(Received for publication, July 25, 1995)
From the
A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1 complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with a ``bubble'' structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication protein A when the DNA contains UV damage.
Xeroderma pigmentosum (XP) ()is a human disease
characterized by a high incidence of actinic cancers and in some cases
neurological abnormalities; it is caused by a defect in excision repair
as a result of mutations in one of seven genes, XPA through XPG(1) . In recent years all of the XP genes required
for excision, with the exception of XPF, have been cloned and
sequenced(2, 3) . Previously it was found that
cell-free extracts (CFEs) from human XP-F and rodent ERCC-4 mutants did
not complement each other in vitro, raising the possibility
that these two complementation groups contained mutations in the human
and rodent homologs of the XPF gene(4, 5) .
In addition, it was found that the complementing activity of XP-F and
ERCC-4 mutant CFEs was tightly associated with the ERCC1
protein(6, 7, 8) . Here we report the
purification of the XPF/ERCC4 protein in complex with ERCC1 to
homogeneity, providing direct evidence that XPF and ERCC4 are identical genes. The tight complex of XPF/ERCC4 and ERCC1 is a
single-stranded DNA-specific endonuclease with weak endonucleolytic
activity on supercoiled DNA. The nicking of supercoiled DNA is
stimulated by human replication protein A (RPA) when the DNA contains
UV damage.
Figure 1: Purification of the XPF-ERCC1 complex. The purified complex was separated on a 10% SDS-polyacrylamide gel and analyzed by silver nitrate staining (lane 1, 240 ng) and immunoblotting with anti-ERCC1 antibodies (lane 2, 40 ng).
It was recently reported that partially
purified ERCC1 fractions complemented both XP-F and ERCC-4 CFEs as well
as ERCC-1 CFE(7, 8) , raising the possibility that the
112-kDa protein, which makes a complex with ERCC1, is XPF and/or ERCC4.
The purified complex was tested for complementing CFEs of mutant cell
lines for excision activity (Fig. 2). As expected, the complex
complemented rodent ERCC-1 mutant extract (lane 6). In
addition, the complex complemented XP-F and ERCC-4 CFEs as well (lanes 4 and 10), while no other mutant
extract (XP-A, ERCC-3 (XP-B), and ERCC-5 (XP-G)) was complemented (lanes 2, 8, and 12). Furthermore, the
complementation of both XP-F and ERCC-4 CFEs by the complex was
inhibited by anti-ERCC1 antibodies (data not shown). This inhibition
was overcome by the addition of the excess amount of the purified
complex, indicating that both complementations were due to the
ERCC1-containing complex but not to a minor contaminant in our
preparation. These results lead us to conclude that the 112-kDa protein
is XPF, which in turn is identical to ERCC4. Previously, it was found
that CFEs from human XP-F and rodent ERCC-4 mutants did not complement
each other in vitro for either excision (4) or repair
synthesis (5, 11) activity, and it was suggested that
the ERCC4 gene might be identical to the XPF gene or
that XPF-ERCC1 was in a complex with
ERCC4(4, 5, 11) . Here we show direct
biochemical evidence that XPF and ERCC4 are identical, ()in
spite of earlier evidence based on somatic cell hybrids suggesting that ERCC4 and XPF might be different genes(12) .
Figure 2: Specific complementation of XP-F, ERCC-1, and ERCC-4 mutants by the XPF-ERCC1 complex. CFEs from different repair complementation groups were incubated in the standard reaction mixture in the absence(-) or presence (+) of the purified XPF-ERCC1 complex (8 ng). Lanes 1 and 2, XP-A (GM02345B); lanes 3 and 4, XP-F (GM08437A); lanes 5 and 6, ERCC-1 (UV20); lanes 7 and 8, ERCC-3 (UV24); lanes 9 and 10, ERCC-4 (UV41); lanes 11 and 12, ERCC-5 (UV135). 28 indicates the position of the main excision product.
Figure 3:
The XPF-ERCC1 complex is an endonuclease. A, degradation of covalently closed single-stranded DNA by the
XPF-ERCC1 complex. Lane 1, DNA alone; lane 2, DNA
with 16 ng of the XPF-ERCC1 complex and 0.5% SDS; lanes
3-6, DNA with 4, 8, 12, and 16 ng of the XPF-ERCC1 complex,
respectively, and the average numbers of nicks per DNA molecule
introduced by the XPF-ERCC1 complex were 0.2, 1.2, 2.2 and 4.0,
respectively. kb, kilobase pairs. B, nicking of
covalently closed supercoiled double-stranded DNA by the XPF-ERCC1
complex. Lanes 1-3, non-irradiated DNA; lanes
4-6, UV-irradiated (1 kJ/m) DNA. Lanes 1 and 4, DNA alone; lanes 2 and 5, DNA
with 8 ng of the XPF-ERCC1 complex; lanes 3 and 6,
DNA with 16 ng of the XPF-ERCC1 complex. The average number of nicks
per DNA molecule introduced by 16 ng of the XPF-ERCC1 complex was 0.12. NC, nicked circular DNA; CC, covalently closed
supercoiled DNA. C, co-elution of nuclease activity with the
XPF-ERCC1 complex. Fractions 21-27 (1 ml each) from the last
purification step on heparin-agarose were concentrated to 350 µl
individually. Toppanel, 20 µl of the
concentrated fractions was analyzed for the XPF-ERCC1 complex by
immunoblotting with anti-ERCC1 antibodies; bottompanel, 4 µl of the same concentrated fractions were
assayed for nuclease activity with single-stranded M13 DNA. On the
average 5.0 nicks/DNA molecule were introduced by the XPF-ERCC1 complex
in fraction 24. C, control DNA with no addition of fraction. D, immunodepletion of nuclease activity by anti-ERCC1
antibodies. The XPA-affinity column-purified fraction was used for this
experiment. Lane 1, DNA alone; lanes 2-4, DNA
plus XPF-ERCC1 fractions treated with protein A alone (lane
2), protein A plus preimmune serum (lane 3), and protein
A plus immune serum (lane 4), respectively. The average
numbers of nicks/DNA molecule in lanes 2-4 as calculated
by the P(0) class of Poisson distribution were >6, 3.6, and 1.0,
respectively.
It has been reported that RAD1-RAD10 specifically cleaves the single strand to double strand junction of Y-shaped DNA only of the strand with the 3` single-stranded terminus, and hence the preferential nuclease activity on single-stranded M13 DNA by the RAD1-RAD10 complex was attributed to cleavage at the junction of stem-loop structures known to exist in M13 DNA(15) . To test for such an activity, we designed a 90-bp partial duplex DNA with a 30-nucleotide ``bubble'' near the center because of non-complementary sequences. As shown in Fig. 4A, the homogeneous fraction containing only the XPF and ERCC1 proteins (Fig. 1) did not show such a preference; rather a ladder in the region corresponding to the ``bubble'' was observed, even though the fractions from the penultimate purification step had a preference for junction (data not shown). The single-stranded DNA-specific S1 nuclease gave a similar pattern of incision, although the two enzymes differed with regard to the sequence effect on cleavage within the single-stranded region. Nevertheless, these results strongly support the idea that XPF-ERCC1 cleaves M13 DNA and bubble structures as a single-stranded DNA-specific endonuclease but not because of its specific attack on stem-loop structures in M13 DNA.
Figure 4:
Characterization of the endonuclease
activity of the XPF-ERCC1 complex. A, cleavage of a 90-bp
duplex DNA with a 30-nucleotide bubble by the XPF-ERCC1 complex. M, size markers. Lane 1, DNA alone; lane 2,
DNA plus the XPF-ERCC1 complex (8 ng); lane 3, DNA plus S1
nuclease (0.1 unit). B, stimulation of the nicking activity of
the XPF-ERCC1 complex on UV-irradiated double-stranded DNA by human
RPA. Lanes 1-4 contain non-irradiated DNA and lanes
5-12 contain UV-irradiated DNA (4 kJ/m). Lane 1, DNA alone; lane 2, DNA plus XPF-ERCC1; lane 3, DNA plus human RPA; lane 4, DNA plus
XPF-ERCC1 and human RPA; lane 5, UV DNA alone; lane
6, UV DNA plus XPF-ERCC1; lane 7, UV DNA plus human RPA; lane 8, UV DNA plus XPF-ERCC1 and human RPA; lane 9,
UV DNA plus yeast RPA; lane 10, UV DNA plus XPF-ERCC1 and
yeast RPA; lane 11, UV DNA plus E. coli SSB; lane
12, UV DNA plus XPF-ERCC1 and E. coli SSB. NC,
nicked circular DNA; CC, covalently closed supercoiled DNA.
All lanes contained 100 ng of DNA, whereas 165 ng of the
XPA-affinity-purified XPF-ERCC1 and 20 ng of the single-stranded DNA
binding proteins were used as indicated. The average numbers of
nicks/DNA molecule in four experiments including the one shown here
were 0.08 for UV-DNA plus XPF-ERCC1 (lane 6) and 0.15 for UV
DNA plus XPF-ERCC1 and human RPA (lane 8). The yeast RPA and E. coli SSB had no significant effect on the level of
nicking.