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Nucleotide excision repair (NER) functions to remove DNA damage
caused by ultraviolet light and by other agents that distort the DNA
helix. The NER machinery has been conserved in structure and function
from yeast to humans, and in humans, defective NER is the underlying
cause of the cancer-prone disease xeroderma pigmentosum. Here, we
reconstitute the incision reaction of NER in Saccharomyces
cerevisiae using purified protein factors. The Rad14 protein, the
Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease,
replication protein A, and the RNA polymerase II transcription factor
TFIIH were purified to near homogeneity from yeast. We show that these
protein factors are both necessary and sufficient for dual incision of
DNA damaged by either ultraviolet light or
N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted
system requires ATP, which cannot be substituted by adenosine
5`-O-(3-thiotriphosphate), suggesting that the hydrolysis of
ATP is indispensable for the incision reaction. The excision DNA
fragments formed as a result of dual incision are in the
24-27-nucleotide range.
Nucleotide excision repair (NER)
Another level of complexity in
understanding the biological roles of NER genes was introduced from the
observation that in addition to their requirement in NER, RAD3 and RAD25 are also essential for cell
viability
(1) . Studies of temperature-sensitive conditional
lethal mutations of these genes have indicated that both are essential
for RNA polymerase II transcription
(7, 12, 13) .
Studies with the rad3 and rad25 mutants defective in
ATPase/DNA helicase activities have indicated that whereas Rad3
ATPase/helicase activity is required for NER, the Rad25 ATPase/helicase
is essential for both NER and polymerase II
transcription
(7, 13, 14) . Rad3 and Rad25
proteins are two of the six subunits of polymerase II transcription
factor TFIIH, and it has been proposed that the entire TFIIH functions
in NER
(15, 16, 17, 18) .
In addition
to the above mentioned protein factors, the single-stranded DNA binding
protein RPA has been suggested to have a role in an early step of
NER
(19, 20) . In a reconstituted system reported
recently, Mu et al.(21) have shown that human RPA is
essential for incision of a DNA substrate containing a cholesterol
adduct.
Our goal has been to reconstitute nucleotide excision repair
in yeast with purified proteins. Because of the amenability of S.
cerevisiae to genetic and biochemical analyses, development of
such a system is essential for a detailed understanding of processes
that effect different steps of NER, including transcription-coupled DNA
repair. Various yeast protein factors that have been implicated in NER
in genetic and biochemical studies have now been purified to near
homogeneity in our laboratory. Here, we present our results, which
indicate that the incision step of NER can be accomplished by combining
the following highly purified protein components: Rad14, Rad4-Rad23
complex, Rad1-Rad10 complex, Rad2, RPA, and TFIIH. The damage-specific
incision reaction has a strict dependence on ATP, and our results imply
that the two incision nicks are made in a highly coordinated fashion.
In this work we have achieved the reconstitution of a system
that mediates dual incision of DNA damaged either by UV or AAF, by
combining the following essentially homogeneous S. cerevisiae factors: Rad1-Rad10 complex, Rad2, Rad4-Rad23 complex, Rad14, RPA,
and TFIIH consisting of Rad3, Rad25, TFB1, SSL1, p55, and p38 subunits.
Since all these purified protein factors are indispensable for
damage-specific incision, they represent the minimum set of factors for
accomplishing this reaction.
In our reconstituted system, incision
of damaged DNA shows a strict dependence on ATP or dATP, whose
hydrolysis is required for the repair reaction because the
non-hydrolyzable analog ATP
It is intriguing that nicking of the damaged DNA does not occur if
either the Rad1-Rad10 endonuclease or the Rad2 endonuclease is present
alone with the remainder of the incision NER components. This finding
strongly suggests that in addition to providing the endonucleolytic
activities for dual incision, the Rad1-Rad10 protein complex and the
Rad2 protein are also required for the proper assembly of the NER
ensemble at the damage site. The involvement of the two endonucleolytic
components in assembling the NER complex may serve to ensure that the
endonucleolytic scissions at the damage site occur in a coordinated
manner, as well as to minimize gratuitous nicking of DNA.
In
summary, the following major conclusions emerge from this work. First,
the damage recognition factor Rad14, the Rad4-Rad23 complex, the
Rad1-Rad10 endonuclease, the Rad2 endonuclease, RPA, and TFIIH are all
essential for the incision step of NER. Second, because of the high
degree of purity of the protein factors used, we infer that the
combination of these proteins is sufficient for the incision reaction.
Third, the incision reaction occurs only in the presence of ATP, and
our results strongly suggest a requirement of ATP hydrolysis in this
reaction. Finally, our observation that the size of the excision
fragment produced by the yeast incision enzyme complex resembles that
in humans indicates that the yeast and human NER machineries act in a
highly similar manner.
We are very grateful to A. Sancar and colleagues for
communication of their results prior to publication and to W. J. Feaver
and R. D. Kornberg for yeast strain YPH/TFB1.6HIS, for the E. coli plasmid that expresses 6-histidine tagged SSL1, and for a sample
of purified 6-histidine tagged SSL1 protein. We thank S. Wilson for
helpful discussions and A. McCullough for anti-SSL1 antibodies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
represents the most important cellular mechanism for the
removal of DNA damage induced by ultraviolet light (UV). Genetic
studies in the yeast Saccharomyces cerevisiae have identified
seven genes, RAD1, RAD2, RAD3, RAD4, RAD10, RAD14, and
RAD25, that are essential for NER
(1) . To begin to
define the biological roles of these genes, we have purified their
encoded proteins from yeast and characterized their biochemical
activities. This undertaking has allowed us to infer the involvement of
these proteins in different steps of the incision process, viz. in damage recognition, in DNA unwinding, and in dual incision of
the damaged DNA strand. Rad14 is a zinc metalloprotein with an affinity
for UV-damaged DNA
(2) . Rad3 is a single-stranded DNA-dependent
ATPase, and it also has DNA helicase and DNA
RNA helicase
activities
(3, 4, 5) . Rad3 exhibits a preference
for binding UV-damaged DNA that is dependent upon ATP and the degree of
negative superhelicity in DNA
(6) . Rad25 also possesses a
single-stranded DNA-dependent ATPase and DNA helicase
activities
(7) . Rad1 and Rad10 exist as a complex in
vivo, and complex formation between these proteins is essential
for their biological action
(8) . Rad1 and Rad10 together
comprise a DNA endonuclease
(9, 10) , and Rad2 also is a
DNA endonuclease
(11) .
UV Irradiation and AAF Treatment of Plasmid
DNA
Replicative form M13 mp18 DNA (90% supercoiled form)
was purified from infected Escherichia coli strain JM101 by
two rounds of cesium chloride banding. The DNA, at a concentration of
100 µg/ml in TE (10 mM Tris-HCl, pH 7.0, 0.2 mM
EDTA), was irradiated in 15-µl droplets for 60 s with a germicidal
lamp emitting at 254 nm and a fluence rate of 5 J/m
/s to
introduce
2.5 photoproducts/1000 base pairs of DNA. For treatment
with N-acetoxy-2-aminoacetylfluorene (AAF), the plasmid DNA,
10 µg or 30 nmol of nucleotides, was incubated at 37 °C for 12
h in the dark with 0.5 nmol of AAF in 200 µl of 50 mM
sodium acetate, pH 5.8. After extraction with diethyl ether, the DNA
was purified by ethanol precipitation and redissolved in TE to 100
µg/ml.
Reconstitution of NER in Vitro
(i) In the incision
assay, reaction mixtures (10 µl, final volume) were assembled in
buffer R (45 mM K-HEPES, pH 7.9, containing 8 mM
MgCl, bovine serum albumin at 120 µg/ml, 1.5
mM dithiothreitol, 2 mM ATP, an ATP-regenerating
system consisting of 30 mM creatine phosphate and 200 ng of
creatine kinase, and 60 mM potassium acetate and 20
mM KCl that were due to addition of the various NER protein
factors) and contained 100 ng of TFIIH, 50 ng of RPA, 8 ng of
Rad1-Rad10 complex, 10 ng of Rad2 protein, 20 ng of Rad4-Rad23 complex,
and 10 ng of Rad14 protein. The Rad1-Rad10 complex for use in these
studies was formed by incubating equimolar amounts of purified Rad1 and
Rad10 proteins for 24 h on ice in 20 mM Tris-HCl, pH 7.5,
containing 1 mM dithiothreitol and 500 µg/ml bovine serum
albumin to give a final concentration of 1 µM of the
protein complex. The combination of NER factors were incubated at 25
°C for 5 min before the undamaged DNA or damaged DNA, 100 ng each,
was added in 1 µl. After incubation at 30 °C for 10 min, SDS
and proteinase K were added to 0.5% and 200 µg/ml, respectively,
followed by a 5-min incubation at 37 °C to deproteinize the
reaction mixtures. Samples were run in 0.8% agarose gels in TAE buffer
(20 mM Tris acetate, pH 7.4, 0.5 mM EDTA). The gels
were treated with ethidium bromide (1 µg/ml in H
O) to
stain DNA, soaked in a large volume of water to reduce background
staining, and then photographed through a red filter.(ii) For
the excision assay, reaction mixtures (50 µl, final volume) were
assembled in buffer R and contained 500 ng of TFIIH, 250 ng of RPA, 40
ng of Rad1-Rad10 complex, 50 ng of Rad2, 100 ng of Rad4-Rad23 complex,
and 50 ng of Rad14. The combination of NER proteins was incubated at 25
°C for 5 min before 1 µg of undamaged or UV-damaged DNA was
added in 10 µl of TE. The complete reaction mixtures were incubated
at 30 °C for 60 min and then deproteinized by extraction with an
equal volume of buffered phenol. The DNA was precipitated by ethanol
and redissolved in 10 µl of TE, and 5 µl of which was treated
with 7.5 units of calf thymus terminal transferase (Boehringer
Mannheim) with 5 µCi of [
-
P]dideoxy ATP
(Amersham Corp.; 5000 Ci/mmol) for 60 min at 37 °C in a final
volume of 20 µl of the buffer supplied by the vendor. The labeling
mixtures were deproteinized by treatment with 0.5% SDS and 300
µg/ml proteinase K for 10 min at 37 °C, followed by the
precipitation of the DNA. The pellets were dissolved in 4 µl of
buffer and analyzed on 15% sequencing gels. The gels were dried and
exposed to x-ray films (Kodak Bio Max MR) to reveal the excision DNA
fragments.
Purification of NER Factors
Rad10 protein was
purified 1,000-fold to near homogeneity (Fig. 1B,
lane1) from yeast strain CMY135 harboring the
overproducing plasmid pSUC8 as described
(22) . The RAD14 gene contains an intron and encodes a protein with a predicted
size of 43 kDa, and it exhibits a size of 48 kDa in
SDS-PAGE
(23) . Rad14 protein has been purified
1,000-fold
to near homogeneity (Fig. 1B, lane 2) from
yeast strain YRP11 harboring the overproducing plasmid pR14.15
containing RAD14 under the control of the yeast alcohol
dehydrogenase I (ADC1) promoter, with the purification scheme
outlined in Fig. 1A. Rad1 protein was purified
2,000-fold to near homogeneity (Fig. 1C, lane1) from yeast strain CMY135 harboring the overproducing
plasmid pRR168 as described previously
(9) . Rad2 protein was
purified
3,000-fold to near homogeneity (Fig. 1C,
lane2) from yeast strain LY2 harboring the
overproducing plasmid pR2.26 as described
(11) . To overproduce
the RAD4-encoded protein in yeast for purification, the
protein coding frame of RAD4 was fused to the yeast ADC1 promoter to yield plasmid pR4.1, which was introduced into yeast
strain YRP11. The scheme presented in Fig. 1A was used
to purify Rad4 protein
4,000-fold to near homogeneity
(Fig. 1C, lane3) from YRP11(pR4.1).
The elution of Rad4 protein from various chromatographic matrices was
monitored by immunoblotting using affinity-purified polyclonal
antibodies raised against an insoluble form of Rad4 protein expressed
in insect cells with the use of baculovirus (data not shown). Rad4
protein has a predicted molecular mass of 87 kDa
(24) , but it
migrates in SDS-polyacrylamide gels with a relative molecular mass of
116 kDa. Throughout the purification of Rad4, we observed a precise
co-elution of a protein with a relative molecular mass of 57 kDa, which
is the same as that described for the RAD23 gene product (25)
that plays an important role in the proficiency of
NER
(1, 25) . By immunoblotting using affinity-purified
anti-Rad23 antibodies
(25) , we have verified that this 57-kDa
species was indeed Rad23 protein. Because in wild type yeast Rad23 is
more abundant than Rad4
(25) , only overproduction of Rad4 was
necessary to purify the Rad4-Rad23 complex (Fig. 1C,
lane3). TFIIH was purified >10,000-fold to near
homogeneity (Fig. 1D) from yeast strain YPH/TFB1.6HIS
that contains a 6-histidine tag in the TFB1 subunit of TFIIH
(26) as outlined in Fig. 1A. During purification,
the elution of TFIIH from various chromatographic matrices was
monitored by immunoblotting using affinity-purified antibodies specific
for the Rad3, Rad25, TFB1, and SSL1 subunits
(23) . The TFIIH
preparation used in this study contained six subunits, Rad25, Rad3,
TFB1, SSL1, p55, and p38 (Fig. 1D). RPA, consisting of
three subunits of 69, 36, and 13 kDa, was purified to near homogeneity
(Fig. 1B, lane3) from yeast strain
LP2749-9B using the procedure of Brill and Stillman
(27) .
Figure 1:
Protein factors for in vitro reconstitution of NER. A, purification schemes for Rad14,
Rad4-Rad23 complex, and TFIIH. B-D, SDS-PAGE of purified
NER factors. B, Rad10 (1 µg in lane1),
Rad14 (1 µg in lane2), and RPA (2 µg in
lane3) consisting of the 69-, 36-, and 13-kDa
subunits each marked by an asterisk were run in a 12%
denaturing polyacrylamide gel and stained with Coomassie Blue.
M, molecular size standards. C, 1 µg each of Rad1
(lane1), Rad2 (lane2), and the
complex of Rad4 (upperband marked by asterisk in lane 3) and Rad23 (lowerband marked by
asterisk in lane3) were run in a 7%
denaturing polyacrylamide gel and stained with Coomassie Blue.
M, molecular size standards. D, TFIIH (500 ng total
protein) was run in an 8% denaturing polyacrylamide gel and
silver-stained with a kit purchased from Bio-Rad. The various subunits
of TFIIH are indicated. The identity of subunits was verified by
immunoblotting using affinity-purified anti-Rad25, anti-Rad3,
anti-TFB1, and anti-SSL1 antibodies.
ATP-dependent Incision of UV-damaged DNA
To
examine whether the purified yeast factors Rad14, Rad4-Rad23 complex,
Rad1-Rad10 complex, Rad2, RPA, and TFIIH are sufficient for
reconstituting NER in vitro, they were incubated at 30 °C
in buffer containing ATP with plasmid DNA previously irradiated with UV
light to introduce photoproducts into the DNA. Reaction mixtures were
deproteinized and analyzed by agarose gel electrophoresis, followed by
staining with ethidium bromide to examine the fate of the DNA
substrate. Fig. 2A shows that when all the purified NER
factors were present, greater than 80% of the UV-damaged supercoiled
plasmid DNA was converted to the open circular form (lane4), indicating that incision of the damaged DNA had
occurred. By contrast, plasmid DNA that had not been subjected to UV
treatment was not acted on by the same protein factors
(Fig. 2A, lane2). Thus, the
combination of NER proteins carries out an incision reaction that is
highly specific for UV damage. Importantly, omission of any of the
components Rad1-Rad10 complex, Rad2, Rad14, Rad4-Rad23 complex, TFIIH,
or RPA from the reaction mixture abolished the formation of the open
circular form (Fig. 2A, lanes 5-10),
indicating that incision of UV-damaged DNA requires all of these
purified protein factors. It is of particular interest that nicking of
the UV-damaged DNA did not occur when either the Rad1-Rad10
endonuclease or the Rad2 endonuclease was omitted from the reaction
mixture (Fig. 2A, lanes5 and
6). This observation strongly suggests that both the
Rad1-Rad10 complex and the Rad2 protein, in addition to being the
endonucleolytic components, have a pivotal role in the proper assembly
of the incision enzyme complex at the damage site, thus ensuring that
the two incision nicks are made in a coordinated fashion.
Figure 2:
ATP-dependent incision of UV-damaged DNA.
A, reconstitution of the incision reaction using purified
protein factors. The DNA used was either not treated
(-UV, lanes1 and 2) or
treated with UV (+UV, lanes 3-10). The
full complement of NER factors (Rad1-Rad10 complex, Rad2, Rad4-Rad23
complex, Rad14, TFIIH, and RPA) was incubated with undamaged DNA
(lane2) and UV-damaged DNA (lane4) in buffer containing ATP at 30 °C for 10 min. One
of the aforementioned NER factors was absent in the reaction mixtures
in lanes 5-10 that contained UV-damaged DNA and ATP;
Rad1-Rad10 complex was omitted in lane 5, Rad2 was omitted in
lane6, Rad14 was omitted in lane7, Rad4-Rad23 complex was omitted in lane8, TFIIH was omitted in lane9, and RPA
was omitted in lane10, as indicated at the top of the figure. Bl, DNA incubated in buffer without any of
the NER factors (lanes1 and 3). B, incision of UV-damaged DNA requires ATP. The full complement of
NER factors (lane2 and lanes4-11) was incubated with either undamaged DNA
(-UV, lane2) or with UV-damaged DNA
(+UV, lanes4-11) in the
presence of ATP (lanes2 and 4), ADP
(lane6), dATP (lane7), ATPS
(lane8), CTP (lane9), GTP
(lane10), UTP (lane11), or
without any nucleotide (lane5) at 30 °C for 10
min. In lanes6 and 8, creatine kinase was
omitted to inactivate the ATP-regenerating system. No NER factors were
added in lanes 1 and 3. SC, supercoiled
form; OC, open circular form.
Because
Rad3 and Rad25 proteins both possess an ATP (dATP)-dependent helicase
activity that is required for NER (7, 13, 14), it was of considerable
importance to determine whether the incision of damaged DNA in our
reconstituted system requires ATP. We found that in the absence of ATP,
the incision of UV-damaged DNA does not occur (Fig. 2B,
compare lanes4 and 5). Whereas dATP is as
effective as ATP in promoting the incision reaction, ADP, CTP, GTP, and
UTP are inactive (Fig. 2B). Interestingly, the
non-hydrolyzable ATP analog ATPS is also inactive
(Fig. 2B, lane8), suggesting that ATP
hydrolysis is in fact required for the incision reaction. Furthermore,
Mg
is also required for incision (data not shown).
Incision of DNA Damaged by AAF
The NER machinery
has specificity for a variety of DNA lesions. We examined whether our
purified NER proteins would also incise DNA damaged by
N-acetoxy-2-aminoacetylfluorene. As shown in
Fig. 3A, plasmid DNA containing the AAF adduct was
incised when all the purified protein factors were present (lane4) but not when any of the factors was omitted from the
reaction mixture (lanes 5-10). The incision of
AAF-modified DNA also has a specific requirement for ATP or dATP
(Fig. 3B). ATPS did not promote incision
(Fig. 3B), again suggesting a requirement for ATP
hydrolysis in the repair of AAF-damaged DNA.
Figure 3:
ATP-dependent incision of DNA containing
AAF adducts. A, the NER factors incise AAF-containing DNA. The
undamaged DNA (lane2) or damaged DNA (lane4) was incubated with the Rad1-Rad10 complex, Rad2,
Rad4-Rad23 complex, Rad14, TFIIH, and RPA at 30 °C for 10 min in
buffer containing ATP. The reaction mixtures in lanes 5-10 contained AAF-damaged DNA and ATP, but one of the NER factors was
omitted; Rad1-Rad10 complex was absent in lane5,
Rad2 was absent in lane6, Rad14 was absent in
lane7, Rad4-Rad23 complex was absent in lane8, TFIIH was absent in lane9, and RPA
was absent in lane10. Bl, DNA incubated in
buffer without any of the NER factors (lanes1 and
3). B, incision of AAF-containing DNA requires ATP.
The full complement of NER factors (lane2 and
lanes 4-11) was incubated with either undamaged DNA
(-AAF, lane2) or with AAF-containing
DNA (+AAF, lanes 4-11) in the presence of
ATP (lanes 2 and 4), ADP (lane6),
dATP (lane7), ATPS (lane8),
CTP (lane9), GTP (lane10), UTP
(lane11), or without any nucleotide (lane5) at 30 °C for 10 min. In lanes6 and 8, creatine kinase was omitted to inactivate the
ATP-regenerating system. No NER factors were added in lanes1 and 3. SC, supercoiled form;
OC, open circular form.
Size of Excision DNA Fragments
NER in humans has
been shown to occur by way of dual incision of the DNA strand that
contains the lesion, resulting in the release of a DNA fragment
27-29 nucleotides in length
(28) . To detect the excision
DNA fragment, we incubated plasmid DNA damaged by UV light in the
reconstituted repair system. Following phenol extraction to remove
repair proteins, the DNA was purified and treated with calf thymus
terminal transferase in the presence of
[-
P]dideoxy ATP to label any excision DNA
fragments that might have been generated. As shown in Fig. 4, a
series of DNA fragments ranging in size from 25 to 28 nucleotides was
detected in the reaction mixture that contained UV-damaged DNA as
substrate (lane5) but not in the control that
contained undamaged DNA (lane2). Importantly, the
DNA fragments were not produced if ATP was absent (Fig. 4,
lane6) or when TFIIH was omitted from the repair
reaction (Fig. 4, lane4). Because the
P-labeling protocol added one nucleotide to the excision
DNA fragments, the actual size range of these fragments is 24-27
nucleotides.
Figure 4:
Size of the excision DNA fragments.
UV-irradiated (lanes3-6) and unirradiated
(lanes1 and 2) plasmid DNAs were incubated
with the full complement of NER factors (lanes2,
5, and 6) in the presence of ATP (lanes2 and 5) or in its absence (lane6). The reaction mixture in lane4 contained ATP but lacked TFIIH. The numbers to the right of the autoradiogram indicate the positions in nucleotides of the
DNA markers used. Bl, DNA incubated in buffer without any of
the NER factors (lanes1 and
3).
S does not promote the incision of
damaged DNA. The requirement for ATP and its hydrolysis in the incision
step of NER is consistent with the results from genetic studies
conducted with mutant variants of Rad3 and Rad25 proteins that are
defective in ATP hydrolysis
(7, 13, 14) . We
suggest that at the expense of ATP hydrolysis, the combined helicase
function of Rad3 and Rad25 creates a single-stranded region at the
damage site for dual incision by the Rad1-Rad10 and Rad2 endonucleases.
S, adenosine
5`-O-(3-thiotriphosphate); AAF,
N-acetoxy-2-aminoacetylfluorene.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.