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J Biol Chem, Vol. 273, Issue 51, 34180-34189, December 18, 1998
From the The nucleotide excision repair (NER) pathway of
eukaryotes involves ~30 polypeptides. Reconstitution of this pathway
with purified components is consistent with the sequential assembly of
NER proteins at the DNA lesion. However, recent studies have suggested
that NER proteins may be pre-assembled in a high molecular weight
complex in the absence of DNA damage. To examine this model further, we
have constructed a histidine-tagged version of the yeast DNA damage
recognition protein Rad14. Affinity purification of this protein from
yeast nuclear extracts resulted in the co-purification of Rad1, Rad7,
Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of
these proteins bound to the affinity resin in the absence of
recombinant Rad14. Furthermore, many of the co-purifying proteins were
present in approximately equimolar amounts. Co-elution of these
proteins was also observed when the nuclear extract was fractionated by
gel filtration, indicating that the NER proteins were associated in a
complex with a molecular mass of >1000 kDa prior to affinity
chromatography. The affinity purified NER complex catalyzed the
incision of UV-irradiated DNA in an ATP-dependent reaction.
We conclude that active high molecular weight complexes of NER proteins
exist in undamaged yeast cells.
The repair of base damage by the eukaryotic nucleotide excision
repair (NER)1 pathway
involves ~30 proteins (1). In the yeast Saccharomyces cerevisiae genes that encode polypeptides required for the events of base damage recognition, damage-specific incision, and
oligonucleotide excision during NER can be divided into two broad
groups. Genes such as RAD1, RAD2,
RAD4, RAD10, and RAD14 are
indispensable for NER but not for cell viability, whereas genes such as
RFA1, RFA2, RFA3, RAD3,
SSL1, SSL2, TFB1, TFB2,
TFB3, and TFB4 are essential for both NER and
cell viability (2-4). In addition, mutations in the RAD7,
RAD16, or RAD23 genes confer moderate sensitivity to killing by ultraviolet (UV) radiation (5, 6), suggesting that their
polypeptide products may either increase the efficiency of NER or
participate in a subpathway(s) of this process.
The characterization of cell-free extracts from mammalian (7) and yeast
cells (8) capable of catalyzing repair synthesis of DNA associated with
NER in vitro has led to important insights into the
molecular mechanism of this process. By using this assay it was
demonstrated that Rad3 protein functions in NER as a component of
TFIIH, a multiprotein complex that is required for transcription initiation by RNA polymerase II (9). The identification of Rad3 as a
subunit of this transcription factor provided an explanation for the
essential function of the RAD3 gene (10). Additionally, the
requirement for core TFIIH in NER predicted that genes encoding the
other six subunits of this complex are also indispensable for this
process. This has now been directly demonstrated for all seven proteins
using a yeast cell-free system (2,
9-13).2 Interestingly,
extracts prepared from rad7, rad16, or
rad23 strains, which exhibit moderate sensitivity to cell
killing by UV radiation (5, 6), are also defective in repair synthesis
in vitro (14). Recent studies have shown that
rad7 and rad16 mutant extracts are proficient in
DNA damage-dependent incision both in vitro and
in vivo but fail to excise the oligonucleotide fragment
containing the DNA lesion (15). Hence, the Rad7 and Rad16 proteins,
which interact with each other (16), appear to be required for
post-incision events during NER.
Distinct biochemical functions have been assigned to a number of the
NER gene products. Rad14 protein recognizes and binds to UV
radiation-induced DNA lesions, in particular (6-4) photoproducts, suggesting a role in the recognition of base damage (17). It appears
that the heterotrimeric protein complex Rpa, which is required for DNA
replication, is also involved in DNA lesion recognition (18-20). Rad3
and Ssl2 proteins, both of which are components of core TFIIH, are DNA
helicases that appear to be involved in unwinding the DNA duplex in the
vicinity of DNA lesions, thereby generating NER "bubbles" that
include sites of base damage (12, 21, 22).
Finally, Rad2 and a heterodimeric complex of Rad1 and Rad10 proteins
are DNA structure-specific endonucleases that are similar in that they
cleave DNA at duplex/single-strand junctions but differ in terms of the
polarity of the single strand that they cleave (23-25). These
junctions are likely generated by TFIIH-mediated "bubble"
formation. Rad2 protein cleaves the 5' single-strand at the junction
and hence operates as a 3' endonuclease with respect to sites of base
damage. Reciprocally, Rad1/Rad10 protein cleaves the 3' single-strand
at the junction and hence operates as a 5' endonuclease with respect to
sites of base damage (23-25). The combined action of these two
endonucleases is responsible for the dual incision events that are
characteristic of NER in all eukaryotes studied and that result in the
removal of DNA lesion-containing oligonucleotides 27-30 nucleotides in
length (20, 26). In addition to the proteins described above, a complex
of Rad4 and Rad23 proteins is required for early events during NER
(20); however, the precise roles of these proteins is not yet understood.
Recently, a relatively stable high molecular weight complex comprising
core TFIIH and multiple NER subunits was identified and extensively
purified from cell extracts prepared from yeast cells not exposed to
exogenous DNA-damaging agents (11). These observations led to the
suggestion that, in the yeast S. cerevisiae and possibly
other eukaryotes, a NER complex is assembled in the absence of
substrate base damage. In this initial study, the NER complex was
called a repairosome although it was not shown to perform any of the
reactions involved in NER. Here we define a repairosome as a stable
complex of proteins that carry out the DNA damage recognition and
incision reactions that are characteristic of NER. Recent studies have
also provided evidence suggesting the existence of a high molecular
weight complex of NER proteins in extracts from undamaged human cells
that catalyze DNA damage-dependent DNA synthesis (27).
Although it is plausible that such preformed multiprotein complexes may
function to constantly monitor the genome for sites of base damage and
catalyze NER in the presence of such damage, there is as yet no direct
biochemical evidence for such functions by the repairosome. It has also
not been established that yeast cells contain only a single
multiprotein complex that comprises both TFIIH and NER polypeptides.
Indeed, the primary question as to whether or not any NER complex is
pre-assembled in yeast (and other eukaryotes) or is assembled in a
step-wise process exclusively when cells are required to carry out NER
is controversial (11, 28) (see "Discussion.")
In initial studies that led to the elaboration of the repairosome
hypothesis, extracts containing histidine (his)-tagged Tfb1 protein
(one of the subunits of TFIIH) were fractionated by assaying for
complementation of defective RNAP II transcription in vitro (11). To further test the hypothesis that a high molecular weight complex of proteins comprising both TFIIH and NER subunits exists in
undamaged yeast cells, we have constructed a his-tagged derivative of
the DNA damage recognition protein Rad14 (which is not required for
RNAP II transcription) and expressed this recombinant protein in a
yeast strain deleted of the chromosomal RAD14 gene. Here we
present evidence that single step affinity chromatography of recombinant Rad14 protein from nuclear extracts results in the co-purification of multiple other NER proteins, including TFIIH subunits, in a complex with a molecular mass >1000 kDa. Additionally, we show that fractions containing this complex incise UV-irradiated DNA
in an ATP-dependent reaction.
Materials--
Purified mouse IgG was purchased from Pierce. The
Rad1 IgG monoclonal antibody, 3E3 (29), and the rabbit polyclonal
antibodies specific for Rad3 (30), Rad10 (31), Rad7, Rad16, and Rad23 (15), have been described previously. Antibodies for Tfb1 (32) and RPB1
were provided by Roger Kornberg, Stanford University. Antibodies for
the 34-kDa subunit of Rpa (4), PCNA, and Rad51 were supplied by Steve
Brill, State University of New Jersey, Peter Burgers, Washington
University, and Tom Kodadek, University of Texas Southwestern Medical
Center, respectively.
Rad1 and Rad10 proteins were purified as described previously (33). RPA
was purified from the yeast strain RKY2275 that overexpresses each of
the Rpa subunits, as described (34). Purified TFIIH containing
his-tagged Tfb1 (35) and RNA polymerase II holoenzyme (36) were
provided by Roger Kornberg. Recombinant XPG protein purified from
baculovirus-infected insect cells was obtained from Rick Wood, ICRF,
United Kingdom (37). Protein concentrations were measured by the
Bradford assay (38) using bovine serum albumin as the standard.
Plasmid Constructs--
A HindIII fragment from the
plasmid pGEM4Z-SP6RAD14, which contains the RAD14
open reading frame (39), was subcloned into pQE-30 (Qiagen) to produce
the plasmid pQE-hisRad14, in which a truncated version of
Rad14 deleted of the amino-terminal 14 amino acids was expressed as a
fusion protein with a poly(his) sequence at the amino terminus. An
EcoRI fragment encoding his-tagged Rad14 was subcloned in
the yeast plasmid pYES2 (Invitrogen) to produce plasmid
pYES2-hisRad14, in which recombinant Rad14 was expressed
from a GAL1-inducible promoter.
Cellular Sensitivity to UV Radiation--
Plasmids pYES2 and
pYES2-hisRad14 were transformed into the yeast strains SX46A
(MATa ade2 his3-52 ura3-52 trp1-289) and SX46A Purification of Recombinant Rad14 Protein--
The
Escherichia coli strain M15 pREP4 (Qiagen) harboring
pQE-hisRad14 was grown at either 30 or 37 °C for
preparation of soluble and insoluble recombinant Rad14, respectively.
Expression of recombinant Rad14 in exponentially growing cultures was
induced by the addition of isopropyl thiogalactoside to a final
concentration of 2 mM. After further incubation for 3 h, cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate (pH 7.8), 300 mM NaCl
containing a mixture of protease inhibitors (33) and lysed by
sonication. Recombinant Rad14 was purified from the clarified lysate by
metal chelating affinity chromatography using Ni-NTA beads (Qiagen) and
then by ion exchange chromatography using FPLC Mono Q and Resource S
columns (Amersham Pharmacia Biotech). Approximately 0.4 mg of Rad14
that was >95% homogeneous was obtained from 1 liter of culture.
The majority of recombinant Rad14, which was present in the insoluble
fraction, was purified by immobilized metal affinity chromatography
according to the manufacturer's protocol (Qiagen). Recombinant Rad14
was eluted from the Ni-NTA beads with 8 M urea, 100 mM Na2H2PO4, 10 mM Tris-HCl (pH 4.5) and was purified to near homogeneity
by electrophoresis through a preparative denaturing polyacrylamide gel
(40). The position of Rad14 in the gel was identified by reverse
staining with copper chloride using the Pro-Green Staining System
according to the manufacturer's protocol (Integrated Separation
Systems). Rad 14 was electroeluted from these gel slices using an
Electro-Eluter 422 (Bio-Rad) as suggested by the manufacturer.
Approximately 1.5 mg of Rad14 was obtained from a 1-liter culture.
In Vitro NER--
Yeast whole cell extract (40 µg) containing
overexpressed Rad2 protein (8), nuclear extract (200 µg) from SX46A
Rad14 Antiserum--
Mice were initially immunized with
recombinant Rad14 purified from the insoluble fraction as described
above, plus Freund's complete adjuvant. Subsequently the animals were
immunized at 2-week intervals with the same antigen plus Freund's
incomplete adjuvant and bled 10 days after each injection.
Preparation of Nuclei--
Stationary phase cultures of yeast
strains grown in uracil dropout media were diluted 1:20 in 8 liters of
2% Bacto-peptone, 1% yeast extract containing 1% galactose, 1%
raffinose as the carbon source. When the cultures reached late
logarithmic phase cells were harvested, washed with 0.1 M
EDTA (pH 8.0), 1 mM DTT and resuspended in the same buffer
at 10 ml/g wet weight of cells. Cells were resuspended in 1% yeast
extract, 2% Bacto-peptone, 1 M sorbitol (1 ml/g wet weight
of cells) containing yeast lytic enzyme (ICN, 1.4 mg/g wet weight of
cells) and incubated with shaking at 30 °C. When >80% of the cells
had been converted to spheroplasts digestion was stopped, and
spheroplasts were collected as described (8). Spheroplasts were
resuspended in 50 mM Tris-HCl (pH 8.0), 20 mM
KCl, 2 mM EDTA, 0.125 M spermidine, 0.05 M spermine, 1% thiodiglycol, 18% Ficoll plus a mixture of
protease inhibitors (33) (5 ml/g wet weight of cells), and after Dounce
homogenization the lysate was cleared by centrifugation at 7000 rpm for
10 min in a Sorvall SS34 rotor. The supernatant was centrifuged twice more at 7000 rpm for 5 min before a final spin at 15,000 rpm for 30 min
which pelleted the nuclei. After removal of the supernatant the pellet
of nuclei was flash-frozen in liquid nitrogen and stored at
Affinity Purification of Rad14 and Associated
Proteins--
Nuclei were resuspended in 20 mM Tris
acetate (pH 8.0), 300 mM potassium acetate, 20% glycerol,
5 mM Immunoblotting--
Polypeptides were separated by denaturing
gel electrophoresis and transferred electrophoretically to a
nitrocellulose membrane. Following incubation with TBST (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween
20) containing 5% dried milk for 30 min at room temperature, the
membrane was incubated overnight at 4 °C with the primary antibody
in TBST containing 2% dried milk overnight. Antigen-antibody complexes
were detected by enhanced chemiluminescence (Pierce) after incubation
for 60 min at room temperature with the appropriate secondary antibody
linked to horseradish peroxidase.
In the repairosome purification scheme described above Rad14-associated
proteins were detected in both the 20 and 50 mM imidazole eluates from the Ni2+-NTA agarose beads. To facilitate
analysis of the repairosome, the 20 mM elution step was
omitted. Rad14 and the putative repairosome subunits were eluted by 50 mM imidazole. To determine the relative amounts of proteins
that co-purify with recombinant Rad14, 5 µg of the 50 mM
imidazole eluate and varying amounts of a purified NER protein were
separated by denaturing gel electrophoresis and detected by
immunoblotting. The amount of chemiluminescent signal was quantitated
by scanning of the x-ray film in a laser densitometer. After arbitrary
densitometer units were plotted against protein concentration to
construct a standard curve for each purified protein, the amount of
that protein in the affinity purified fraction was estimated.
Analytical Gel Filtration--
Aliquots (200 µl) were applied
to an FPLC Superose 6 HR10/30 column (Amersham Pharmacia Biotech)
equilibrated with buffer A. Proteins were eluted with buffer A at a
flow rate of 0.4 ml/min. Fractions (400 µl) were collected,
flash-frozen in liquid nitrogen, and stored at Nuclease Assays with DNA Substrates Containing
Duplex/Single-strand Junctions--
Radiolabeled partial duplex DNA
molecules with non-complementary 3' and 5' extensions at the same end
(Y structures) were constructed by end-labeling one of the
oligonucleotides with T4 polynucleotide kinase prior to annealing as
described previously (29). These molecules were purified by
electrophoresis through a preparative 15% polyacrylamide gel (29).
Reaction mixtures (20 µl, final volume) contained 50 mM
Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM
DTT, labeled DNA substrate with a Y structure (250 pmol), and aliquots
of fractions from the affinity column (1 µg of protein). In some
cases the affinity column fractions were preincubated with either the
Rad1 monoclonal antibody, 3E3 (27), or purified mouse IgG for 10 min at
37 °C. Reactions were incubated at 37 °C for 40 min. Proteinase K
(100 µg) and SDS (2 µl of a 1% solution) were added to
deproteinize the reaction mixtures, and incubations were continued at
37 °C for 10 min. Samples were electrophoresed through a
non-denaturing 15% polyacrylamide gel. After drying, labeled
oligonucleotides in the gel were detected by autoradiography.
Endonuclease Assays with UV-irradiated and Undamaged Plasmid DNA
Substrates--
Supercoiled plasmid DNA was irradiated with UV light
at 1.3 J/m2/s. Reaction mixtures (15 µl) contained 45 mM Hepes-KOH (pH 7.9), 8 mM MgCl2,
30 mM potassium acetate, 120 µg/ml nuclease-free bovine serum albumin, 1.5 mM DTT, damaged or undamaged supercoiled
plasmid DNA, and aliquots of fractions from the affinity columns as
indicated. In some cases the affinity column fractions were
preincubated with either the Rad1 monoclonal antibody, 3E3 (29), or
purified mouse IgG for 10 min at 37 °C. When indicated, an
ATP-regenerating system composed of 2 mM ATP, 30 mM creatine phosphate, and 200 ng of creatine kinase was
added. After incubation at 37 °C for 30 min proteinase K was added
to a final concentration of 200 µg/ml and incubation continued for a
further 10 min. Samples were electrophoresed through 0.8% agarose gels
in 20 mM Tris acetate (pH 7.5), 5 mM EDTA.
Plasmid DNA was detected by staining with ethidium bromide (1.5 µg/ml
in distilled H2O). After destaining in 1-2 liters of
distilled water gels were photographed through a red filter.
His-tagged Rad14 Protein Complements Both the UV Radiation
Sensitivity and Defective NER of a rad14 Deletion Mutant--
We
constructed a version of the DNA damage recognition protein Rad14 (39)
in which the amino-terminal 14 amino acids were replaced by a poly(his)
sequence. A plasmid that expresses this version of Rad14 protein from a
GAL1 promoter restored the UV radiation sensitivity of a
RAD14 deletion mutant to wild type levels under both
inducing conditions (galactose medium, Fig. 1A) and non-inducing
conditions (glucose medium, Fig. 1B). To monitor the
expression of Rad14 protein, we generated polyclonal antibodies to
recombinant Rad14 purified from E. coli. In glucose medium,
the cellular level of recombinant Rad14 was similar to that of native
Rad14 in the wild type strain, whereas in galactose medium the cellular
level of recombinant Rad14 was about 100-fold higher (Fig.
2). As expected, no immunoreactivity was
detected with similarly prepared nuclear extracts from the
Although the majority of recombinant his-tagged Rad14 protein expressed
in E. coli is insoluble, we purified recombinant Rad14 from
the soluble fraction to >95% homogeneity by affinity and ion exchange
chromatography. Addition of purified recombinant Rad14 protein to
cell-free extracts prepared from a Affinity Purification of Rad14 and Associated Proteins from Yeast
Nuclear Extracts--
When a nuclear extract from the
The repairosome complex described previously was isolated by affinity
chromatography using his-tagged Tfb1 protein (11). We therefore asked
whether Tfb1 co-purified with recombinant his-tagged Rad14. Nuclear
extracts prepared from the
Since TFIIH is required for transcription initiation by RNA polymerase
II (RPB1), we asked whether this enzyme co-purified with recombinant
Rad14. Some RPB1 was retained by the affinity beads in the presence of
recombinant Rad14 and eluted in the 20 mM imidazole eluate
(Fig. 3C). Unlike the situation with Tfb1, however, this
represented only a small fraction of the RPB1 protein in the nuclear
extract. To identify other members of this putative complex, we
analyzed the same fractions for other proteins known or believed to be
involved in NER. Since Tfb1 is one of seven polypeptides that
constitute the core TFIIH subcomplex (2, 10), we anticipated that other
subunits of this transcription factor would co-purify with Rad14
protein. In agreement with this notion we observed
Rad14-dependent binding of Rad3 protein to the affinity
beads (Fig. 3D).
The products of the RAD1 and RAD10 genes are
known to form a highly stable heterodimeric complex that is responsible
for incisions 5' to sites of base damage during NER (24, 25). Both Rad1 and Rad10 proteins co-eluted with recombinant his-tagged Rad14 during
affinity chromatography (Fig. 3D). Replication protein A
(Rpa) is a single-strand DNA-binding protein initially identified as an
essential factor for DNA replication (4, 41, 42). Subsequent studies
have shown that this protein is also required for the reconstitution of
the DNA damage recognition and incision steps of the eukaryotic NER
pathway (1, 20). The 34-kDa subunit of Rpa, which is encoded by the
RFA2 gene (and presumably the 70- and 11-kDa subunits of
Rpa), was also eluted from the affinity beads in the presence of
recombinant Rad14 protein (Fig. 3D).
The Rad7 and Rad16 proteins also form a stable heterodimeric complex
(16). Recent studies have demonstrated that these proteins are required
for post-incision events during NER (15). Both proteins co-fractionate
with recombinant Rad14 (Fig. 3D), suggesting that they are
also components of the repairosome. Rad23 has been purified as a
component of a stable Rad23·Rad4 complex which is required for the
reconstitution of NER with purified yeast proteins (20). Hence, the
co-fractionation of Rad23 (and presumably Rad4) with recombinant Rad14
is consistent with the participation of this protein in NER (Fig.
3D).
Co-elution of the proteins discussed above was not observed using
extracts lacking Rad14 protein. Thus we consider it unlikely that the
co-fractionation of multiple NER proteins is purely fortuitous. It is
conceivable, however, that the association of multiple NER proteins is
mediated by their independent binding to DNA in nuclear extracts. To
address this issue nuclear extracts were treated with DNase I prior to
incubation with the affinity beads. This treatment had no effect on the
Rad14-dependent binding of the proteins shown in Fig. 3 to
the affinity column (data not shown).
It is possible that the co-purification of NER proteins described above
is a consequence of Rad14 overexpression. To address this question, we
performed the same fractionation procedure with nuclear extracts
prepared from cells grown in media with glucose as the carbon source.
Under these growth conditions, the cellular level of recombinant Rad14
is similar to that of native Rad14 in wild type cells (Fig. 2). In
these experiments Rad23, Rad7, Tfb1, RPB1, and Rad10 were retained by
the affinity beads in a Rad14-dependent manner (data not
shown), indicating that the association of these proteins is not a
consequence of the abnormally high levels of recombinant Rad14.
It has been demonstrated that PCNA is required for repair synthesis, a
late step in the NER process in eukaryotic cells (43). Remarkably, we
did not observe co-fractionation of PCNA protein, either in the
presence or absence of recombinant Rad14 (Fig. 3E). This
result suggests that the yeast repairosome we have identified does not
include components required for repair synthesis of DNA, an event that
is in fact not specific for NER. Gratuitously, this result also
indicates that the binding of Rad14 to the affinity beads does not
facilitate nonspecific binding of other proteins in nuclear extracts.
To support this notion further, we examined the binding of Rad51, a
relatively abundant nuclear protein that is not involved in NER, to the
affinity beads. As expected, we did not detect binding of this protein
either in the presence or absence of recombinant Rad14 protein (data
not shown).
Stoichiometry of the Putative Repairosome Components and Molecular
Mass of the Repairosome Complex--
Since NER proteins were
consistently detected in both the 20 and 50 mM imidazole
eluates, we omitted the 20 mM imidazole elution step in
subsequent experiments. When the affinity column was eluted with a
single step of 50 mM imidazole, the same NER proteins
co-purified (data not shown). In quantitative immunoblotting
experiments (Fig. 4), we estimate that 5 µg of the 50 mM eluate contains 0.2 pmol of Tfb1, 0.1 pmol of RPB1, 0.1 pmol of Rpa2, 0.1 pmol of Rad10, and 0.1 pmol of
Rad23. Thus, several of the putative repairosome subunits are present
in approximately equimolar quantities.
It is prudent to consider the possibility that the association of
proteins with recombinant Rad14 is a biochemically specific but
biologically irrelevant consequence of the binding of Rad14 to the
affinity beads. To determine whether NER proteins are associated in the
absence of affinity chromatography, we applied nuclear extracts
directly to an FPLC Superose 6 gel filtration column. In Fig.
5 we show the elution profiles of Rad14
and several NER proteins that co-purified with recombinant Rad14 on the
affinity column. In these experiments extracts were treated with DNase I prior to filtration. Similar results were obtained with extracts that
were not treated with DNase I (data not shown). Recombinant Rad14 and
other NER proteins co-eluted in fractions corresponding to a molecular
mass of 1000-1500 kDa, indicating that they are associated in a high
molecular weight complex in the nuclear extract prior to the
binding of recombinant Rad14 to the affinity beads.
DNA Structure-specific Endonuclease Activity Associated with the
Repairosome--
The results presented thus far support the notion
that NER proteins exist in a high molecular weight complex in yeast
cells not exposed to exogenous DNA damaging agents. For this complex to
be designated as a functional repairosome, it should support some, if
not all, of the reactions that transpire during the DNA damage
recognition and incision steps of NER in vivo. We examined the ability of affinity purified Rad14 and associated proteins to
specifically cleave Y-shaped DNA substrates that contain
duplex/single-strand junctions known to be cleaved by the
junction-specific Rad1/Rad10 and Rad2 endonucleases (23-25). Fractions
containing Rad14 and associated NER proteins cleaved a Y-shaped
substrate radiolabeled at the duplex end to produce a partial linear
duplex product (Fig. 6A). In
contrast, equal amounts of protein from a comparable fraction purified
from a
Cleavage of the radiolabeled Y-shaped substrate described above by
either Rad1/Rad10 or Rad2 endonuclease generates products that are
indistinguishable by gel electrophoresis. However, if the
non-complementary 5' single-strand of the Y structure is end-labeled to
produce the substrate shown in Fig. 5B, cleavage by the Rad2 endonuclease will produce a unique end-labeled single-strand
oligonucleotide ~20 residues in length (23). In assays with such a
substrate, fractions containing Rad14 and associated NER proteins
generated both the partial linear duplex expected from cleavage by the
Rad1/Rad10 endonuclease and a novel product with increased
electrophoretic mobility (Fig. 6B). This product was not
observed in assays with a comparable fraction purified from a
Nicking of UV-irradiated Supercoiled DNA by the
Repairosome--
In reconstitution experiments with purified NER
proteins, specific nicking of UV-irradiated DNA is
ATP-dependent (20). We asked whether the fraction from the
affinity column that contains recombinant Rad14 and other NER proteins
could perform the same reaction. ATP-dependent nicking of
UV-irradiated plasmid DNA was indeed observed in assays with this
fraction, whereas no nicking of undamaged or damaged plasmid DNA
occurred in the absence of ATP (Fig.
7A, lane 6). Similarly, no
nicking was observed with an equivalent amount of protein from a
comparable fraction purified from the nuclear extract lacking
recombinant Rad14. Fractions containing the repairosome supported some
nicking of undamaged plasmid DNA in the presence of ATP (Fig.
7A, lane 5) but to a significantly lower extent
than that observed with UV-irradiated DNA.
Since the Rad1 monoclonal antibody 3E3 inhibits both the cleavage of Y
structures by the Rad1/Rad10 endonuclease and in vitro NER
by yeast cell-free extracts (29), we examined the effect of this
antibody on the ATP-dependent nicking of UV-irradiated plasmid DNA. Preincubation of an affinity-purified fraction containing Rad14 and associated NER proteins with antibody 3E3 resulted in >50%
inhibition of endonuclease activity (Fig. 7B, lane 5). In contrast, preincubation of the same fraction with mouse IgG had no such
effect (Fig. 7B, lane 6).
In the experiment shown in Fig. 8, we
employed reaction conditions essentially identical to those described
for the reconstituted NER reaction that includes the Rad7·Rad16
complex (16). UV-irradiated plasmid DNA containing approximately one UV
photoproduct/molecule was rapidly cleaved such that ~50% of the
substrate was converted to the nicked circular form in 2 min in the
presence of 100 fmol of repairosome (assuming a molecular weight of
1.6 × 106 from all the identified subunits) (Fig. 8,
A and B). The rate of endonucleolytic cleavage
was estimated to be 0.2 nicks/min/repairosome. After 30 min essentially
all the supercoiled plasmid DNA was nicked (Fig. 8, A and
B). Undamaged plasmid molecules were nicked at a similar
initial rate (Fig. 8B). However, the reaction reached a
plateau when ~30% of the substrate molecules were nicked (Fig. 8B). This reaction profile could be explained if a
proportion of the putatively undamaged plasmid molecules contain some
sort of DNA lesion that is recognized by the repairosome. In the NER reaction reconstituted from purified subcomplexes (16), the rate of
cleavage of the UV-irradiated substrate was about 0.006 nicks/min/repairosome subcomplex. After subtracting the nicking of
undamaged DNA, it appears that the affinity-purified repairosome is at
least 10-fold more efficient at cleaving the UV-irradiated DNA
substrate than the combination of purified NER subcomplexes (16). This
may reflect the higher specific activity of the preformed repairosome
complex and/or the presence of additional factors in the
affinity-purified fraction that enhance the rate of DNA damage-dependent incision. Alternatively, it is possible
that one or more of the subcomplexes may not have been purified in a
fully active form, reducing the efficiency of the reconstituted reaction.
During purification of TFIIH containing a his-tagged Tfb1 subunit,
it was observed that this RNA polymerase II basal transcription factor
co-purified with NER proteins not involved in RNAP II transcription (11). Based on these results it was suggested that a large multiprotein complex dedicated to NER (nucleotide excision repairosome) exists in
yeast cells, the assembly of which does not require prior exposure of
cells to DNA damaging agents. Subsequent independent studies, which
employed different column resins and different elution protocols during
the initial purification steps, led to the conclusion that NER is
mediated by the sequential assembly of subcomplexes at sites of base
damage (28). If NER proteins are indeed associated in a large
multiprotein complex prior to the time that cells sustain bulky base
damage of the type that is a substrate for NER, it should be possible
to identify such a complex by affinity purification regardless of which
NER protein is tagged with poly-histidine residues, with the obvious
caveat that the tagged protein is accessible for binding to appropriate
affinity matrices.
By using a his-tagged version of the DNA damage recognition protein
Rad14, we have partially purified a multiprotein complex by a single
affinity chromatography step. The NER proteins Rad1, Rad10, Rad7,
Rad16, Rad23, the 34-kDa subunit of Rpa, the catalytic subunit of RNA
polymerase II holoenzyme, RPB1, and at least two components of TFIIH
bound to the affinity column exclusively in the presence of recombinant
Rad14. These results confirm and extend previous observations and are
consistent with other studies describing a high molecular weight
complex including both TFIIH and NER proteins in yeast and human cell
extracts (11, 27). In contrast to this study, the Rad7 and Rad16
proteins were not detected in the repairosome that was affinity
purified using his-tagged Tfb1 (11, 13). This is presumably a
consequence of the different purification schemes that were used to
isolate the repairosome complexes.
It is formally possible that our results reflect the fortuitous
independent binding to DNA of multiple proteins involved in NER. We
consider this unlikely since treatment of the nuclear extract with
DNase did not affect the behavior of these proteins in subsequent
fractionation steps of either gel filtration or affinity
chromatography. We also consider it unlikely that the binding of
overexpressed recombinant Rad14 to the affinity column specifically
promotes the association of NER (and possibly other) proteins in the
nuclear extract for the following reasons: (i) when nuclear extracts
were applied directly to a gel filtration column many of the NER
proteins, which co-purified with recombinant Rad14 by affinity
chromatography, co-eluted with Rad14 in fractions that correspond to a
molecular mass of >1000 kDa; (ii) although the NER proteins that
co-purify with recombinant Rad14 were present in approximately
equimolar amounts, the relatively abundant nuclear proteins PCNA and
Rad51, which are not involved in the DNA damage recognition and
incision steps of NER (events that are specific to NER), were not
retained by the affinity column in either the presence or absence of
recombinant Rad14; (iii) a similar spectrum of NER proteins was
retained by the affinity column in a Rad14-dependent manner
when recombinant Rad14 was expressed at the same level as the native protein.
A high molecular weight complex of NER proteins was initially detected
after affinity purification of a his-tagged version of Tfb1 expressed
at wild type levels (11). The putative repairosome containing
his-tagged Tfb1 complemented the repair defect in extracts from several
yeast strains mutated in NER genes (11). However, this complex was not
investigated for its ability to directly support reactions involved in
NER. Here we have demonstrated that the multiprotein complex partially
purified by affinity chromatography of his-tagged Rad14 protein incises
UV-irradiated DNA in an ATP-dependent reaction. Hence, we
conclude that the high molecular complex of NER proteins constitutes a
functional repairosome.
Unlike the complex of human proteins purified by XPA affinity
chromatography (27), we failed to detect co-purification of PCNA with
NER proteins. Thus, it is unlikely that the affinity purified complex
of yeast NER proteins can support DNA damage-dependent DNA
synthesis. In fact the yeast NER complex is similar to another high
molecular weight transcription-repair complex isolated from human cells
in that it contains the catalytic subunit of RNA polymerase II
(45).
Although our results provide support for the notion that repairosomes
exist in undamaged yeast cells, it is not known what proportion of NER
proteins are present in such complexes. Nor can we eliminate the
possibility that affinity chromatography identifies several independent
complexes containing NER proteins, some of which contain additional
polypeptides dedicated to other cellular responses to DNA damage.
However, the relative ease of genetic manipulation in yeast make this
an attractive organism to identify proteins that play important roles
in the assembly and/or stability of multiprotein complexes. Finally,
these results do not exclude the possibility that some, if not most,
DNA lesions are in fact removed by the sequential assembly of NER
subcomplexes at or near the site of DNA damage in vivo.
It seems reasonable to speculate that a preformed repairosome is
dedicated to continual surveillance of the genome in eukaryotic cells
for sites of base damage normally processed by NER. We suggest that
when (and only when) such sites are encountered, the multiprotein complex undergoes specific conformational changes that strengthen the
protein-DNA interactions. Additionally, these and/or further conformational changes may both facilitate localized unwinding of the
DNA that flanks sites of base damage by the Rad3 and Ssl2 DNA helicases
and position the Rad2 and Rad1/Rad10 endonucleases at the
duplex/single-strand junctions generated by this unwinding, specifically on the damaged DNA strand.
Several observations suggest that the amount and/or composition of the
nucleotide excision repairosome may change when yeast cells are exposed
to UV radiation or other DNA-damaging agents that generate substrates
for NER. For example, genes that encode several subunits of the
repairosome are up-regulated under these conditions. Notable examples
include RAD2 (46), RAD7 (47), RAD16
(48), and RAD23 (49). Additionally, recent studies in one of
our laboratories (E. C. Friedberg) have demonstrated significant inhibition of RNAP II transcription in vitro in the presence
of active NER (50). This inhibition can be relieved by supplementing in vitro reactions with purified holo-TFIIH, a form of TFIIH
specifically required for transcription initiation. Hence, when cells
are required to carry out NER, subunits of the repairosome such as
TFIIH and Rpa which are indispensable for transcription and
replication, respectively, may be preferentially sequestered by the
repairosome. This would not only enhance the cellular repair capacity
but would also inhibit transcription and replication until the genome
is repaired.
We thank Steve Brill, Peter Burgers, Tom
Kodadek, Roger Kornberg, and Rick Wood for purified proteins and
antibodies and Richard Kolodner for the yeast strain overexpressing
RPA. We are grateful to Patrick Sung for advice on destaining and
photographing agarose gels.
*
This work was supported by U. S. Public Health Service
Grants CA-12428 (to E. C. F.) and CA-67978 (to Z. W.) and Grant 3786 from the Council for Tobacco Research, U. S. A. (to A. E. T.).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.
The abbreviations used are:
NER, nucleotide
excision repair; DTT, dithiothreitol; FPLC, fast protein liquid
chromatography; his, histidine; TFIIH, transcription factor IIH; NTA, nitrilotriacetic acid; PCNA, proliferating cell nuclear antigen.
2
W. J. Feaver, W. Huang, and E. C. Friedberg, unpublished results.
Affinity Purification and Partial Characterization of a Yeast
Multiprotein Complex for Nucleotide Excision Repair Using
Histidine-tagged Rad14 Protein*
,,,
Department of Molecular Medicine, Institute
of Biotechnology, The University of Texas Health Science Center,
San Antonio, Texas 78245, the § Laboratory of Molecular
Pathology, Department of Pathology, The University of Texas
Southwestern Medical Center, Dallas, Texas 75225, and the
¶ Graduate Center for Toxicology, University of Kentucky,
Lexington, Kentucky 40536-0305
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RAD14::HIS3. Cells were grown at
30 °C in the appropriate drop-out media with either 2% glucose or
1% galactose, 1% raffinose as the carbon source. When the
A600 of the cultures reached ~1.0 cells were
collected by centrifugation and serially diluted in sterile water.
Aliquots (100 µl) of the dilutions were plated onto the appropriate
minimal drop-out agar plates containing either glucose or
galactose/raffinose as the carbon source. The plates were irradiated
with 254 nm UV light (Sylvania Germicidal 15 watts) at a rate of 1.3 J/m2/s. Survival was scored after 3-4 days of incubation
in the dark at 30 °C.
RAD14::HIS3 cells, and recombinant
Rad14 were incubated for 2 h at 26 °C with 300 ng each of
N-acetoxy-2-acetylaminofluorene-treated pUC18 DNA and
undamaged pGEM3Zf(+) DNA, 45 mM Hepes-KOH (pH 7.8), 7.4 mM MgCl2, 0.9 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 µM each of
dATP, dCTP, dGTP, and dTTP, 1 µCi of [
-32P]dCTP
(3,000 Ci/mmol), 40 mM phosphocreatine, 2.5 µg of bovine serum albumin, and 5% polyethylene glycol 8000 in a final volume of 50 µl. Plasmid DNA was purified and processed as described previously
(8).
80 °C.
-mercaptoethanol and a mixture of protease
inhibitors (buffer A), lysed by Dounce homogenization, and centrifuged
at 15,000 rpm for 30 min. In some cases, MgCl2 and DNase I
were added to the cleared lysate to final concentrations of 10 mM and 20 µg/ml, respectively, prior to incubation at
25 °C for 30 min. The cleared lysate (25 mg) was incubated with
rotation at 4 °C with 2 ml of a 50% slurry of Ni2+- NTA
agarose beads (Qiagen) for 1 h. This mixture was poured into a
column, and the beads were washed extensively with buffer A containing
5 mM imidazole. Bound proteins were eluted in a stepwise manner with 20, 50, 100, and 200 mM imidazole in buffer A. Protein-containing fractions from each eluate were detected using the
Bradford assay (38). In some cases, the protein-containing fractions
were pooled and concentrated by ultrafiltration using Centricon-10
(Amicon). The 20 mM eluates from the extracts with and
without recombinant Rad14 contained 1.2 and 0.4 mg, respectively. The
50 mM eluates from the extracts with and without
recombinant Rad14 contained 0.4 and 0.1 mg, respectively. Fractions
were flash-frozen in liquid nitrogen and stored at 80 °C.
80 °C. NER proteins
were detected by immunoblotting. The column was calibrated by
determining the elution position of molecular mass standards
(thyroglobulin 670,000, gamma globulin 158,000, ovalbumin 44,000, myoglobin 17,000, and vitamin B12 1,350, Bio-Rad) under identical
chromatography conditions.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
RAD14 strain transfected with the plasmid vector (Fig.
2). Taken together these results demonstrate that the his-tagged
version of Rad14 functions in NER in vivo.
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Fig. 1.
Complementation of the UV-sensitive phenotype
of a RAD14::HIS3 mutant strain by
expression of his-tagged Rad14. The strains SX46A (MATa ade2
his3-52 ura3-52 trp1-289) pYES2 (filled circles), SX46A
RAD14::HIS3 pYES2 (open
squares), and SX46A RAD14::HIS3
pYES2-hisRad14 (filled squares) were grown at
30 °C in appropriate drop-out media with either 1% galactose, 1%
raffinose (A) or 2% glucose (B) as the carbon
source. Cells were diluted, plated, and irradiated with UV light as
described under "Experimental Procedures." Survival was scored
after 3-4 days of incubation in the dark at 30 °C.
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Fig. 2.
Analysis of the levels of recombinant and
native Rad14 protein in yeast nuclear extracts by immunoblotting.
Yeast strains were grown in media with either glucose (glu)
or galactose/raffinose (gal) as the carbon source, and
nuclear extracts were prepared as described under "Experimental
Procedures." Rad14 in the nuclear extracts was detected by
immunoblotting. 32 µg of SX46A RAD14 (WT)
nuclear extract from cells grown in either glucose or galactose medium.
35 and 4 µg of SX46A RAD14::HIS3
pYES2 (RAD14 pYes) nuclear extract from cells grown in glucose and
galactose medium, respectively. 37 and 4 µg of SX46A
RAD14::HIS3 pYES2 (RAD14 pYes)
nuclear extract from cells grown in glucose and galactose medium,
respectively. Purified recombinant Rad14 (0.4 µg, lane C)
was included as a positive control.
RAD14 mutant restored
NER activity as measured by DNA damage-dependent DNA synthesis (data not shown). Thus, his-tagged recombinant Rad14 is
active both in vivo and in vitro.
RAD14 pYES2-hisRad14 strain was fractionated
by immobilized metal affinity column chromatography, recombinant Rad14
was not detected in the flow-through fraction (Fig.
3A). However, substantial
amounts of the protein were eluted in the 20 and 50 mM
imidazole eluates (Fig. 3A). Smaller quantities were also
detected in 100 and 200 mM imidazole eluates (data not shown). No immunoreactivity was detected when the nuclear extract from
the RAD14 pYES2 strain was fractionated by the same
procedure (Fig. 3A).
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Fig. 3.
Affinity purification of recombinant Rad14
and associated proteins from yeast nuclear extracts. The
yeast strains SX46A
RAD14::HIS3 pYES2 and SX46A
RAD14::HIS3
pYES2-hisRad14 were grown and nuclei prepared as
described under "Experimental Procedures." The nuclei were lysed by
Dounce homogenization, and the cleared lysates were fractionated by
affinity chromatography as described under "Experimental
Procedures." Equal amounts of protein from the two extracts in the
cleared lysate (20 µg, Load), pass-through fractions (15 µg, Pass), 20 mM imidazole eluates (23 µg,
20 mM), and 50 mM imidazole eluates (7 µg, 50 mM) were separated by denaturing gel electrophoresis and
transferred to nitrocellulose membranes. Antigen-antibody complexes
were detected by enhanced chemiluminescence. Extracts were prepared
from SX46A RAD14::HIS3 pYES2 ()
and SX46A RAD14::HIS3
pYES2-hisRad14, (+) cells. A, immunoblot with
Rad14 antiserum. B, immunoblot with Tfb1 antibody.
C, immunoblot with RPB1 antibody. D, immunoblots
with antibodies specific for the indicated NER proteins. E,
immunoblot with PCNA antibody.
RAD14 pYES2 and the RAD14 pYES2-hisRad14 strains contain
equivalent amounts of Tfb1 protein (Fig. 3B). In the absence
of recombinant Rad14 protein Tfb1 was detected in the column
flow-through fraction but not in the 20 or 50 mM imidazole
fractions. In contrast, Tfb1 protein was quantitatively bound by the
affinity beads in the presence of recombinant Rad14 and eluted in the
20 and 50 mM imidazole eluates (Fig. 3B). The
observations of Rad14-dependent retention of Tfb1 protein
by the affinity resin and the co-elution of Rad14 and Tfb1 suggest that
these proteins are associated in a complex.
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Fig. 4.
Quantitation of the proteins that co-purify
with recombinant Rad14. Proteins (5 µg, S) that
co-purified with recombinant Rad14 when the metal chelating column was
eluted with single step of 50 mM imidazole and varying
amounts of the indicated purified protein were separated by denaturing
gel electrophoresis and transferred to nitrocellulose membranes.
Antigen-antibody complexes were detected by immunoblotting with the
indicated antibody and quantitated as described under "Experimental
Procedures."
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Fig. 5.
Fractionation of yeast NER proteins in a
nuclear extract by gel filtration. An aliquot (200 µl) of a
nuclear extract from the yeast strain SX46A
RAD14::HIS3
pYES2-hisRad14 was fractionated by gel filtration as
described under "Experimental Procedures." Fractions
(FXN) (400 µl) were collected, flash-frozen in liquid
nitrogen, and stored at 80 °C. NER proteins were detected by
immunoblotting. The column was calibrated by determining the elution
position of molecular mass standards (Bio-Rad) under identical
chromatography conditions. The void volume of this column corresponded
to fraction 5. The largest molecular mass standard (thyroglobulin,
molecular mass 670 kDa) eluted in fractions 18-20.
RAD14 pYES2 nuclear extract did not contain
junction-specific cleavage activity, although low levels of nonspecific
nuclease activity were observed (Fig. 6A). Preincubation of
the repairosome fraction with 6 µg of Rad1 monoclonal antibody 3E3,
which inhibits the Rad1/Rad10 endonuclease (29), reduced cleavage of
the Y structure by ~80%, whereas purified mouse IgG had no
inhibitory effect (data not shown). These results indicate that the
Rad1 and Rad10 proteins that co-purify with recombinant Rad14 are
catalytically active.
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Fig. 6.
Cleavage of single-strand/duplex junctions in
DNA by the putative repairosome. Nuclease assays with labeled Y
structures were carried out as described under "Experimental
Procedures." After separation by polyacrylamide gel electrophoresis
labeled oligonucleotides were detected by autoradiography. Fractions
from SX46A RAD14::HIS3 pYES2
(50) and from SX46A
RAD14::HIS3
pYES2-hisRad14 (50+) extracts are indicated.
A, Y structure 5' end-labeled at the complementary duplex
end. The positions of the labeled Y structure substrate and partial
duplex products are shown on the left. B, Y
structure 5' end-labeled at the non-complementary single strand. This
substrate was also incubated with recombinant XPG protein as indicated
(XPG). The positions of the labeled Y structure substrate
and partial duplex and linear single-strand products are shown on the
right.
RAD14 pYES2 nuclear extract. However, it was detected in
reactions with XPG protein, the human homolog of Rad2 protein (23, 25,
37, 44) (Fig. 6B). Thus, we are led to the conclusion that
the affinity purified fractions containing recombinant Rad14 possess
Rad2 activity. Antibodies to Rad2 protein were not available to
demonstrate specific inhibition of this activity. However,
preincubation with 6 µg of the Rad1 monoclonal antibody, 3E3,
resulted in significant inhibition (~80%) of the putative Rad2
activity (data not shown). Since the Rad1 antibody is known to inhibit
the NER reaction catalyzed by yeast extracts (29), it is possible that
binding of the antibody to the Rad1 component of the repairosome
precludes the activity of the Rad2 endonuclease.
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Fig. 7.
Nicking of UV-irradiated and undamaged
supercoiled DNA by the repairosome; effect of ATP. A,
supercoiled plasmid DNA was irradiated with 300 J/m2 of UV
light from a germicidal lamp. Reactions (15 µl) contained 500 ng of
damaged (+) or undamaged ( ) supercoiled plasmid DNA and aliquots (1 µg) of affinity purified fractions from the SX46A
RAD14::HIS3 pYES2 (50) and SX46A
RAD14::HIS3
pYES2-hisRad14 (50+) extracts. An ATP-regenerating system
(ATP) was added as indicated. Incubations were performed as
described under "Experimental Procedures." B, affinity
purified fractions (50+) were preincubated with the Rad1
monoclonal antibody 3E3 or purified mouse IgG as indicated, prior to
the addition of the other reaction components including UV-irradiated
DNA and the ATP-regenerating system. Similar assays were performed
without addition of the affinity purified fraction () to demonstrate
the absence of endonuclease activity in the antibody preparations.
After electrophoresis through a 0.8% agarose gel plasmid DNA was
detected by staining with ethidium bromide. The positions of
supercoiled DNA (ccc) and nicked circular DNA
(nc) are shown on the right.
View larger version (34K):
[in a new window]
Fig. 8.
Time course of endonucleolytic cleavage of
damaged and undamaged supercoiled DNA by the nucleotide excision
repairosome. A, reaction mixtures containing 200 ng of
UV-irradiated plasmid DNA (30 J/m2), an ATP-regenerating
system, and an aliquot (160 ng) of the affinity purified fraction from
the SX46A RAD14::HIS3
pYES2-hisRad14 extract were incubated at 30 °C for the
times indicated. B, graphic representation of the data shown
in A (squares) and a similar assay with undamaged
supercoiled plasmid (circles).
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
210-567-7327; Fax: 210-567-7324; E-mail: tomkinson{at}uthscsa.edu.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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