(Received for publication, October 13, 1994; and in revised form, December 27, 1994)
From the
Recent studies have revealed that the general transcription
factor TFIIH is also a general excision repair factor which, along with
several other proteins, is required for transcription-independent
excision reaction. As a general transcription factor, TFIIH is
recruited to RNA polymerase II-promoter complex by another general
transcription factor called TFIIE. We were interested in knowing
whether TFIIE is also involved in recruiting TFIIH to the excision
repair complex. We found that cell-free extract depleted of TFIIE
carried out excision repair at a normal rate, leading us to conclude
that TFIIE is not involved in recruiting TFIIH to the damage site and
has no role in general excision repair. In contrast, the human damage
recognition protein XPA specifically binds to TFIIH and apparently
recruits it to the damage site. The carboxyl-terminal half of XPA is
responsible for specific interaction with TFIIH. The C261S/C264S mutant
of XPA bound the ERCC1XPF complex normally, but failed to bind
TFIIH and failed to complement an XP-A mutant cell-free extract
indicating that the XPA-TFIIH interaction is essential to effecting the
excision reaction. Interestingly, XPA also binds to the p34 subunit of
TFIIE specifically and in competition with the p56 subunit of TFIIE.
This latter interaction has no apparent role in general excision repair
but may be relevant in the transcription-coupled repair reaction.
In humans bulky DNA lesions such as cyclobutane pyrimidine
dimers and cisplatin 1,2-d(GpG) intrastrand cross-links are removed by
an excision nuclease system which incises at the 4-5th
phosphodiester bond 3` and the 22nd-24th phosphodiester bond 5` to the
lesion and thus releases 27-29 nucleotide-long oligomers carrying
the DNA damage (Huang et al., 1992; 1994a, 1994b). Defective
excision repair causes xeroderma pigmentosum (XP), ()a
disease characterized by photodermatoses including skin cancers, and in
some cases neurological abnormalities (Cleaver and Kraemer, 1989).
Somatic cell genetics have identified 8 complementation groups, XPA-XPG
and ERCC1 of mammalian excision repair proteins (see Prakash et
al.(1993) and Tanaka and Wood(1994)). However, recent biochemical
studies have revealed that the entire TFIIH general transcription
factor complex (at least 8 subunits including the XPB and XPD proteins)
is a repair factor suggesting that additional proteins, not defined by
XP complementation groups, may play essential roles in excision repair
(Drapkin et al., 1994a; Schaeffer et al., 1993, 1994)
and that there may be an intimate relation between transcription
initiation by RNA Pol II and transcription-independent excision repair.
This raised the possibility that other general transcription factors
may participate in excision repair. An obvious candidate is TFIIE
because it appears that all TFIIH transactions in transcription are
mediated through specific interactions with TFIIE (Flores et
al., 1989; Goodrich and Tjian, 1994; Ohkuma and Roeder, 1994).
During transcription initiation TFIIE interacts with several
proteins assembled at the promoter, including RNA Pol II itself, before
recruiting TFIIH (Conaway and Conaway, 1993; Zawel and Reinberg, 1993;
Maxon et al., 1994); thus we reasoned that it may function in
an analogous manner in repair by first interacting with the damage
binding proteins at the lesion site and then recruiting TFIIH to carry
out its repair function. Therefore, we tested the interactions of the
human damage binding protein XPA with TFIIE and TFIIH and the
requirement for these interactions to carry out the excision repair
reaction. We found that XPA binds to the p34 subunit of TFIIE and to
TFIIH directly. Cell-free extracts (CFEs) depleted of both subunits of
TFIIE are capable of full strength excision repair indicating that the
interaction of XPA with TFIIE plays no role in general
(transcription-independent) excision repair. In contrast, the XPA-TFIIH
interaction is crucial for general excision repair. Two point mutations
in the carboxyl-terminal half of XPA protein do not affect the binding
of the ERCC1XPF complex to XPA but abolish the binding of TFIIH
to XPA and the excision activity of the in vitro system
reconstituted with the mutant XPA protein. We conclude that, most
likely, the XPA protein recruits TFIIH to the lesion site and that
TFIIE is not involved in the general nucleotide excision reaction.
The carboxyl-terminal deletion mutants were constructed as follows. The N138 was made by digestion of pGEX18-XPA with HindIII and self-ligation to generate pGEX18-XPAN138. The N192 was constructed by digesting pGEX18-XPA with BsaI and NdeI, filling-in with Klenow fragment, and religating it to give pGEX18-XPAN192. The N256 was made as follows: pGEX18-XPA was digested with NdeI, filled-in with Klenow fragment, redigested with SacI, and then ligated with SacI and RsaI-digested fragment of pGEX18-XPA to generate pGEX18-XPAN256.
The point mutant XPA2CS (C261S, C264S) was constructed in the plasmid pCHA801 (Park and Sancar, 1993) by site-directed mutagenesis using the oligonucleotide 5`-TACCGTAAGACTAGTACTATGAGTGGCCATGAACTG-3` as the mutagenic primer (Sambrook et al., 1989). The mutation was confirmed by restriction digestion of the newly introduced restriction site. An 857-base pair fragment carrying the entire mutant XPA2CS was amplified by polymerase chain reaction and subcloned into pMAL-c2 to generate pMAL-XPA2CS, with which pGEX18-XPA2CS was constructed as described for pGEX18-XPA.
Anti-XPB antibodies were raised against Keyhole limpet
hemocyanin-coupled peptide (NH-DLFDFYEQMDKDEEEE-COOH)
corresponding to amino acids 247-262 of the deduced XPB
polypeptide (Weeda et al., 1990) with an additional cysteine
at the amino terminus of the peptide to aid in coupling. Anti-ERCC1
antibodies were raised against MBP-ERCC1 protein expressed in Escherichia coli (Park and Sancar, 1993). Anti-p62(TFIIH),
anti-p56E(TFIIE), and anti-p34E(TFIIE) antibodies were purchased from
Santa Cruz Biotechnology, Inc.
Competition of XPA with p56E for binding to p34E was studied using
glutathione-agarose beads containing 4 µg of GST-p56E, 5 µl of
the in vitro translated product of p34E, and the indicated
amounts of MBP--galactosidase
or MBP-XPA.
Interaction of XPA with TFIIH was assayed using glutathione-agarose beads containing 10 µg of GST or 2-4 µg of wild-type or mutant GST-XPA proteins and 50 µg of HeLa CFE or 2 ng of purified TFIIH. The bound proteins were visualized by Western blotting using antibodies against XPB or p62 subunits of TFIIH or anti-ERCC1 antibodies.
Figure 1: XPA binds to p34 (TFIIE) but not to the other subunits of TFIIE and TFIIH. Radiolabeled proteins (three subunits of TFIIH, XPB, XPD, and p62; and two subunits of TFIIE, p56E and p34E) were mixed with 4 µg of GST-XPA linked to glutathione-agarose beads and the unbound and bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. I, input proteins; U, unbound proteins; B, bound proteins.
Figure 2: XPA binds to p34 subunit of TFIIE but not to the p56-p34 (TFIIE) complex. The individual subunits of TFIIE (p56E and p34E) were expressed separately or co-expressed in the in vitro transcription-translation system and incubated with 10 µg of GST or 4 µg of GST-XPA linked to glutathione-agarose beads. The unbound and bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. I, input proteins; U, unbound proteins; B, bound proteins.
Figure 3:
XPA competes with p56E for binding to
p34E. Radiolabeled p34E was incubated with 4 µg of GST-p56E linked
to glutathione-agarose beads in the presence of the indicated amounts
(µg) of MBP--galactosidase
(
gal
) or
MBP-XPA proteins. The unbound and bound p34E were separated by SDS-PAGE
and visualized by autoradiography. I, input protein; U, unbound protein; B, bound
protein.
To find out if p34E is directly involved in excision repair, we tested the activities of immunodepleted CFE by the excision assay. Fig. 4shows that the extracts, immunodepleted of p34E by either anti-p56E or anti-p34E antibodies, carry out normal excision repair. In contrast, the transcription activity in the immunodepleted extract was reduced by two-thirds (data not shown). Hence, it appears that TFIIE or its individual subunits are not involved in transcription-independent (general) excision repair.
Figure 4: Immunodepletion of TFIIE from HeLa cells does not affect its excision activity. HeLa CFE was depleted of TFIIE using anti-p56E or anti-p34E antibodies linked to protein A-agarose beads. The immunodepleted CFE (50 µg) was tested in Western blotting and excision assay. A, Western blot after immunodepletion. The immunodepleted CFE was resolved by SDS-PAGE and analyzed by Western blot with anti-XPB, anti-p56E, and anti-p34E antibodies. B, excision assay with immunodepleted HeLa CFE. The immunodepleted CFE was tested for excinuclease activity. 28 indicates the major excision product.
Figure 5: XPA binds directly to TFIIH. HeLa CFE (50 µg) or purified TFIIH (2 ng) were incubated with 10 µg of GST or 4 µg of GST-XPA linked to glutathione-agarose beads. The unbound and bound proteins were separated by SDS-PAGE and analyzed by Western blot with anti-XPB antibody. The Western blot with anti-p62 (TFIIH) antibody gave identical results (data not shown). I, input proteins; U, unbound proteins; B, bound proteins. The diffuse bands in the 25-30 kDa region of lanes 5 and 10 are GST-XPA products which react nonspecifically with anti-XPB antibodies.
Figure 6: The carboxyl-terminal domain of XPA is responsible for binding to TFIIH. A, schematic diagram of XPA deletion and point mutants and summary of binding analyses. B, binding of XPA mutants to TFIIH or ERCC1. Deletion or point mutants of XPA were expressed as GST fusion proteins and coupled to glutathione-agarose beads. Aliquots of the resins were analyzed by SDS-PAGE to confirm the concentration and integrity of bound protein (data not shown). HeLa CFE (50 µg) was incubated with 10 µg of GST or 2-4 µg of GST-XPA derivatives linked to glutathione-agarose beads. The bound proteins were separated by SDS-PAGE and analyzed by Western blot with anti-XPB and anti-ERCC1 antibodies.
Figure 7:
Wild-type and mutant XPA proteins used in
complementation and inhibition experiments. MBP-XPA and MBP-XPA2CS
(C261S, C264S) were purified through amylose and heparin-agarose
columns. Lane 1, molecular mass markers; lane 2, 2
µg of MBP-XPA; lane 3, 2 µg of MBP-XPA2CS; the asterisk indicates a minor degradation product of the mutant
protein which contains about 10% of the XPA protein from the
amino-terminal half fused to MBP. It is not expected to interact with
either TFIIH or the ERCC1XPF complex.
Figure 8: Complementation of XP-A CFE with wild-type and mutant XPA proteins. The indicated amounts of MBP-XPA or MBP-XPA2CS proteins were incubated with 50 µg of XP-A mutant CFE and the excision reaction was conducted under standard conditions. Lane 1, no addition; lanes 2 and 3, 50 and 100 ng of MBP-XPA, respectively; lanes 4-7, 50, 100, 200, and 400 ng of MBP-XPA2CS, respectively.
Figure 9: Inhibition of HeLa CFE by mutant XPA protein. The indicated amounts of MBP-XPA or MBP-XPA2CS proteins were added to the reaction mixtures containing 25 µg of HeLa CFE and the excision reaction was conducted under standard conditions. A, autoradiographic analysis of excision products. Lane 1, no addition; lanes 2-4, 0.8, 1.2, and 1.6 µg of MBP-XPA, respectively; lanes 5-7, 0.8, 1.2, and 1.6 µg of MBP-XPA2CS, respectively. B, quantitative analysis of inhibition. The excision products were quantitated by PhosphorImager (Molecular Dynamics).
Recent research in several laboratories has revealed that TFIIH contains XPB and XPD as two of its 8-9 subunits and that the entire factor, but not a subassembly of it, is an essential component of nucleotide excision repair (Drapkin et al., 1994a; Schaeffer et al., 1994; Wang et al., 1994). In fact, since TFIIH is required only for some promoters (Parvin et al., 1992; Parvin and Sharp, 1993; Tyree et al., 1993) yet is absolutely required for any excision repair, it might be more appropriate to consider it as a repair factor first and as a transcription factor second. In transcription, TFIIH is recruited to the promoter by TFIIE (Maxon et al., 1994; Zawel and Reinberg, 1995) and its helicase, ATPase, and RNA Pol II carboxyl-terminal domain kinase activities are modulated by TFIIE (Lu et al., 1992; Drapkin et al., 1994a; Ohkuma and Roeder, 1994). Therefore, it was hypothesized that TFIIH may be recruited to the excision complex by TFIIE. Surprisingly, we found that not to be the case. General excision repair occurs normally in the absence of TFIIE and XPA plays a crucial role in recruiting TFIIH to the damage site. However, it must be pointed out that the two assays used in this study, Western blotting for TFIIE and the excision assay for excinuclease activity, may be differentially sensitive to the presence of TFIIE. In other words, it is possible that sufficient TFIIE for excision repair remains following immunodepletion even though the remaining amount is not seen in the Western blot. To eliminate this possibility we tested the immunodepleted extract for RNA Pol II transcription and found that its transcription activity was severely reduced. In addition, we tested our recently reconstituted excinuclease system for TFIIE activity in a defined transcription system. We found that the defined excision repair system contained less than 5% of the TFIIE activity in a TFIIE/TFIIH-dependent transcription system and that the TFIIH from the defined excision repair system gave full TFIIH transcription activity when the transcription system was supplemented with TFIIE (data not shown).
Thus, taken together, these data indicate that either alone or possibly in combination with other XP proteins, XPA is a functional substitute of TFIIE in excision repair. However, in contrast with the TFIIE-TFIIH interactions which appear to be mediated by binding of XPB to both subunits of TFIIE (Drapkin et al., 1994a; Maxon et al., 1994), the subunit of TFIIH binding to XPA is not known. Only three subunits of TFIIH (XPB, XPD, and p62) were available to us for testing for potential interaction and these subunits did not bind to XPA. It is possible that one of the other subunits mediates XPA-TFIIH binding or that the binding interface is created by several subunits. Further experiments with the other subunits of TFIIH will resolve this issue.
The second specific interaction detected in our experiments,
the XPA-p34 (TFIIE) binding, remains to be explained at present. It is
conceivable that this interaction may play a role in coupling
transcription to repair. Based on the interaction of TFIIE with
XPB/ERCC3, Maxon et al.(1994) proposed a model which involves
TFIIE-TFIIH interaction in transcription-repair coupling. Even though a
defined system for RNA Pol II transcription has been available for some
time, a defined system for excision repair has only recently been
developed (Mu et al., 1995) and hence this model and several
others that have been proposed by other groups (see Hanawalt and
Mellon(1993) and Drapkin et al. (1994b)) can now be tested
under specific conditions. However, the following observations are
pertinent to this question. First, mutations in XPB and XPD which are
components of TFIIH (Schaeffer et al., 1993, 1994; Drapkin et al., 1994a) and in XPG which is loosely associated with
TFIIH (Mu et al., 1995), often but not always, are associated
with Cockayne's syndrome (CS) which is biochemically
characterized by lack of excision repair of transcribed sequences.
Second, CSA and CSB mutations which have only minor effects on excision
repair are totally defective in gene specific repair (Venema et
al., 1990). The CSB/ERCC6 gene has been cloned and it corrects the
defect in gene specific repair and is thought (perhaps in conjunction
with CSA protein) to act as a transcription-repair coupling factor
(Troelstra et al., 1992). Third, as reported in this paper,
XPA bound to a lesion is necessary for directing TFIIH to the site in
transcription-independent excision repair. Currently, it is difficult
to propose a transcription-repair coupling mechanism that incorporates
all these, at times contradictory, observations. In the past, various
groups including ours, have proposed that XPB/ERCC3, CSB/ERCC6, or
TFIIE play a role analogous to that of the Mfd protein in E. coli which displaces stalled RNA Pol from the lesion site and targets
the damage recognition subunit to the site of damage (Selby and Sancar,
1993). Clearly all of these models cannot be correct. We believe that
an explicit model can be made only after reconstitution of a defined
system capable both of excision repair and transcription. However, the
data presented in this paper along with the data on the functional
yeast homologs of XPG protein (Rad 2) and the ERCC1XPF complex
(Rad 10/1) enables us to propose the following working model for human
excinuclease system (Fig. 10). XPA binds to the damage site and
forms a stable complex with HSSB (Coverley et al., 1992; Mu et al., 1995); then it recruits the ERCC1
XPF complex and
the TFIIH factor with the associated proteins XPC and XPG. The XPE
protein may enter the assembly at some point. In the assembled complex
XPG makes the 3` incision (Habraken et al., 1993; Harrington
and Lieber, 1994; O'Donovan et al., 1994) (
)and ERCC1/XPF makes the 5` incision (Tomkinson et
al., 1993; Bardwell et al., 1994; Habraken et
al., 1994). The replication proteins PCNA (proliferating cell
nuclear antigen) (Nichols and Sancar, 1992; Shivji et al.,
1992), RFC (replication factor C), and Pol
/
dissociate the
postincision complex, release the excised oligomer, and fill-in the gap
which is then ligated.
Figure 10:
Model for excision repair in humans. The
damage recognition protein XPA binds to the lesion (i) and
interacts with HSSB to make a stable complex. This complex recruits the
ERCC(XPF) complex in an ATP-independent reaction (ii)
and the TFIIH factor in a reaction requiring ATP hydrolysis (iii). The XPC and XPG proteins are recruited with TFIIH and
at one point XPE presumably enters the complex. The DNA is partially
unwound and bent by the limited helicase activity of TFIIH. This
distortion enables XPG to make the 3` incision and the ERCC1
XFP
complex makes the 5` incision (iv). Following the dual
incisions the replication proteins displace the excinuclease subunit (v), fill-in and seal the gap (vi).