(Received for publication, June 10, 1997, and in revised form, July 7, 1997)
From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
In eukaryotes, nucleotide excision repair of
ultraviolet light-damaged DNA is a highly intricate process that
requires a large number of evolutionarily conserved protein factors.
Genetic studies in the yeast Saccharomyces cerevisiae have
indicated a specific role of the RAD7 and RAD16
genes in the repair of transcriptionally inactive DNA. Here we show
that the RAD7- and RAD16-encoded products exist
as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation
constant (Kd) of <4 × 1010
M. The Rad7-Rad16 complex has been purified to near
homogeneity in this study and is shown to bind, in an
ATP-dependent manner and with high specificity, to DNA
damaged by ultraviolet light. Importantly, inclusion of the Rad7-Rad16
complex in the in vitro nucleotide excision repair system
that consists entirely of purified components results in a marked
stimulation of damage specific incision. Thus, Rad7-Rad16 complex is
the ATP-dependent DNA damage sensor that specifically
functions with the ensemble of nucleotide excision repair factor (NEF)
1, NEF2, NEF3, and replication protein A in the repair of
transcriptionally inactive DNA. We name this novel complex of Rad7 and
Rad16 proteins NEF4.
In eukaryotes, nucleotide excision repair (NER)1 of ultraviolet light-damaged DNA occurs by dual incision of the DNA strand that contains the UV lesion, excising the damage in the form of a short DNA fragment ~30 nucleotides in length (1). Mutational inactivation of NER in humans results in the cancer-prone syndrome xeroderma pigmentosum, which underscores the importance of this repair system in neutralizing the genotoxicity of UV light. The dual incision event in NER is accomplished by the concerted action of a large number of evolutionarily conserved proteins, and our studies in the yeast Saccharomyces cerevisiae have indicated an organization of these proteins into distinct subassemblies: nucleotide excision repair factor I, or NEF1, consisting of the damage recognition protein Rad14 and the Rad1-Rad10 endonuclease (2), NEF2, containing the Rad4 and Rad23 proteins (3), and NEF3, comprising the endonuclease Rad2 together with the RNA polymerase II transcription factor TFIIH (4). The combination of NEF1, NEF2, NEF3, and the heterotrimeric replication protein A (RPA) is sufficient for ATP-dependent dual incision to occur, indicating that the basic NER machinery is composed of these protein subassemblies (3). Dual incision of UV-damaged DNA can also be accomplished by combining the human equivalents of the aforementioned yeast repair proteins (5-7).
At the genomic level, two functionally distinct modes of NER have been described, the first concerns with the repair of the transcribed strand in transcriptionally active chromosomal DNA, which involves the stalling of RNA polymerase II at the DNA lesion, and requires the CSA and CSB gene products (8). The second mode of excision repair is specific for the nontranscribed strand and for genomic regions that are transcriptionally inactive. Interestingly, in the rad7 and rad16 mutants of S. cerevisiae, the nontranscribed strand is not repaired, while the repair of the transcribed strand is not affected (9, 10), and transcription-independent NER is impaired in rad7 and rad16 mutant extracts (11, 12). Thus, the repair of the nontranscribed strand has a specific dependence on the RAD7 and RAD16 genes.
Here we describe our biochemical studies that help elucidate the molecular function of the Rad7 and Rad16 proteins in the repair of the nontranscribed strand. We show by co-immunoprecipitation that Rad7 and Rad16 proteins exist as a stable complex in yeast cells. The Rad7-Rad16 complex, which we have named NEF4, has been purified to near homogeneity from extract of a yeast strain co-expressing the two proteins. Using a DNA mobility shift assay, we demonstrate that NEF4 binds with high specificity and avidity to UV lesions and that this damage binding reaction has a strong dependence on ATP. Importantly, the addition of NEF4 to the reconstituted NER reaction results in a marked stimulation of damage-specific incision. Taken together, our results suggest that NEF4 is an ATP-dependent DNA damage sensor that functions specifically to target the basic NER machinery (viz. NEF1, NEF2, NEF3, and RPA) to the repair of nontranscribed DNA.
The Rad7 protein coding frame from 20 nucleotides upstream of the ATG initiating codon to 130 nucleotides downstream of the TAG stop codon was placed under the control of the GAL-PGK promoter in plasmid pPM231, yielding plasmid pR7.8 (2µ, GAL-PGK-RAD7). To overproduce the RAD16-encoded product, the RAD16 gene from 30 nucleotides upstream of the ATG initiating codon until 130 nucleotides downstream of the TAG stop codon was fused to the ADC1 promoter in plasmid pSCW231, yielding plasmid pR16.15. Immunoblot analyses of yeast extracts with affinity-purified antibodies specific for Rad7 and Rad16 proteins revealed that these proteins are individually overexpressed at least 20-fold compared with the level seen in wild type extract.
Production of AntibodiesThe portion of Rad7 protein
encompassing amino acid residues 1-485 was expressed as a fusion with
the 12 amino-terminal residues of LacZ. The portion of Rad16 protein
from amino acid residues 273-534 was fused to the
NH2-terminal 48 residues of the Escherichia coli
transcription terminator . Both of these fusion proteins are
insoluble in E. coli, and they were purified from inclusion bodies by preparative SDS-polyacrylamide gel electrophoresis. The
purified LacZ-Rad7 and
-Rad16 hybrid polypeptides were used as
antigens for the production of polyclonal antisera in rabbits. Antibodies were purified from rabbit sera using antigens cross-linked to cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech Inc.).
Extract was prepared from yeast strain LY2 harboring pR7.8, pR16.15, or both of these plasmids, as described (13). Clarified extract (0.5 ml) was mixed gently at 4 °C for 2 h with 10 µl of protein A-agarose beads containing covalently conjugated, affinity-purified anti-Rad57 (13), anti-Rad7, and anti-Rad16 antibodies (2 mg of antibodies/ml of matrix). Immunoprecipitates were treated with 30 µl of 3% SDS to elute the bound proteins, and aliquots (10 µl) of the eluates were subjected to immunoblot analysis.
Purification of NEF4Extract was prepared from 450 g
of strain LY2 co-harboring pR7.8 and pR16.15 using a French press (14),
clarified by centrifugation (100,000 × g, 90 min), and
the Rad7-Rad16 complex was precipitated by the addition of ammonium
sulfate to 0.22 g/ml. The ammonium sulfate pellet was dissolved in 200 ml of K buffer (20 mM KH2PO4, pH
7.4, 10% glycerol, 1 mM dithiothreitol, and 0.5 mM EDTA) and dialyzed against 2 liters of K buffer + 50 mM KCl for 12 h. The dialysate (Fraction I) was
applied onto a column of Q-Sepharose (2.5 × 9 cm; 44-ml matrix)
equilibrated in K buffer + 100 mM KCl. The flow-through
from Q-Sepharose (Fraction II; 250 ml) was loaded directly onto a
SP-Sepharose column (2.5 × 9 cm; 44-ml matrix), which was
developed with a 400-ml gradient of 100-700 mM KCl in K
buffer, collecting 50 fractions. The fractions containing the peak of
the Rad7-Rad16 complex, eluting at 420 mM KCl, were
identified by immunoblotting and pooled (Fraction III; 40 ml). The SP
pool was dialyzed against K buffer for 2 h to lower the ionic
strength to 50 mM KCl, before being fractionated in a
column of Q-Sepharose (1.5 × 8 cm; 14-ml matrix) using a 150-ml
gradient from 50 to 300 mM KCl in buffer K, collecting 50 fractions. The pool of Rad7-Rad16 complex (Fraction IV; 12 ml), eluting
at about 160 mM KCl from the Q-Sepharose column, was
applied onto a Bio-Gel HTP hydroxyapatite column (1 × 3 cm; 2.3 ml), which was eluted with a 20-ml gradient of 10-300 mM
KH2PO4, pH 7.4, in buffer K, and 20 fractions
were collected. The peak of the Rad7-Rad16 complex (Fraction V; 2 ml), eluting at about 280 mM KH2PO4 from
hydroxyapatite, was diluted with 6 ml of K buffer and then fractionated
in Mono S (HR5/5) with a 20-ml, 100-600 mM KCl gradient in
buffer K, collecting 40 fractions. The Rad7-Rad16 complex elutes from
Mono S at about 425 mM KCl, the pool of which (Fraction VI;
1.5 ml containing 65 µg of protein) was concentrated to 0.15 ml in a
Centricon-30 microconcentrator (Amicon) and stored in small portions at
70 °C.
The 130-base pair
HindIII-SalI fragment used in mobility shift
experiments was isolated from plasmid pTB402, labeled with 32P, and irradiated with UV light (254 nm), as described
previously (14). Fraction VI NEF4 was incubated with the
32P-labeled UV-irradiated DNA fragment (1.2 ng) and 20 ng
of unlabeled, unirradiated X174 dsDNA (linearized with
PstI) in 10 µl of reaction buffer (30 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM
ATP, 100 µg/ml bovine serum albumin, and 1 mM
dithiothreitol) at 30 °C for 10 min. Reaction mixtures were run in
3.6% polyacrylamide gels in TAE buffer (40 mM Tris
acetate, pH 7.0, 0.1 mM EDTA) at 30 mA and 4 °C for
1 h. Gels were dried onto Whatman No. 3MM paper and exposed to
Kodak MS films. The autoradiograms were subjected to image analysis in
a Bio-Rad GS670 densitometer to obtain data points for graphical
representation of the results.
The NER reaction was carried out as described (2-4, 15). Briefly, 50 ng of RPA, 30 ng of NEF1, 30 ng of NEF2, 120 ng of NEF3, and 30 ng of NEF4 (when added) were incubated in 10 µl of reaction buffer with 120 ng of M13mp18 DNA that had been irradiated under a UV germicidal lamp emitting at 254 nm and a fluence rate of 1 J/m2/s for 30 s. Reaction mixtures were incubated at 30 °C for varying times, deproteinized, and analyzed in 0.8% agarose gels, as described (3).
The RAD7-encoded protein was overproduced in yeast by
placing the RAD7 gene under the control of the
GAL-PGK promoter-yielding plasmid pR7.8 (Fig.
1A). To overproduce the Rad16
protein in yeast, the RAD16 gene was fused to the alcohol
dehydrogenase I (ADC1) promoter, yielding plasmid pR16.15
(Fig. 1A). When extract from the protease-deficient yeast
strain LY2 co-harboring pR7.8 and pR16.15 was subjected to
immunoprecipitation with protein A-agarose beads containing covalently
conjugated anti-Rad7 and anti-Rad16 antibodies, co-precipitation of the
Rad7 and Rad16 proteins was observed (Fig. 1A, lanes 8 and
9); no precipitation of either Rad7 or Rad16 protein was
seen with beads containing antibodies specific for the Rad57 protein
(Fig. 1A, lane 7), which functions in recombinational repair
(13). Control experiments showed that the precipitation of Rad7 by the
anti-Rad16 immunobeads requires the presence of Rad16 protein and,
likewise, that the precipitation of Rad16 protein by anti-Rad7
immunobeads is dependent upon Rad7 protein (Fig. 1A). In
immunoprecipitation experiments using extract from wild type yeast
cells, co-precipitation of the Rad7 and Rad16 proteins was again
observed. Thus, Rad7 and Rad16 proteins are complexed to each other in
yeast cells.
For purifying the Rad7-Rad16 protein complex, extract from LY2
co-overexpressing Rad7 and Rad16 proteins was subjected to the
chromatographic fractionation scheme described under "Materials and
Methods." The Rad7 and Rad16 proteins remained quantitatively associated throughout all the purification steps, even in column fractions that contained as little as 4 × 1010
M of the protein complex, indicating a dissociation
constant of the complex that is considerably lower than this protein
concentration. When the Rad7-Rad16 protein complex from the last step
of purification in Mono S (Fraction VI) was subjected to
SDS-polyacrylamide gel electrophoresis and staining with Coomassie
Blue, only the Rad7 and Rad16 bands were seen (Fig. 1, B and
C), indicating a high degree of purity of the complex. Image
analysis of the gel in Fig. 1B revealed a one to one
stoichiometry of the Rad7 and Rad16 proteins in the complex.
We speculated that NEF4 might effect the repair of transcriptionally
inactive DNA by sensing the DNA damage located in such regions and then
recruiting the basic NER machinery to initiate the repair reaction. As
a first test of this hypothesis, we examined whether NEF4 would bind
UV-damaged DNA using a DNA mobility shift assay. The DNA fragment was
labeled with 32P, irradiated with UV doses ranging from 1 to 12 kJ/m2, and then incubated with NEF4 in the presence
of ATP and an excess of unlabeled X174 DNA at 30 °C. After
running the reaction mixtures in polyacrylamide gels under
nondenaturing conditions, the gels were dried and exposed to x-ray
films to reveal the radiolabeled DNA species. As shown in Fig.
2, incubation of the UV-irradiated DNA
probe with NEF4 resulted in the formation of slower migrating forms of
DNA, indicating binding of the damaged DNA by NEF4; no binding of NEF4
to the undamaged DNA was seen. The level of nucleoprotein complex
formation was proportional to the amount of NEF4 (Fig. 2, A
and B) and to the UV dose (Fig. 2, C and
D). Multiple nucleoprotein complexes were detected in these
experiments, with the slower migrating nucleoprotein species being more
prevalent at higher UV doses and with increasing concentrations of NEF4
(Fig. 2), suggesting that these species contained multiple NEF4
molecules bound to different damage sites in the DNA probe.
Importantly, when ATP was omitted from the reaction, binding of the
UV-damaged DNA decreased dramatically, such that only ~6% of the
UV-irradiated DNA was bound by NEF4 in the absence of the nucleotide,
as compared with greater than 50% binding in its presence (Fig.
2E). Although NEF4 possesses an ATPase activity that is
activated by either single-stranded or double-stranded
DNA,2 ATP binding by NEF4 is
apparently sufficient for damage recognition, because the
nonhydrolyzable ATP analogue ATP
S is nearly as effective as ATP in
promoting damage binding (Fig. 2E).
Having established that NEF4 has high affinity for UV-damaged DNA, we
then tested if NEF4 might improve the efficiency of the damage-specific
incision reaction mediated by the basic NER machinery comprising NEF1,
NEF2, NEF3, and RPA (2-4, 15). To do this, we examined the rate of
incision of supercoiled M13 DNA that had been exposed to 30 J/m2 UV light to introduce on average 1.8 photoproducts per
plasmid DNA molecule (approximately 7.3 kilobase pairs long) by the
basic NER machinery with or without added purified NEF4. Interestingly, addition of NEF4 to the in vitro NER reaction resulted in a
marked stimulation of the conversion of the UV-irradiated supercoiled M13 plasmid DNA to the open circular form (Fig.
3, A and B), while no incision of the nondamaged M13 DNA was observed whether or not NEF4
was added to the repair reaction (Fig. 3A). These
observations indicate that NEF4 helps promote the damage-specific
incision reaction. We also subjected the NER reaction to labeling with [-32P]ddATP and calf thymus terminal transferase to
detect the excision DNA fragments (3, 15). In agreement with the
results from the agarose gel method, the amount of excision fragments
generated was also higher when NEF4 was included in the repair reaction (data not shown).
Our findings with NEF4 suggest that eukaryotes employ different mechanisms of damage recognition for the nontranscribed versus the transcribed strand. NEF4, an ATP-dependent damage recognition factor, is essential for the repair of the nontranscribed strand, whereas the products of the CSA and CSB/RAD26 genes are essential for preferential repair of the transcribed strand. NEF4 may be pivotal in locating the damage on the nontranscribed strand and in transcriptionally inactive regions of the genome, and it may do so by a tracking mechanism that utilizes the energy of ATP hydrolysis. Subsequent to binding the DNA lesion, NEF4 may serve as the nucleation site for the assembly of the other repair components for dual incision to occur. An additional role of NEF4 in the turnover of the incision enzyme complex is also possible.
We thank Yvette Habraken for help in preparation of the manuscript and E. J. Miller and T. Johnson for technical assistance.