From the Sealy Center for Molecular Science, University of Texas
Medical Branch, Galveston, Texas 77555-1061 and
Institute
of Biotechnology, Department of Molecular Medicine, University of Texas
Health Science Center, San Antonio, Texas 78245
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INTRODUCTION |
Nucleotide excision repair
(NER)1 of ultraviolet-damaged
DNA in eukaryotes is a complex process requiring the products of a large number of genes. The structure and function of the nucleotide excision repair genes are highly conserved among eukaryotes from yeast
to humans (1, 2). Biochemical fractionation of yeast cell extracts have
revealed the organization of the NER proteins into distinct functional
subassemblies called nucleotide excision repair factors or NEFs.
Specifically, we have shown that NEF1 contains the damage recognition
protein Rad14 and the Rad1-Rad10 endonuclease (3), NEF2 comprises the
Rad4 and Rad23 proteins (4), and that the Rad2 endonuclease and the
six-subunit RNA polymerase II transcription factor TFIIH form NEF3 (5).
Our biochemical studies have further demonstrated that the combination of NEF1, NEF2, NEF3, and the heterotrimeric single-stranded DNA (ssDNA)
binding factor replication protein A (RPA) is sufficient for dual
incision of UV-damaged DNA to occur (3-6). These studies have
suggested that the basic yeast NER machinery consists of NEF1, NEF2,
NEF3, and RPA. Human equivalents of these yeast NER proteins also carry
out the dual incision of damaged DNA (7-9).
In addition to the aforementioned NER factors, genetic studies have
indicated a role of four additional genes, RAD7,
RAD16, RAD26, and MMS19, in NER (1,
2). The MMS19-encoded protein affects cell viability and
functions in RNA polymerase II transcription, probably as a regulatory
component by modulating the activity of TFIIH and of proteins that
function in other cellular processes (10). The Rad26 protein is a
member of the Swi2/Snf2 family of proteins, possesses a
DNA-dependent ATPase activity (11), and is required for the
preferential repair of the transcribed DNA strand (12). The
RAD7 and RAD16 genes, on the other hand, are
specifically required for the nucleotide excision repair of nontranscribed DNA (13-16). The RAD7 gene product does not
possess significant homology to any other known protein, whereas the
RAD16-encoded product is another member of the
Swi2/Snf2 protein family (17). Recently, we showed that the Rad7
and Rad16 proteins are associated in a stoichiometric complex, which we
have named NEF4. NEF4 has high affinity for UV-damaged DNA, and
addition of NEF4 to the reconstituted NER system consisting of NEF1,
NEF2, NEF3, and RPA results in marked stimulation of damage-specific
incision (18). Here we describe our studies demonstrating a
DNA-dependent ATPase activity in NEF4. Interestingly, UV
irradiation of the DNA cofactor results in a marked down-regulation of
the NEF4 ATPase activity. We suggest a model in which the free energy
from ATP hydrolysis is utilized to fuel the translocation of NEF4 on
DNA to search for DNA lesions. Binding of NEF4 to a DNA lesion results
in suppression of ATP hydrolysis, and the stable NEF4-DNA damage
complex serves as the nucleation site for the assembly of other NER
factors.
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MATERIALS AND METHODS |
NEF4 Purification--
Extract was prepared from 500 g of
cells of strain LY2 co-harboring pR7.8 (2 µm,
GAL-PGK-RAD7) and pR16.15 (2 µm, ADCI-RAD16) using a
French press and clarified by centrifugation (100,000 × g for 120 min). The clarified extract was subjected to
ammonium sulfate precipitation (35% saturation) to precipitate NEF4
and approximately 15% of the total protein. The precipitate was
harvested by centrifugation (20,000 × g, 30 min) and
dissolved in K buffer (20 mM
KH2PO4, pH 7.4, 10% glycerol, 1 mM
dithiothreitol, 0.5 mM EDTA) followed by sequential
fractionation in columns of Q-Sepharose, SP-Sepharose, hydroxyapatite,
and Mono S to yield fraction VI NEF4 (18). The final yield of NEF4 was
90 µg with a recovery of ~15%. When fraction VI NEF4 was analyzed
in an SDS-polyacrylamide gel and stained with Coomassie Blue,
only the Rad7 and Rad16 proteins were seen (18), indicating that the
preparation was nearly homogeneous. Fraction VI NEF4 was used in all
the biochemical studies described below.
ATPase Assay--
Purified NEF4, 120 ng, was mixed with 250 ng
of
X ssDNA or 250 ng of
X double-stranded DNA (dsDNA), 0.5 mM ATP and 1 µCi of [
-32P]ATP (3,000 Ci/mmol; Amersham Life Science, Inc.) in buffer R (30 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM
dithiothreitol, 100 µg/ml bovine serum albumin). The reaction
mixtures were incubated at 30 °C for 30 min or as indicated. For
quantifying ATP hydrolysis, a 0.5-µl portion of the reaction mixtures
were spotted onto a polyethyleniminecellulose sheet, which was
developed with 1 M formic acid containing 0.3 M
LiCl. Quantification was done by phosphoimage analysis in a Molecular
Dynamics PhosphorImager.
DNA Helicase Assay--
The substrate for the DNA helicase assay
was obtained as described previously (19). A 5' 32P-labeled
17-base DNA fragment was hybridized to the viral (+) strand of M13mp18
to generate a partial duplex. The nonhybridized labeled DNA fragment
was separated from the partial duplex DNA by gel filtration in
Sepharose G-50 matrix equilibrated in 50 mM Tris-HCl, pH
7.5, 0.1 M NaCl at 4 °C. Fractions containing the peak
of partial duplex DNA were pooled and concentrated to a small volume.
The helicase substrate, 5 ng, was mixed with the indicated amount of
NEF4 protein in 10 µl of buffer R containing 2 mM ATP.
After incubation for 45 min at 30 °C, the reaction was stopped by
adding 5 µl of 1% SDS, 50 mM EDTA, 20% glycerol, 0.02% bromphenol blue. The reaction mixture was run in 12% polyacrylamide gels, which were then dried and subjected to autoradiography. DNA
helicase assay employing Rad3 protein was carried out as described (20).
DNA Mobility Shift Assay--
A 0.9-kilobase AT-rich DNA
fragment from plasmid pS288 (21) was cloned into pUC19 to generate the
plasmid pTB402. A 130-base pair HindIII-SalI
fragment that contained eight consecutive thymines was isolated from
plasmid pTB402 and labeled with 32P at the 3' end by
treatment with Klenow polymerase (22). UV irradiation of the fragment
was carried out using a germicidal UV lamp emitting at 254 nm, as
described previously (22). Purified NEF4, 60 ng, was incubated with 2 ng of the 32P-labeled DNA fragment and 20 ng of linear
X
dsDNA as cold competitor in 10 µl of reaction buffer R that contained
2 mM ATP or ATP
S. After the addition of 2 µl of
loading buffer (40% glycerol, 0.1 mM Tris acetate, pH 7.0, 10 mM EDTA, 0.02% acridine orange), the reaction mixtures
were run in 4% polyacrylamide gels at 30 mA and 4 °C for 1 h.
The gels were dried onto Whatman 3MM paper and then exposed to Kodak MR
film or subjected to phosphoimage analysis to quantify the binding
reaction.
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RESULTS |
By immunoprecipitation and other criteria, we have shown recently
that Rad7 and Rad16 are associated in a stable complex
(Kd
4 × 10
10 M)
termed NEF4, which has been purified to near homogeneity (18). The
Rad16 protein contains Walker types A and B nucleotide binding motifs
(23) suggestive of an ability to bind and hydrolyze ATP. To examine
whether purified NEF4 has ATPase activity, it was incubated with
[
-32P]ATP in the presence of Mg2+, and the
reaction mixtures were analyzed by thin layer chromatography in
polyethylenimine cellulose sheets using lithium chloride and formic
acid as the developing solvent (11, 24). However, without DNA, we did
not detect significant hydrolysis of ATP (
2%) over the pH range of
6.0-9.0 (data not shown). Since NEF4 functions in DNA repair and binds
DNA, we tested whether in the presence of DNA NEF4 would hydrolyze ATP.
Interestingly, the addition of either ssDNA or dsDNA resulted in
substantial hydrolysis of ATP by NEF4 (see below). When the column
fractions from the last step of NEF4 purification in Mono S were
subjected to immunoblotting with anti-Rad7 and anti-Rad16 antibodies to
determine their NEF4 content and also assayed for ssDNA- and
dsDNA-activated ATP hydrolysis, we found that both the ssDNA- and
dsDNA-dependent ATPase activities closely paralleled the
level of NEF4 in these fractions (Fig. 1). This precise co-elution of the
DNA-dependent ATPase activity with NEF4 together with the
high degree of purity of the NEF4 preparation (18) strongly suggests
that this activity is an intrinsic property of NEF4. We also examined
the kinetics of ATP hydrolysis, and as shown in Fig.
2, the hydrolysis of ATP increased with
the incubation time and was proportional to the amount of NEF4 used.
From the results presented in Fig. 2, the turnover number for ATP
hydrolysis was calculated to be 150 for the
X 174 ss DNA and 380 for
the
X dsDNA. NEF4 hydrolyzes dATP with the same efficiency as
ATP.

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Fig. 1.
DNA-dependent ATPase activity
co-elutes with NEF4. Mono S fractions 9-21 from the last step of
NEF4 purification were subjected to immunoblot analyses with anti-Rad7
and anti-Rad16 antibodies and also analyzed for
dsDNA-dependent ( ) and ssDNA-dependent ( )
ATPase activities. For the immunoblot analyses, 1.0 µl of the
fractions was used, and for the ATPase assays, 4.0 µl of the fractions were used.
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Fig. 2.
Characterization of the NEF4 ATPase activity.
A, dsDNA-dependent ( ) and
ssDNA-dependent ( ) ATPase activities of NEF4 (120 ng) as
a function of the reaction time. B,
dsDNA-dependent ( ) and ss DNA dependent ( ) ATPase
activities as a function of the NEF4 amounts. The incubation time was
30 min. C, NEF4 does not exhibit DNA helicase activity.
NEF4, at 25, 50, 100, and 300 ng was incubated with 5 ng of the 17-base
pair partial duplex substrate for 45 min at 30 °C (lanes
3-6). Lane 1, DNA substrate. In lane 2, the
DNA substrate was boiled for 2 min to release the annealed
32P-labeled DNA fragment. nt, nucleotides.
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Consistently, we found that single-stranded
X DNA was about 50% as
effective as double-stranded
X DNA in activating ATP hydrolysis by
NEF4 (Fig. 2 and Table I). To investigate
the possibility that ATP hydrolysis seen with
X ssDNA might have
been due to the presence of secondary structure in the DNA, we examined
whether the single-stranded homopolymer poly(dA), which is relatively free of secondary structure, could also activate ATP hydrolysis by
NEF4. As shown in Table I, poly(dA) was also effective in activating
ATP hydrolysis. Taken together, these results indicate that NEF4
hydrolyzes ATP upon binding either ssDNA or dsDNA, with the latter
being the preferred DNA cofactor. No ATP hydrolysis was detected with
the polyribonucleotides poly(A) and RNA.
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Table I
DNA-dependent ATPase activity of NEF4
NEF4, 120 ng, was incubated with various DNA cofactors for 30 min as
described under "Materials and Methods." 100% activity corresponds
to the hydrolysis of 65% of the input ATP.
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Although the Rad7 protein does not show any discernible conserved
sequence motifs, the Rad16 protein belongs to the Swi2/Snf2 family of proteins whose members function in diverse chromosomal processes including transcription, transcription-coupled repair, post-replication repair, and recombinational repair (see Ref. 17 for
discussion). All of the Swi2/Snf2 family of proteins possess the
seven consensus helicase-like motifs that may be primarily involved in
coordinating DNA binding, ATP binding, and the hydrolysis of ATP, as
none of them has yet been shown to have DNA helicase activity (11,
25-27). To determine whether Rad16 protein was an exception, we
hybridized a 17-base 32P-labeled DNA fragment to circular
M13 ssDNA and used the resulting partial duplex as substrate to test
whether NEF4 had helicase activity. Incubation of the helicase
substrate with as much as 300 ng of NEF4 for 45 min did not result in
displacement of the hybridized fragment from the partial duplex (Fig.
2C), whereas 100 ng of Rad3 protein, a known helicase (20),
resulted in >80% displacement of the hybridized fragment (data not
shown). NEF4 also did not unwind a forked DNA substrate that contained
20-nucleotide-long 3'- and 5'-overhanging tails adjoining a 30-base
pair duplex region (data not shown). Thus, it appears that, like the
other Swi2/Snf2 family proteins that have been examined to date
(11, 25-27), ATP hydrolysis by Rad16 is utilized for purposes other
than extensive disruption of base pairing in dsDNA.
Rad16 protein contains two potential zinc binding, DNA binding motifs,
a C4 motif and a C3HC4 motif (23),
which could confer damage-specific DNA binding activity to NEF4.
C4 motifs are present in other known damage recognition
factors including the Escherichia coli UvrA protein and the
yeast Rad14 and human XPA proteins (22, 28-31). The DNA binding
activity of NEF4 was examined by a DNA mobility shift assay in which
purified NEF4 was incubated with a 130-base pair DNA fragment that was
end-labeled with 32P in the presence of cold
X 174 dsDNA
as competitor to titrate out the nonspecific binding and then analyzing
the reaction mixtures on nondenaturing polyacrylamide gels followed by
autoradiography of the dried gels to detect nucleoprotein complexes. As
reported in our recent work (18) and reiterated here in Fig.
3, in the presence of ATP, NEF4 binds
specifically to UV-irradiated DNA. For instance, whereas less than 2%
of the nondamaged DNA fragment was bound by NEF4 (Fig. 3A,
lane 4; Fig. 3B), UV irradiation of the DNA
fragment with doses of 2 and 10 kJ/m2 resulted in binding
of about 40 and 90% of the DNA fragment (Fig. 3A,
lanes 8 and 12; Fig. 3B),
respectively. The omission of ATP from the binding reaction diminished
damage-specific DNA binding markedly, such that only 5 and 24% of the
fragments irradiated with 2 and 10 kJ/m2 were bound by
NEF4, respectively (Fig. 3).

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Fig. 3.
Effects of ATP and ATP S on damage
recognition by NEF4. A, NEF4, 60 ng, was incubated with
nondamaged DNA and with DNA irradiated with 2 and 10 kJ/m2
of UV light in the absence or presence of 2 mM ATP or
ATP S, as indicated. N/P, no protein; F, free
DNA probe; C, nucleoprotein complexes. B, the gel
in A was subjected to phosphoimage analysis to quantify the
binding results.
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Previously, we showed that the nonhydrolyzable ATP analogue ATP
S
promotes damage binding by NEF4 (18), indicating that damage
recognition can occur in the absence of ATP hydrolysis. To determine if
ATP hydrolysis modulates the level of damage recognition, here we
examine the effect of ATP and ATP
S on the ability of NEF4 to bind
DNA that had been irradiated with 2 or 10 kJ/m2 of UV
light. As shown in Fig. 3, even though ATP
S promotes damage binding,
the level of damage binding achieved with ATP
S (26 and 67% for the
2- and 10 kJ/m2-irradiated fragments) was lower than that
seen with ATP (40 and 90% binding of the 2- and the
10-kJ/m2 irradiated fragments). These results suggest that
ATP hydrolysis increases the efficiency of damage recognition by
NEF4.
To further analyze the relationship between damage recognition and ATP
hydrolysis by NEF4, we incubated NEF4 with
X dsDNA that has been
exposed to increasing doses of UV light (0.1-10 kJ/m2) and
32P-labeled ATP. Unexpectedly, we found that UV irradiation
of the dsDNA cofactor in fact results in a marked inhibition of the
NEF4 ATPase activity (Fig. 4). For
instance, irradiating the dsDNA with a dose of 500 J/m2,
which introduced approximately five photoproducts per kilobase pair,
resulted in 30% inhibition of the ATPase activity, with more
inhibition occurring at progressively higher UV doses (Fig. 4). Thus,
the results indicate that the binding of NEF4 to UV lesions is
accompanied by an attenuation of ATP hydrolysis.

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Fig. 4.
UV irradiation of the DNA cofactor attenuates
ATP hydrolysis. NEF4, 120 ng, was incubated for 30 min with
32P-labeled ATP and with 250 ng of X dsDNA that had been
irradiated with increasing doses of UV light. ATP hydrolysis was
quantified by thin layer chromatography and phosphoimage analysis as
described under "Materials and Methods."
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DISCUSSION |
Consistent with the presence of nucleotide binding motifs in the
Rad16 protein, purified NEF4 has an ATPase activity that requires
either ssDNA or dsDNA for its activation. In this regard, dsDNA is
approximately twice as effective as ssDNA in activating ATP hydrolysis.
NEF4 binds preferentially to UV damaged DNA (Ref. 18 and this work),
and ATP is required for maximal damage-specific binding. Even though
ATP
S promotes damage-specific DNA binding, this reaction occurs more
efficiently with ATP than with ATP
S. Furthermore, we find that UV
irradiation of the dsDNA cofactor results in an attenuation of the
ATPase activity. Taken together, our results strongly suggest that ATP
hydrolysis by NEF4 is primarily utilized in steps before the binding of
NEF4 to the UV damage in the target DNA.
The strong dependence of the NEF4 damage recognition ability on ATP
distinguishes it from two other known damage recognition factors, Rad14
and RPA, which are integral components of the NER machinery (4). The
yeast Rad14 protein and its human counterpart XPA both contain a zinc
finger motif and have affinity for UV damage, but the damage binding
activity of these yeast and human NER factors is not influenced by ATP
(22, 30). RPA has also been shown to have an affinity for UV damage,
but it does so in the absence of ATP (32). From the results of genetic
and biochemical studies, we surmise that NEF4, by utilizing the free
energy from ATP hydrolysis, translocates on chromosomal DNA to survey
the DNA for the presence of bulky damages such as UV photoproducts (Fig. 5). Stable binding of NEF4 to the
DNA lesion results in an attenuation of the ATPase activity, and the
now damage-associated NEF4 serves as the nucleation site for the
recruitment of other NER factors including the aforementioned damage
recognition proteins to initiate the assembly of the NER machinery.
This working model for NEF4 action predicts that Rad7 and Rad16
interact with one or more protein components in NEF1, NEF2, NEF3, and
RPA to effect the coupling of the damage recognition process to the
assembly of the basic NER machinery comprising the latter protein
complexes.

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Fig. 5.
A model of NEF4 action in damage
recognition. ATP hydrolysis fuels the translocation of NEF4 on
DNA. At the damage site, NEF4 ATPase is attenuated, allowing for the
assembly of other NER factors.
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