(Received for publication, December 7, 1995; and in revised form, February 3, 1996)
From the
In yeast and humans, nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA requires a large number of highly conserved protein factors, which include the multisubunit RNA polymerase II transcription factor TFIIH. Here, we examine whether NER occurs by sequential assembly of different repair factors at the site of DNA damage or by the placement there of a ``preformed'' repairosome containing TFIIH and all the other essential NER factors. Contrary to the recent report (Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D.(1995) Cell 80, 21-28), our results provide no evidence for a pre-assembled repairosome; instead, they support the sequential assembly model. By several independent criteria, including co-purification, immunoprecipitation, and gel filtration of homogeneous proteins, we show that the damage recognition factor Rad14 exists in a ternary complex with the Rad1-Rad10 nuclease. We also find that Rad14 interacts directly with Rad1, but only slightly with Rad10, and that it interacts with the Rad1-Rad10 complex much more efficiently than with Rad1 alone. In the reconstituted NER system, a higher level of incision of UV-damaged DNA is achieved with the Rad1-Rad10-Rad14 complex, which we designate as nucleotide excision repair factor-1, NEF-1.
Nucleotide excision repair (NER) ()represents the
most important pathway for the removal of DNA damage inflicted by
ultraviolet (UV) light and by a variety of other DNA-damaging agents
that distort the DNA helix. NER is accomplished by dual incision of the
damage containing DNA strand, thus excising the damage in the form of a
short DNA fragment (reviewed in (1) and (2) ). A
defect in NER results in xeroderma pigmentosum (XP) in humans; XP
patients are extremely sensitive to sunlight, and they suffer from a
high incidence of skin cancers. Cells derived from XP individuals show
elevated mutation rates because of misincorporation of bases opposite
the unexcised UV lesions by DNA polymerases during replication of the
damaged DNA template.
Studies in the yeast Saccharomyces cerevisiae have been instrumental in defining the functions of NER genes in different stages of the repair reaction and in revealing their roles in cellular processes other than NER. Seven genes, namely RAD1, RAD2, RAD3, RAD4, RAD10, RAD14, and RAD25, are essential for the incision step of NER, and four other genes, namely RAD7, RAD16, RAD23, and MMS19, affect the proficiency of the repair reaction(1) . In addition to their NER function, RAD3 and RAD25 genes are essential for viability because of their requirement in RNA polymerase II transcription(3, 4, 5) . Rad3 and Rad25 constitute two of the subunits of the transcription factor TFIIH(6) . RAD1 and RAD10 also function in mitotic recombination in a pathway that is distinct from the RAD52 recombination pathway(7, 8) .
The products encoded by the seven RAD genes indispensable for the incision of UV damaged DNA have been purified to near homogeneity and their biochemical activities have been determined. Rad14 is a zinc metalloprotein that binds preferentially to UV-damaged DNA(9) . Rad3 protein is a DNA helicase (10) and it binds UV-damaged DNA preferentially in an ATP-dependent manner(11) . The Rad25 protein also possesses a DNA helicase activity(5) . Rad1 and Rad10 proteins form a complex that has single-stranded DNA endonuclease activity(12, 13) . Rad2 protein is also a single-stranded DNA endonuclease(14) . The Rad4 protein exists in a stoichiometric complex with Rad23, and available evidence suggests a role of this complex in the assembly of the repair factors at the damage site(15, 16) .
Using purified protein factors, we have recently reconstituted the damage-specific incision reaction of NER(16) . This work has indicated the requirement for the Rad1-Rad10 complex, Rad2 protein, the Rad4-Rad23 complex, Rad14 protein, replication protein A (RPA), and the RNA polymerase II transcription factor TFIIH in the incision reaction. ATP-dependent dual incision of the UV-damaged DNA by the combination of purified factors results in the release of 24-27-nucleotide-long DNA fragments. No incision of the damaged DNA occurs if any of the aforementioned protein factors is omitted(16) . The requirement for such a large body of protein factors in the incision reaction indicates that this reaction likely involves a series of highly coordinated events dictated by stringent temporal and spatial assemblies of the protein factors at the damage site. One of our main goals now is to define the manner and sequence by which these proteins are assembled onto the DNA damage site. These studies, however, will be greatly facilitated by a prior understanding of how the NER proteins are organized into functional subassemblies, since such a determination not only would enable us to begin dissecting the biological functions and significance of each subassembly, but it would also be a prerequisite for logical experimental designs in working toward the goal of defining the order and manner of recruitment of the subassemblies at the DNA damage site.
From previous work, core TFIIH is known to contain the following six
subunits, Rad3, Rad25, TFB1, SSL1, p55, and
p38(6, 16, 17) . RPA is a heterotrimer of
69-, 36-, and 13-kDa subunits(18) . Rad1 and Rad10 form a
complex(19, 20) , as do Rad4 and Rad23(16) .
It is not known whether or not these multimeric NER factors are
physically associated with one another and with the other NER proteins
in one complex forming a repair holoenzyme. Should there be a higher
order complex consisting of the aforementioned NER factors, however,
co-purification of these factors will be observed upon chromatographic
fractionation of cell extract; precisely the same approach has been
used in identifying various functional subassemblies that help mediate
transcription and DNA replication. Here, we find co-purification of the
damage recognition protein Rad14 with the Rad1-Rad10 complex. Results
from additional experiments indicate that Rad14 binds directly to the
Rad1-Rad10 complex with a dissociation constant (K) of < 5.3
10
M, and that Rad1-Rad10-Rad14 function together as a
subassembly in the NER reaction.
Recently, it has been reported that TFIIH exists in a physical complex with products of NER genes RAD1, RAD2, RAD4, RAD10, and RAD14, forming a nucleotide excision ``repairosome''(21) . Contrary to this report, we find no evidence for a pre-assembled complex of TFIIH with Rad1, Rad4, Rad10, and Rad14 proteins. Our data support the model wherein different repair factors assemble at the site of DNA damage in a sequential manner.
Buffers A, B, C, and D were as described
in (26) . All the column fractionation steps were carried out
at 4 °C, and all of the dialysis steps were done on ice. The
extract (Fraction I; 1,200 ml) was clarified by centrifugation (100,000
g for 60 min) and dialyzed against 10 liters of buffer
A for 14 h. The ionic strength of the dialysate was adjusted to 120
mM KOAc with buffer A before it was applied onto a column of
Bio-Rex 70 (5
10.2 cm; 200 ml matrix total) equilibrated in
buffer A + 120 mM KOAc. The column was washed with 600 ml
each of buffer A + 120 mM KOAc and buffer A + 200
mM KOAc, before the bound proteins were eluted with buffer A
+ 600 mM KOAc, collecting 10-ml fractions. The Bio-Rex 70
protein pool (Fraction II; 120 ml) was dialyzed against 2 liters of
buffer B + 100 mM KOAc for 14 h and then applied onto a
DEAE-Sephacel column (2.5
10.2 cm; 50 ml matrix total)
equilibrated in buffer B + 100 mM KOAc. After loading,
the DEAE column was washed with 100 ml of buffer B + 100 mM KOAc, 150 ml of buffer B + 200 mM KOAc, and then
eluted with buffer B + 500 mM KOAc, collecting 4-ml
fractions. The protein pool (Fraction III; 30 ml) from the 500 mM KOAc wash was dialyzed against 1 liter of buffer C without EDTA
for 14 h. The dialysate was clarified by centrifugation (20,000
g, 15 min) and applied onto a column of hydroxyapatite
(Bio-Gel HTP from Bio-Rad; 1.6
5 cm; 10 ml matrix total)
equilibrated in buffer C and which was developed with a 120-ml gradient
from buffer C to buffer D, collecting 2.4-ml fractions. The
hydroxyapatite fractions were screened by immunoblotting for the Rad1,
Rad4, Rad10, Rad14, and for the following TFIIH subunits: Rad3, Rad25,
SSL1, and TFB1.
Figure 2: TFIIH is not physically associated with Rad1, Rad10, or Rad14. A, purified Rad1, Rad10, and Rad14 proteins were mixed individually with nickel-agarose. The starting material (ST), the supernatant (SP) obtained after separation of the nickel matrix by centrifugation, and the 10, 20, 30, 40, and 100 mM imidazole eluates were examined by immunoblotting for their content of the Rad proteins. B, the 20 and 30 mM imidazole washes from the nickel-agarose binding step (see Fig. 1) were combined, concentrated, and subjected to molecular sizing in a column of Sephacryl S-300 HR as described under ``Materials and Methods.'' The column onput (OP) and fractions 24-48 from the column were subjected to immunoblotting to locate TFIIH and Rad1, Rad10, and Rad14 proteins.
Figure 1: Affinity binding of TFIIH to nickel NTA-agarose. A, fractionation scheme used. Details for the chromatographic steps are given under ``Materials and Methods.'' B, the hydroxyapatite pool (ST) containing TFIIH, Rad1, Rad10, and Rad14 proteins was mixed with nickel-agarose at 4 °C for 150 min and then centrifuged to collect the matrix. After decanting the supernatant (SP), the nickel matrix was transferred to a column and washed with 10, 20, 30, 40, and 100 mM imidazole to elute the bound proteins. The different fractions were run along with molecular size markers (M) in 9% denaturing polyacrylamide gels, which were either stained with Coomassie Blue in B or subjected to immunoblotting with the indicated affinity purified antibodies in C. The Rad3, Rad25, and TFB1 subunits of TFIIH in the 100 mM imidazole wash are marked in B.
In the experiment described in Fig. 4A,
purified Rad14 protein, 6.9 µg or 160 pmol, in 400 µl of buffer
F also containing 60 µg of BSA was applied onto a column of
Sephacryl S-200 HR (1 42 cm; total 33 ml) in buffer F
containing 100 mM KOAc, at a flow rate of 0.1 ml/min and
collecting 0.5 ml fractions. In the experiment described in Fig. 4B, 20 µg of Rad1, 3.9 µg of Rad10, and
6.9 µg of Rad14, 160 pmol each of protein, were incubated on ice in
150 µl of buffer F with 60 µg of BSA for 12 h, diluted to 400
µl with buffer F, and then subjected to sizing analysis in the
S-200 HR using the same conditions described for Rad14 protein. The
fractions were subjected to immunoblot analyses to locate Rad1, Rad10,
and Rad14 proteins.
Figure 4:
Assembly of a Rad1-Rad10-Rad14 complex in vitro. A, purified Rad14 protein was subjected to
sizing analysis in Sephacryl S-200 HR. The column onput (OP)
and fractions 20-48 from the column were analyzed by
immunoblotting for their content of Rad14. B, purified Rad1,
Rad10, and Rad14 proteins were incubated for 12 h on ice and then
subjected to sizing analysis in the same S-200 HR column. The column
onput (OP) and fractions 20-48 from the column were
analyzed by immunoblotting for their content of the three Rad proteins. C, the S-200 HR fractions 26-30 described in B that contained the peak of the Rad1-Rad10-Rad14 complex were
combined and a portion of the pool was mixed with protein A agarose (lane 1) or with protein A-agarose bearing anti-Rad1 (1; lane 3), anti-Rad10 (
10; lane 4), anti-Rad14
(
14; lane 5), or anti-Rad51 (
51; lane 2)
antibodies. The immunoprecipitates were washed and then treated with 2%
SDS to elute the bound proteins. The various eluates were analyzed by
immunoblotting for their content of the three Rad
proteins.
Figure 3:
Rad14 is physically associated with the
Rad1-Rad10 complex. Sephacryl S-300 HR fractions 36-40 containing
the peak of the Rad1, Rad10, and Rad14 proteins were combined and a
portion of the pool was mixed with protein A-agarose (lane 1)
or with protein A-agarose bearing covalently conjugated anti-Rad1
(1; lane 2), anti-Rad10 (
10; lane 3),
anti-Rad14 (
14; lane 4), or anti-Rad51 (
51; lane
5) antibodies. After washing, the immunoprecipitates were treated
with 2% SDS to elute the bound proteins, and the eluates were subjected
to immunoblotting to examine their content of the Rad
proteins.
Figure 5:
The Rad1-Rad10 complex is the molecular
entity for Rad14 recruitment. S-Labeled Rad14 protein
obtained by in vitro translation was incubated with Rad1
protein (lanes 1 and 3), with Rad10 protein (lanes 4 and 6), with the Rad1-Rad10 complex (lanes 7-9), or without any added protein (lanes 2 and 5) and then subjected to immunoprecipitation with
protein A-agarose beads bearing anti-Rad1 (
1; lanes 2, 3,
and 8), anti-Rad10 (
10; lanes 5, 6, and 9), or anti-Rad51 (
51; lanes 1, 4, and 7). Proteins were eluted from the immunoprecipitates with 2%
SDS and then subjected to SDS-polyacrylamide gel electrophoresis and
fluorography to reveal the content of radiolabeled Rad14 in each
case.
Figure 6:
Effect
of Rad1-Rad10-Rad14 complex formation on incision of UV damaged DNA. A, replicative form M13mp18 DNA (>90% supercoiled) with or
without UV treatment was incubated with the collection of NER factors
consisting of purified TFIIH, RPA, Rad2, the Rad4-Rad23 complex, and
the Rad1, Rad10, and Rad14 proteins, which were added either as
pre-assembled Rad1-Rad10-Rad14 complex (panel I), or as the
Rad1-Rad10 complex plus free Rad14 (panel II). After
incubation at 30 °C for the indicated times, the reaction mixtures
were deproteinized and subjected to electrophoresis in 0.8% agarose
gels, which were stained with ethidium bromide to reveal the
proportions of supercoiled form (SC) and open circular form (OC) in each sample. B, the gels shown in A were subjected to imaging analysis to obtain data points for a
graphical representation of the results. The first three time points (lanes 4-6 in A) were plotted for
Rad1-Rad10-Rad14 complex () and Rad1-Rad10 and free Rad14
(
).
Densitometric analysis of the immunoblot in Fig. 1C revealed that essentially all (>95%) of the
TFIIH in the onput was immobilized on the nickel-agarose, and that
15% and
75% of the bound TFIIH was eluted by 40 mM and
100 mM imidazole, respectively, consistent with specific
binding of this fraction of the transcription factor to the nickel
matrix through the 6-histidine tag on TFB1. Approximately 3 and 7% of
the total TFIIH was found in the 20 and 30 mM imidazole
washes, respectively, which might correspond to immobilization of TFIIH
through nonspecific interaction of one or more of the subunits of TFIIH
with the nickel matrix. Essentially all (>95%) of Rad1, Rad10, and
Rad14 proteins in the starting material also bound to the
nickel-agarose, and they were co-eluted from 10 to 30 mM imidazole, with 3, 48, and 47% of the total found in the three
washes with increasing imidazole, respectively. Considering the large
number of proteins in the onput that bound nonspecifically to
nickel-agarose (Fig. 1B), most plausibly, the
Rad1-Rad10 complex and Rad14 protein were also retained via nonspecific
interactions with the matrix. However, though highly unlikely, it could
not be formally excluded that the bound Rad1, Rad10, Rad14 were
associated with the small amount of TFIIH found in the 10-30
mM imidazole washes (Fig. 1B).
To establish
whether or not there was a physical association between Rad1, Rad10,
and Rad14 with TFIIH, we conducted the following two experiments.
First, purified Rad1, Rad10, Rad14 proteins, all greater than 95%
homogeneous(12, 16, 23) , were mixed
individually with nickel-agarose, which was then eluted with increasing
concentrations of imidazole. The various fractions with increasing
imidazole were subjected to immunoblotting, which indicated that, with
the exception of Rad10 protein, Rad1 and Rad14 proteins by themselves
bind quantitatively to nickel-agarose, with the bulk of the bound
proteins eluting from 10 to 40 mM imidazole in both instances (Fig. 2A). In fact, we found that bovine serum albumin
also binds nickel-agarose and is eluted from 10 to 20 mM imidazole (data not shown). Second, the 20 and 30 mM imidazole washes from the nickel-agarose step (Fig. 1, B and C) were combined, concentrated to a small
volume, and subjected to molecular sizing in a column of Sephacryl
S-300 HR (exclusion size limit: 1.5 10
daltons),
which was selected because it is particularly suited for separating
macromolecules in the size range relevant to TFIIH and the NER
proteins. If the Rad proteins were indeed associated with TFIIH,
precise co-elution of the NER factors would occur. On the other hand,
if, as was suspected, the presence of the Rad proteins and TFIIH in the
20 and 30 mM imidazole eluates was due to nonspecific
retention of these factors on nickel-agarose, we should see separation
of the Rad proteins from TFIIH because of their size difference. The
fractions from the S-300 column were subjected to immunoblotting, which
revealed the peak of TFIIH in fractions 30-34, corresponding to
45.5% to 51.5% of the column volume, and that Rad1, Rad10, and Rad14
co-eluted, away from TFIIH, in fractions 36-40, corresponding to
54.5% to 60.6% of the column volume. Thus, the results from this
molecular sizing analysis indicated that there was no physical
association of TFIIH with any of Rad1, Rad10, and Rad14 proteins (Fig. 2B). The retention of Rad1 and Rad14 on
nickel-agarose during purification of TFIIH (Fig. 1B)
can be best explained not by an association with TFIIH, but by direct,
nonspecific binding of these two proteins to the nickel matrix.
By molecular sizing, we examined whether Rad14 combines
stably with the Rad1-Rad10 complex in the absence of any additional
component. To do this, we first subjected purified Rad14 protein (M 43,000) to sizing analysis in a column of
Sephacryl S-200 HR (exclusion size limit: 2.5
10
daltons). As shown in Fig. 4A, the peak of Rad14
protein emerged in fractions 34-38, corresponding to
51.5-57.5% of the column volume; bovine serum albumin (M
67,000) emerged from the same column in
fractions 32-36 (data not shown). Thus, the elution pattern of
Rad14 protein from the S-200 sizing column is consistent with it being
a monomer under the conditions employed. Next, purified Rad1, Rad10,
and Rad14 proteins were incubated in buffer on ice for 12 h to allow
for protein-protein interactions to occur, and the mixture was
subjected to sizing analysis in the same S-200 column. In this case,
essentially all of the Rad14 protein co-eluted with the Rad1 and Rad10
proteins in fractions 26-32, corresponding to 39.4-48.5% of
the column volume (Fig. 4B). Again, Rad1-Rad10-Rad14
proteins in the S-200 fractions co-immunoprecipitated specifically (Fig. 4C). Taken together, our results indicated that
Rad14 protein forms a complex with Rad1-Rad10 in the absence of any
other NER component. Since the majority of the Rad14 protein remained
associated with the Rad1-Rad10 protein in the S-200 column fractions
over the course of >24 h (Fig. 4C), the kinetic
constant for dissociation (K
) of the
Rad1-Rad10-Rad14 complex is at or below the concentration of 5.3
10
M of the proteins present in the
S-200 column fractions.
As shown in Fig. 5, Rad1
protein alone interacts with Rad14 protein, as indicated by the fact
that 12% of the input Rad14 co-precipitated with the added Rad1
protein by the anti-Rad1 immunobeads (lane 3), but not by the
same immunobeads in the absence of Rad1 protein (lane 2), nor
by beads bearing anti-Rad51 antibodies in the presence of Rad1 (lane 1). By contrast, less than 2% of the input Rad14
co-precipitated with Rad10 protein (Fig. 5, lanes
4-6), thus indicating a much weaker interaction of Rad14
with Rad10 than with Rad1. Interestingly, we found that greater than
70% of the input Rad14 protein co-precipitated with the Rad1-Rad10
complex by either anti-Rad1 or anti-Rad10 immunobeads (Fig. 5, lanes 7-9). Thus, the amount of Rad14 that
co-precipitated with the Rad1-Rad10 complex was much greater
(
5-fold) than the sum of Rad14 protein that co-precipitated with
Rad1 and Rad10 singly. The finding that Rad14 combines with the
Rad1-Rad10 complex much more efficiently than with either Rad1 or Rad10
protein alone was verified in two other independent experiments (data
not shown). Taken together, our results indicate that the Rad1-Rad10
complex is the physiologically relevant entity for interaction with
Rad14.
Since the agarose gel method (16; see ``Materials and Methods'') represents a highly sensitive means for determining the initial rate of incision of the UV damaged substrate, we used it to examine the relevance of the Rad1-Rad10-Rad14 complex to the damage specific incision reaction. To do this, the standard protocol was modified so that all the protein components except for Rad1, Rad10, and Rad14 were still subjected to preincubation, and then the latter three proteins were added, together with the DNA substrate, either as the Rad1-Rad10 complex plus free Rad14 or as pre-assembled Rad1-Rad10-Rad14 complex. The incision reaction was then allowed to proceed at 30 °C and terminated at 3, 6, 9, 12, and 15 min by adding SDS and proteinase K. The reaction samples were run in an agarose gel and then stained with ethidium bromide to reveal the proportions of open circular and supercoiled forms in the various samples. The gel in Fig. 6A was also subjected to image analysis to obtain data points for a graphical representation of the results (Fig. 6B). As reported in our recent work (16) and shown here in Fig. 6, ATP-dependent incision of the UV-damaged plasmid DNA occurred, as indicated by the accumulation of the open circular form with increasing reaction time. Importantly, a higher level of damage specific incision occurred where Rad1, Rad10, and Rad14 were added as a preformed complex than when they were added as the Rad1-Rad10 complex and free Rad14 (Fig. 6, A and B). This observation was confirmed in two other independent experiments. Thus, complex formation between Rad1-Rad10 nuclease and Rad14 represents an important step that has a direct influence on incision proficiency.
The presence
of the 6-histidine tag in the TFB1 protein allowed the immobilization
of TFIIH and, theoretically, any protein that is physically associated
with TFIIH on nickel-agarose. To test whether the presence of Rad1,
Rad10, and Rad14 proteins in the hydroxyapatite TFIIH pool was due to a
simple partial overlap of the peaks of the Rad proteins and TFIIH or
actually reflected a physical association between the Rad proteins and
TFIIH, we mixed the TFIIH pool with nickel agarose and then washed the
nickel matrix with increasing concentrations of imidazole to elute the
bound proteins. From examining the protein makeup and content of TFIIH
and Rad proteins in the various fractions (Fig. 1, B and C), it was clear that (i) a large number of the
protein species in the starting material bound nonspecifically to the
nickel matrix and were eluted from 10 to 30 mM imidazole, (ii)
the bulk of TFIIH bound specifically through the 6-histidine tag to the
nickel matrix and was eluted by 100 mM imidazole; the 100
mM imidazole eluate was 50-fold enriched in TFIIH, but
contained no Rad1, Rad10, and Rad14 proteins, and (iii) a small
fraction (
10%) of TFIIH and almost all (
95%) of the Rad1,
Rad10, and Rad14 were found in the 20 and 30 mM imidazole
eluates. The co-existence of the three Rad proteins and TFIIH in the 20
and 30 mM imidazole washes as noted in iii was due to
fortuitous, nonspecific interaction of these protein factors with the
nickel matrix as indicated in two additional experiments. First, the
three Rad proteins were well separated from TFIIH when the 20 and 30
mM imidazole washes were combined and subjected to molecular
sizing in Sephacryl S-300 HR (Fig. 2B). This sizing
analysis demonstrated clearly that TFIIH was not physically associated
with the Rad proteins in these imidazole fractions, because if they had
been associated, precise co-elution of these factors would have been
seen. Second, when purified Rad1 and Rad14 proteins that do not possess
a 6-histidine tag were mixed with nickel agarose in the absence of any
other protein component, quantitative nonspecific binding of these
proteins to the matrix occurred (Fig. 2A). Because
Rad10 protein by itself does not show any affinity for nickel-agarose (Fig. 2A), its retention on nickel-agraose (Fig. 1) was effected via its association with Rad1 and Rad14
proteins (see below), both of which bind the nickel matrix (Fig. 2A). Taken together, our results indicate that
TFIIH is not physically associated with any of Rad4, Rad1, Rad10, and
Rad14 proteins in a stable form that can be isolated by column
chromatography.
Our results differ from the recent report (21) that TFIIH, Rad1, Rad4, Rad10, and Rad14 proteins existed
in a physical complex, which the authors called
``repairosome.'' In deriving their conclusion, these workers
used the same yeast strain as was used in our work and observed that
when a phosphocellulose column fraction enriched in TFIIH
(TFB1-6His) and Rad proteins was mixed with nickel-agarose,
retention of Rad1, Rad10, Rad14, and other Rad proteins occurred and
that all of the bound Rad proteins were eluted by lower concentrations
of imidazole than was needed to elute the bulk of TFIIH. Svejstrup et al.(21) also subjected the 20 mM imidazole eluate from the nickel-agarose step that contained Rad
proteins and some TFIIH to molecular sizing in a column of Sepharose
CL-2B and found co-elution of these factors. However, our studies
indicate that Rad1 and Rad14 proteins bind nickel-agarose by
themselves. Also, since the molecular mass of TFIIH and that of the
other NER proteins, as well as the size differences between the former
and latter, are rather insignificant relative to the large pore size of
Sepharose CL-2B (exclusion size limit: 4 10
daltons), all of these protein factors will be retarded to very
similar degrees in the Sepharose matrix, resulting in poor separation
of factors. In fact, when we subjected the combined 20 and 30 mM imidazole eluates from nickel-agarose to sizing in Sepharose
CL-4B, which has a smaller pore size (exclusion size limit: 2
10
daltons) than that of Sepharose CL-2B, even then the
poor resolution of TFIIH from the Rad1-Rad10-Rad14 complex resulted in
substantial overlap of the peaks of the two entities, creating the
impression that they might be physically associated (data not shown).
For this reason, we selected Sephacryl S-300 HR (exclusion size limit:
1.5
10
daltons) in our study (Fig. 2B) for effective separation of TFIIH from the
Rad1-Rad10-Rad14 complex. In summary, our results do not support the
premise that TFIIH is physically associated with all the essential NER
proteins in a ``repairosome.''
To
gain insight into how assembly of the Rad1-Rad10-Rad14 ternary complex
occurs, S-labeled Rad14 protein obtained by in vitro coupled transcription-translation was incubated with Rad1, with
Rad10, or with the Rad1-Rad10 complex. In each case, the amount of
Rad14 that associated with the other component was determined by a
combination of immunoprecipitation, gel electrophoresis, and
fluorography. We found that Rad14 interacts directly with Rad1 protein
but very weakly with Rad10 protein. Importantly, a much higher level of
Rad14 was bound by the Rad1-Rad10 complex than could be accounted for
by the sum of Rad14 amounts that were found associated with free Rad1
and Rad10 proteins (Fig. 5). These observations indicate that
formation of the Rad1-Rad10-Rad14 ternary complex is mediated via
interaction of Rad14 with the Rad1-Rad10 binary complex. It remains to
be determined whether Rad14 protein makes substantial contacts with
both Rad1 and Rad10 in the ternary complex, or when present in the
Rad1-Rad10 binary complex, Rad1 protein adopts a conformation that is
conducive for binding Rad14.
The human Rad1 homolog XPF associates with the Rad10 homolog ERCC1, and like Rad1-Rad10, the XPF-ERCC1 complex is a DNA endonuclease(27) . ERCC1, and the XPA protein, which is the counterpart of Rad14, were shown to interact in a yeast two-hybrid system(28) , and ERCC1 protein was found to bind to amylose-agarose containing a hybrid polypeptide of maltose-binding protein-XPA(28, 29) . The results with ERCC1 and XPA proteins are not necessarily incompatible with our finding that Rad10 interacts with Rad14 only very weakly, because the two-hybrid system and the affinity binding method used in detecting ERCC1-XPA interaction are designed to reveal even weak and transient protein-protein interactions. Our results would suggest that XPA exists as a complex with ERCC1 and XPF proteins in vivo and that XPF has a major role in the formation of this complex.
While the order and the manner by which the NER machinery is assembled at the DNA damage site remains to be determined, our studies provide support to the notion that NER involves the sequential assembly of different protein factors at the damage site, rather than the placement of a ``preformed'' repairosome containing all the factors required for incision.