(Received for publication, November 23, 1994; and in revised form, December 18, 1994)
From the
Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.
Xeroderma pigmentosum (XP) ()is a hereditary disease
characterized by photosensitivity, increased frequency of skin cancers,
and, in some cases, neurological abnormalities(1) . The disease
is caused by a defect in nucleotide excision repair (2) as a
result of mutations in one of several genes, XPA through XPG. In
addition to the proteins defined by the XP complementation groups,
genetic and biochemical studies in mammalian systems indicate that the
ERCC1 protein, the transcription factor TFIIH (which contains the XPB
and XPD proteins and six other proteins), and the replication proteins
PCNA (proliferating cell nuclear antigen) and HSSB (human single
stranded DNA-binding protein, also known as RPA) are required for
excision repair(3, 4) . These studies raised the
possibility that additional proteins, not previously identified, may be
involved in the initial reaction of excision repair which consists of
the incisions of the 22nd through 24th phosphodiester bonds 5` and the
4th through 6th phosphodiester bonds 3` to the
lesion(5, 6) .
In this report, we purified the components known to be required for the incision reaction and here we provide strong evidence that these components are sufficient for this reaction. Moreover, we show that the excised damage-containing fragment is not released by this reconstituted excinuclease system, suggesting that additional helicase activity is required to displace the excised oligomer before repair synthesis can take place.
Replication protein A (also known as HSSB) was a kind gift from Drs. B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and J. Hurwitz (Sloan-Kettering Cancer Center, New York, NY).
The XPB (p89) and XPD (p80) proteins make a tight complex (14) within the transcription factor TFIIH, which contains six other subunits (p62, p50, p44, p41, p38, and p34) of which p62, p44, and p34 are also known to be required for excision repair(15, 16, 17) . Therefore, in order to purify TFIIH and other repair proteins, we followed the purification scheme outlined in Fig. 1. The purified TFIIH (Fraction II) contained the p89 (XPB), p80 (XPD), p62, p50, p44, p41, p38, and p34 subunits visible in silver-stained SDS-PAGE (Fig. 2B) as well as some higher molecular weight proteins also seen in other TFIIH preparations(8, 18, 19) , one of which we identified as the XPG protein (see below). The identities of the p89, p80, and p62 subunits were confirmed by Western blotting using a mixture of antibodies made against the XPB, XPD, and p62 proteins (Fig. 2B, lane 3). Furthermore, the TFIIH preparation complements the excinuclease activity in the CFEs of XP-B and XP-D mutants as well as the CFE of an XP-G mutant (Fig. 3). This is reminiscent of the findings in yeast, as it is known that the yeast homologs of XPC (Rad 4) and XPG (Rad 2) are loosely associated with the yeast TFIIH counterpart, Factor b(20) .
Figure 1: Purification scheme for the reconstitution fractions of the human excinuclease.
Figure 2:
Analyses of the five reconstitution
fractions by silver staining and/or immunoblotting. Approximately
1-5 ng of each reconstitution fraction was subjected to 10%
SDS-PAGE analyses. A, SDS-PAGE of Fraction I
(MBP-XPA). B, lane 1 shows SDS-PAGE of Fraction II
(TFIIH/XPG) (the TFIIH subunits are indicated by arrows); lane2 shows the immunoblotting with anti-XPG
antibodies; lane3 shows immunoblotting with
anti-XPB, anti-XPD and anti-p62 antibodies. C, immunoblotting
of Fraction III (partially purified XPC) with anti-XPC polyclonal
antibodies. D, lanes1-3, SDS-PAGE of
load (L), flow-through (FT), and elution (E)
of fractions of ERCC1XPF complex on a glutathione S-transferase-XPA affinity column. Lanes 4-6, Western blot of the same fraction. Lanes 3 and 6 contained 30 µl of the eluted (E) fraction (<1 ng
of protein). E, SDS-PAGE of Fraction V (HSSB). F,
SDS-PAGE of a highly purified TFIIH preparation using a seven-column
purification protocol(8) . This purification procedure yields
TFIIH complementing XP-B, XP-D, and XP-C but not any of the other XP
complementation groups(8) . Lane 2 shows the
immunoblotting using anti-XPC polyclonal
antibodies.
Figure 3:
Complementation of mutant CFEs by the
purified fractions. Top panel, schematic drawing of the
incision pattern of human excinuclease on the P-labeled
140-mer DNA-containing a cholesterol adduct in the center. The arrows indicate the major incision sites, which generate a
28-nt-long oligomer. Bottom panel, A, complementation
of XP-A mutant CFE (50 µg) by Fraction I (MBP-XPA, 50 ng); B, complementation of rodent mutant CFE defective in XPB, XPD,
and XPG by Fraction II (2 ng); C, complementation of XP-C
mutant CFE by fraction III (60 ng); D, complementation of
ERCC-1 and ERCC-4 mutant CFEs by Fraction IV (<1 ng). Comparable
complementation of XP-F mutant CFE by Fraction IV was observed (not
shown).
In fact, a different purification scheme yielded TFIIH associated with XPC protein(8) . In this current purification scheme, XPC was separated from TFIIH on the third column and designated reconstitution Fraction III (see below).
The ERCC1 and XPF proteins are in a
complex (14, 21, 22) and can be purified by
following ERCC1. The complex was purified as outlined in Fig. 1.
After chromatography on Sephacryl S-300, the fractions eluting at
approximately 200 kDa were found to contain ERCC1 by Western analysis.
Highly purified ERCC1XPF complex was obtained by passing the
sample through a glutathione S-transferase-XPA affinity
column. This procedure removed all of the HeLa cell derived
contaminants from the preparation (12) (Fig. 2D, lanes 1-3).
Protein bands were not visible by silver staining using 3% of our last
purification fraction (Fig. 2D, lane 3), but
immunoblotting (lanes 4-6) using anti-ERCC1 indicated
quantitative recovery of ERCC1 in this fraction (lane 6).
Furthermore, this fraction complemented both ERCC-1 and XP-F mutant
cell-free extracts (Fig. 3D). As shown in Fig. 2D (lane 6), this is our reconstitution
Fraction IV.
Fraction V is HSSB. This three-subunit protein was purified from E. coli cells co-expressing the three subunits(23) .
Figure 4: Reconstitution of human excinuclease. Lane 1, Fractions I-IV; lane 2, Fractions I-V; lane 3, HeLa CFE; lane 4, Fractions I, II, IV, V, and TFIIH/XPC (TFIIH/XPC was from Dr. D. Reinberg); lane 5, Fractions I, II, IV, and V. The excision assays were carried out as described under ``Experimental Procedures'' except that dNTPs were omitted. The total amount of proteins present in each reaction from lane1 to lane 5 is approximately 75 ng, 165 ng, 50 µg, 147 ng, and 145 ng, respectively.
Since the XPC protein (Fraction III) used in
reconstitution (Fig. 4, lanes 1 and 2) was an
early purification fraction, we tested another XPC preparation purified
as part of the TFIIH factor to a high level of purity by a different
scheme (8) to ascertain that only XPC in Fraction III was
needed for reconstitution. Fig. 4(lane 4) shows that
this substitution also led to reconstitution of the excinuclease
activity, whereas no signal was detected with the mixture lacking the
highly pure TFIIH/XPC fraction (lane 5). In addition, we have
obtained highly purified XPC free of any of the other XP proteins by
the method of Masutani et al. (25) and have
reconstituted the excinuclease activity with this protein (data not
shown). The results in Fig. 4thus lead us to propose that these
five fractions are necessary and sufficient for the excision step of
human nucleotide excision repair. Of special note, none of these five
fractions complemented an XP-E mutant cell extract (which has residual
excision activity), suggesting that the XPE protein is not necessary
for making the dual incisions. ()Thus, we hypothesize that
XPE and the proteins defined by the moderately UV-sensitive rodent
complementation groups 6-11 (see (26, 27, 28) ) are accessory factors, which
may stimulate excision repair but are not required for the basal
reaction.
Figure 5: The excised oligomer remains in the post-incision complex. Excision reaction was carried out with the reconstituted system, and the reaction mixture was loaded onto 12% polyacrylamide native gel following the indicated additions/treatments. The positions of the markers (24- and 27-mers), the substrate (140-mer), and the excision product (28-mer) are indicated by arrows. Heating was achieved at 95 °C for 2 min.
In conclusion, we have reconstituted the human excinuclease in a highly defined system. We believe that the proteins known to exist in our system (XPA, TFIIH (XPB and XPD), XPC, XPF, XPG, ERCC1, and HSSB) constitute a minimal set necessary for dual incision. The possibility that some of the other proteins that exist in our system as ``contaminants'' participate in excision cannot be eliminated until the entire system is reconstituted with recombinant proteins. However, the availability of this highly defined system now makes it possible to investigate the roles of individual XP proteins in excision and to dissect the individual steps of human excision repair. Indeed, this system has enabled us to determine that proteins in addition to those known to exist in our system are required to dissociate the post-incision complex and release the excised oligomer.