (KRF-2001-042-D00065) and in
part by the SRC fund to the IRC at University of Ulsan from the
Korea Science and Engineering Foundation and the Korean
Ministry of Science and Technology.
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