(Received for publication, March 22, 1995; and in revised form, July 6, 1995)
From the
Human DNA repair excision nuclease removes DNA damage by
incising on both sides of the lesion in a precise manner. The activity
requires participation of 16-17 polypeptides. Of these, the
XPFERCC1 complex and XPG were predicted to carry the nuclease
active sites based on studies with the recombinant proteins and the
yeast homologs of these proteins. Furthermore, recent work with model
(undamaged) substrates have led to predictions of the roles of these
proteins in incising 5` or 3` to the lesion. We have used damaged DNA
substrates and antibodies to XPG and ERCC1 to test these predictions.
Our results reveal that anti-XPG antibodies change the site of 3`
incision and at high concentration inhibit the 3` incision without
significantly affecting the 5` incision, indicating that XPG makes the
3` incision and further that under this condition 5` incision can occur
without 3` incision. In contrast, anti-ERCC1 antibodies inhibit both
the 3` and 5` incisions. Using a defined system for excision repair we
also demonstrate that the 3` incision can occur without the 5`
incision, leading us to conclude that under certain conditions the two
incisions can occur independently.
In humans and yeast bulky DNA lesions are removed from DNA by an
ATP-dependent multisubunit enzyme system which excises
27-29-nt()-long oligomers by incising the 3-5th
phosphodiester bonds 3` and the 22-24th phosphodiester bonds 5`
to the lesion (Huang et al., 1992; Reardon et al.,
1993). Of the 16-17 polypeptides required for excision, studies
with the yeast homologs of human proteins (Prakash et al.,
1993; Friedberg et al., 1995) have proposed two strong
candidates for making the dual incisions. RAD2 which is the homolog of
XPG is a single-strand specific endonuclease (Habraken et al.,
1993). The RAD1
RAD10 complex which is the homolog of
XPF
ERCC1 is an endonuclease with preference for single-stranded
and supercoiled DNA (Tomkinson et al., 1993; Sung et
al., 1993). Furthermore, human XPG protein has been also shown to
have single-strand specific DNA endonuclease activity using recombinant
protein from baculovirus system (O'Donovan et al.,
1994a; Habraken et al., 1994a).
Recently, model DNA
structures have been used with these proteins in an effort to identify
which subunit in the excision nuclease complex makes which incision.
Using partial duplexes with single-stranded tails or a flap structure
as substrates and a truncated RAD2 protein, Harrington and Lieber(1994)
concluded that RAD2 makes the 3` incision and suggested that
RAD1RAD10 complex makes the 5` nick. In apparent agreement with
this it was found that purified recombinant XPG protein cleaved the
junction of a duplex with single-stranded arms only in the strand with
the 5` single-stranded tail (O'Donovan et al., 1994b)
and the yeast RAD1
RAD10 complex cleaved only the strand with the
3` single-stranded tail (Bardwell et al., 1994).
However,
two recent studies have raised some questions regarding the roles of
these RAD and XP proteins in the dual incisions of the excinuclease.
First, it was found that the RAD1 protein alone was capable of incising
at the Holliday junction (Habraken et al., 1994b), revealing
that RAD1 contained the nuclease active site of the RAD1RAD10
complex. Second, it was also found that RAD2 and XPG possessed
5`
3` exonuclease activity (Habraken et al., 1994c),
leading to the suggestion that scission on the 3` side was made by
RAD1
RAD10 (XPF
ERCC1) followed by 5` incision by RAD2 (XPG).
Furthermore, it was proposed that RAD2 (XPG) digests the excised
oligomer 5` to 3` to create a single-stranded gap necessary for the
helicase functions of RAD3 (XPD) and RAD25 (XPB).
We wished to
address the question using the excision assay of high specificity and
sensitivity as well as incision assays and damaged DNA as substrate.
Carrying out experiments with cell-free extracts (CFEs) and a defined
system, and employing anti-XPG and anti-ERCC1 antibodies, lead us to
conclude that in humans XPG makes the 3` and XPFERCC1 makes the
5` incision. Furthermore, we suggest that in human excinuclease the two
incisions can occur independently under certain conditions.
Amylose resin, T4 polynucleotide kinase, and T4 DNA ligase were from New England Biolabs. Klenow fragment of Escherichia coli DNA polymerase I and T4 DNA polymerase were purchased from Boehringer Mannheim and prestained protein markers were from Life Technologies, Inc.
Figure 1:
Substrates for the excision and
incision assays. A, internally labeled substrate was assembled
from 6 oligomers after 5` labeling of a cholesterol-containing oligomer
(12-mer). For terminally (5` or 3`) labeled substrates, two 140-nt-long
single-stranded oligomers were used and the 5` end of the 140-mer
damaged strand contains T in place of G. B, internally
labeled, 3`-labeled, and 5`-labeled substrates were used for excision
assay, 3` and 5` incision assay, respectively. The predicted products
after repair reaction with each substrate are shown schematically. The X and asterisks indicate the positions of cholesterol
and the P label, respectively.
Depending on the type of experiment performed we
used internally labeled, 5`-labeled or 3`-labeled substrates (Fig. 1B). Internally labeled substrate was assembled
from 6 oligonucleotides as shown in Fig. 1A (Huang et al., 1994). The P label was at the 6th
phosphodiester bond 5` to the lesion. The 5`-labeled substrate was
prepared by labeling the 140-nt-long single-stranded oligomer with the
lesion using [
-
P]ATP and T4 polynucleotide
kinase, followed by annealing to the complementary oligonucleotide. For
3` end-labeling the two 140-nt oligomers were annealed and incubated
with [
-
P]dCTP, dATP, and Klenow fragment of E. coli DNA polymerase I. Filling-in the one nucleotide gap
generates a duplex of 141 bp with blunt ends. The unmodified and
cholesterol-containing oligomers were synthesized by Operon
Biotechnologies and Midland Certified Reagents.
Figure 2:
Specificity of anti-XPG and anti-ERCC1
antibodies. CFEs (100 µg each) from HeLa (lane 1) and
various XP cell lines (lanes 2-9) as described under
``Experimental Procedures'' were separated by 8% (A)
or 10% (B) SDS-polyacrylamide gel electrophoresis and analyzed
by immunoblotting with -XPG or
-ERCC1, respectively. In
experiment A, XP-G CFE prepared from GM03021A (lane
9) in addition to AG08802 (lane 8) was used. The
positions of prestained protein markers in kDa are
indicated.
Figure 3:
Effects of anti-XPG and anti-ERCC1
antibodies on dual incisions. A, the indicated amounts of
antibodies (Ab), preimmune, -XPG, or
-ERCC1 were
preincubated (for 10 min on ice) with HeLa CFE (50 µg) in the
absence (lanes 1-5, 8, and 9) or presence of
antigen (Ag), MBP-XPG (lanes 6 and 7), or
MBP-ERCC1 (lanes 10 and 11), and then added to the
reaction mixtures containing the internally labeled substrate (140-bp).
The excision products were resolved on a 10% polyacrylamide sequencing
gel and the positions of the substrate (140-mer) and the products (27-
and 29-mers) are indicated. B, the excision products were
resolved on a 10% polyacrylamide sequencing gel for longer periods to
increase the resolution of larger fragments. Two µl of
-XPG
were used (lane 3) and the positions of DNA size markers
(
X174/HinfI) are indicated in the left margin. Right
panel, schematic drawings of the incision pattern of human
excinuclease on the internally labeled 140-bp substrate. In the absence
of antibodies, 7.1% of the damage was excised as determined by the
intensity of the bands.
Figure 4:
Perturbation of the 3` incision site by
anti-XPG antibodies. After excision reaction with HeLa CFE in the
presence of -XPG, the products were separated on a 10%
polyacrylamide sequencing gel, and the 27-mer major excision product
and the 29-mer induced by
-XPG were cut out from the gel. The
eluted products were resolved on a 10% polyacrylamide sequencing gel
untreated (lanes 4 and 6) or after treatment with T4
DNA polymerase 3`
5` exonuclease (lanes 5 and 7). For comparison, the products from excision reaction
without (lane 2) or with (lane 3)
-XPG (1
µl) were also examined. The positions of the markers (30-mer) and
the products (26-, 27-, and 29-mers) are indicated. The excised
fragments containing a cholesterol molecule migrate 1 nt slower than
unmodified DNA of the same length (see Fig. 6).
Figure 6:
Effects of anti-XPG and anti-ERCC1
antibodies on the 3` incision. HeLa CFE (50 µg) was preincubated
(for 10 min on ice) with the indicated amounts of antibodies (Ab), preimmune (lanes 2 and 3), -XPG (lanes 5 and 6), or
-ERCC1 (lanes 7 and 8), and then added to the reaction mixture containing 3`
end-labeled substrate (30 °C, 60 min). The products were resolved
by electrophoresis on a 10% polyacrylamide sequencing gel. Lane 4 shows the Maxam-Gilbert reaction of G+A. The closed
arrows and triangles indicate the major 3` incision sites
and the open arrows and triangles indicate the
uncoupled 5` incision site. The position of the substrate (141-mer) is
also indicated.
We reasoned that looking at the
particular incision sites under -XPG inhibitory conditions would
help answer these questions. Indeed, higher resolution gels reveal that
-XPG, in addition to the two effects observed in Fig. 3A, also caused the appearance of a 95 nt-long
fragment with this internally labeled substrate (Fig. 3B). This fragment corresponds to incision at the
25th phosphodiester bond 5` to the lesion and hence represents an
``uncoupled'' 5` incision (
)(see below). Our
polyclonal antibodies must have at least two types of antibodies. One
binds to XPG and changes its specificity; the other inhibits its
function and causes the excinuclease to make only the 5` incision.
Taken together these data are consistent with XPG making the 3`
incision.
Figure 5: Incision assay with 3`-labeled substrate (3` incision assay). Fifty µg of HeLa CFE (lane 2) or mutant CFEs (lanes 3, 4 and 6-8) were incubated with 3` end-labeled substrate at 30 °C for 60 min. In complementation reactions (lanes 5 and 9-11), 25 µg from each cell line was premixed and then added to the reaction mixture. The products were resolved by electrophoresis on a 10% polyacrylamide sequencing gel. Lane 1 shows the Maxam-Gilbert reaction of G+A. The arrow and triangles indicate the major 3` incision site. The position of the substrate (141-mer) is also indicated. As determined by the intensity of the bands relative to the total amount of the DNA, 9.3% of the total radioactivity was detected in the 67-70-nt region with HeLa CFE (lane 2), in contrast to 0.2-0.8% (background) with mutant CFEs (lanes 3, 4, and 6-8). In complementation reactions (lanes 5, 10, and 11), 4.8-5.6% of that was detected in this region (i.e. approximately 50-60% complementation). CHO, Chinese hamster ovary.
Having thus
shown that the 3` incision can be detected in this assay with
3`-labeled DNA, we wished to confirm the observations we made with the
internally labeled substrate using the 3` incision assay. Fig. 6shows the results of such an assay. The -XPG has two
specific effects in addition to overall inhibition of 3` incision (lanes 5 and 6, 71 and 88% inhibition, respectively):
first, they reverse the frequency of incisions at the 3rd and 5th
phosphodiester bonds 3` to the lesion (lane 5, ratio of
incisions is 1 to 1.7) compared to the control (lane 1, ratio
of 1 to 0.4); second, they give rise to a band at position 96
(corresponding to position 95 in the 140-bp substrates) which can only
be generated from a 5` incision uncoupled from 3` incision (Fig. 1B). These results are consistent with the data
obtained with the internally labeled substrate. In contrast,
-ERCC1 inhibits 3` incision entirely but does not produce the
uncoupled 5` incision, indicating that
-ERCC1 possibly inhibits 5`
incision as well. Further experiments were conducted with 5`-labeled
substrate to ascertain our conclusions regarding the roles of XPG and
XPF
ERCC1 on 5` incision.
Figure 7: Incision assay with 5`-labeled substrate (5` incision assay). Fifty µg of HeLa CFE (lane 3) or mutant CFEs (lanes 4, 5, and 7-9) were incubated with 5` end-labeled substrate at 23 °C (to reduce nonspecific degradation) for 60 min. In complementation reactions (lanes 6 and 10-12), 25 µg from each cell line was premixed and then added to the reaction mixture. The products were resolved by electrophoresis on a 10% polyacrylamide sequencing gel. The products of PvuII restriction digestion (lane 1) and the Maxam-Gilbert reaction of G+A (lane 2) were used for determining the incision sites. The closed arrow and triangles indicate the major 5` incision site in human CFE and the open arrow and triangles indicate that in Chinese hamster ovary (CHO) CFE. The position of the substrate (140-mer) is also indicated. As determined by the intensity of the bands relative to the total amount of the DNA, 9.8% of the total radioactivity was detected in the 44-49-nt region with HeLa CFE (lane 3), in contrast to 1.6-2.2% (background) with mutant CFEs (lanes 4, 5, and 7-9). In complementation reactions (lanes 6, 11, and 12), 3.7-5.1% of that was detected in this region (i.e. approximately 37-52% complementation).
Having established a 5` incision assay, we then
investigated the effects of antibodies on the 5` incision. Fig. 8 shows that -XPG inhibits 5` incision only moderately (lanes 5 and 6, 6 and 53% inhibition, respectively)
compared to the control preimmune serum (lanes 3 and 4, 4 and 21%, respectively), whereas
-ERCC1 inhibits the
5` incision as efficiently as the 3` incision (lanes 7 and 8, 69 and 80%, respectively). Hence, we conclude that a subset
of
-XPG inhibits the 3` incision but not the 5` incision, while
-ERCC1 inhibits both the 3` and 5` incisions. Our data up to this
point strongly suggest that XPG makes the 3` incision and XPF (in the
form of XPF
ERCC1 complex) makes the 5` incision and further that
the 5` incision can occur without the 3` incision. The inhibition of
both 5` and 3` incision by
-ERCC1 could be by two mechanisms.
First, the 5` incision is made initially and inhibition of this
incision indirectly prevents 3` incision; second, the XPF
ERCC1
complex is essential for the assembly of the excinuclease complex and
prevention of binding of XPF
ERCC1 to the other subunits of the
excinuclease results in complete inhibition of any nicking activity.
Figure 8:
Effects of anti-XPG and anti-ERCC1
antibodies on the 5` incision. HeLa CFE (50 µg) was preincubated
(for 10 min on ice) with the indicated amounts of antibodies (Ab), preimmune (lanes 3 and 4), -XPG (lanes 5 and 6), or
-ERCC1 (lanes 7 and 8), and then added to the reaction mixture containing 5`
end-labeled substrate (23 °C, 60 min). The products were resolved
by electrophoresis on a 10% polyacrylamide sequencing gel. Lane 1 shows the Maxam-Gilbert reaction of G+A. The arrows indicate the 5` incision sites.
Figure 9:
Effects of anti-XPG antibodies on the
reconstituted excision repair system. A: lane 1,
Fractions I-IV; lane 2, Fractions I-V; lane 3,
Fraction V (replication protein A, RPA) alone. The excision reaction
with the internally labeled substrate (140-bp) was carried out as
described under ``Experimental Procedures'' except that dNTPs
were omitted. The products were resolved by electrophoresis on a 10%
polyacrylamide sequencing gel. The positions of DNA size markers
(X174/HinfI and
X174/HaeIII) are
indicated on the left margin. The closed and open arrows show the major excision product (27-mer) and the novel band
(72-mer) observed in lane 2, respectively. B,
-XPG (1 µl) was added to the mixture of Fractions I-V and
preincubated (for 10 min on ice). The analysis of excision products was
carried out as described in A. The positions of DNA size
markers (
X174/HinfI) and the excision products (27- and
29-mers) are indicated. Right panel, schematic drawings of the
incision pattern of human excinuclease on the internally labeled 140-bp
substrate.
Nucleotide excision repair, in general, is similar in E.
coli and humans: a multisubunit-ATP dependent enzyme system
removes damage by a dual incision mechanism. However, there are no
sequence homologies between the proteins involved in the two systems.
Furthermore, whereas at least 16 polypeptides are required for dual
incisions in humans (Mu et al., 1995), in E. coli 3
subunits are necessary and sufficient for dual incisions (Grossman and
Thiagalingam, 1993; Sancar, 1994). In contrast to the differences
between E. coli and humans, it appears that the excision
repair systems in Saccharomyces cerevisiae and humans are very
similar in all aspects including number and sequences of proteins
involved (Prakash et al., 1993; Friedberg et al.,
1995). Hence the discovery that yeast RAD2 protein (XPG homolog) had
single-strand specific endonuclease activity (Habraken et al.,
1993) and also acted as a 5`3` exonuclease (Harrington and
Lieber, 1994; Habraken et al., 1994c) is relevant to the human
system. Indeed, XPG has both activities (O'Donovan et
al., 1994a; Habraken et al., 1994a, 1994c). Similarly,
the XPF
ERCC1 complex which is the putative homolog of the
RAD1
RAD10 endonuclease is expected to be an endonuclease
(Tomkinson et al., 1993; Sung et al., 1993).
Since
in E. coli UvrB has the 3` incision activity and UvrC makes
the 5` incision (Lin and Sancar, 1992; Lin et al., 1992), the
discovery of endonuclease activities in RAD2 and RAD1 RAD10 led
to the expectation that each of these nucleases made one of the
incisions. Recent work with model substrates has led to specific
predictions. Thus it was found that a truncated RAD2 incised a DNA with
a single-stranded flap only when the flap had a 5` terminus, leading to
the prediction that RAD2 and therefore XPG must make the 3` incision
(Harrington and Lieber, 1994). A study with Y-shaped DNA and
recombinant XPG protein also found that only the strand with 5`
single-stranded terminus was nicked and furthermore, that a DNA with a
single-stranded bubble was nicked at the 3` end of the bubble in the
particular strand analyzed (O'Donovan et al., 1994b)
leading to the conclusion that XPG must make the 3` incision. On the
other hand, studies with RAD1
RAD10 complex and forked DNA showed
that the strand with 3` single-stranded terminus was nicked at the
junction with the double-stranded region (Bardwell et al.,
1994) and hence it was suggested that RAD1
RAD10 (and
XPF
ERCC1) must make the 5` incision. However, an alternative
model was advanced based on the finding that both XPG and RAD2 have
intrinsic 5`
3` exonuclease activity; it was, therefore, suggested
that this finding was consistent with XPG making the 5` incision and
then partially degrading the excised oligomer to enable helicases to
displace the excision product (Habraken et al., 1994c). Two
other observations are consistent with this model. First, the excision
gap is not enlarged from the 3` incision site (Huang et al.,
1992); second, the excised oligomer is degraded in 5`
3` direction
in CFE (Svoboda et al., 1993). Hence, even though studies with
model systems have been highly informative, they are not conclusive.
Experiments with damaged substrates are needed to assign the roles of
XPG and XPF
ERCC1 in the two incision reactions. In the present
study, we used the excision and incision assays with damaged DNA and
specific antibodies for the incision enzymes, and demonstrated that XPG
makes a major incision at the 3rd phosphodiester bond 3` to the
lesion and XPF
ERCC1 complex incises mainly at the 25th
phosphodiester bond 5` to the lesion.
Two reports in the
literature deal with substrates containing DNA lesions and CFEs from
mutant cell lines to analyze roles of the subunits on incision
reaction. In one, it was shown that CFEs from XP-C, -D, and -G incised
weakly at the 3rd phosphodiester bond 3` to a single thymine dimer
(Tateishi et al., 1993), leading one to suspect that perhaps
these subunits were not needed for 3` incision. The other report
presented data showing an incision by HeLa CFE at about 420 nt from the
3` end of a fragment which contained a GTG-cisplatin adduct at position
424. It was also stated that extract from an ERCC-1 mutant cell line
(43-3B) incised weakly at this position whereas CFE from an XP-G
cell line showed little or no damage-dependent incision at this
position (O'Donovan et al., 1994b), suggesting that XPG
but not ERCC1 was needed for 3` incision. These contradictory results
can be reconciled by assuming that the ``incisions'' detected
in these types of experiments may sometimes be the products of
nonspecific 3`5` or 5`
3` exonucleases which degrade DNA
starting from an end or a nonspecific nick in the substrate. Most such
nucleases are blocked by lesions either at the lesion site or 1 to 2
nucleotides prior to the lesion giving the appearance of a specific
nick. Indeed such an effect is seen in our Fig. 5and Fig. 7. This phenomenon is not unique to a particular cell line;
it shows considerable variability between extracts from the same cell
line. In this paper, we show that the incisions are specific to
excinuclease activity based on the complementation experiments and
specific inhibition by the relevant antibodies.
The second
mechanistically significant finding of our work deals with the order of
incision. In E. coli if UvrB (because of active site mutation)
is unable to make the 3` incision, UvrC fails to make the 5` incision.
On the other hand if active-site mutant UvrB is loaded onto damaged DNA
containing the 3` incision, addition of UvrC to the UvrB-preincised DNA
complex leads to quantitative 5` incision and hence excision. In
contrast, when active site mutant UvrC binds to UvrBDNA complex,
UvrB does make (uncoupled) 3` incision quantitatively. Thus, it was
concluded that in UvrB
UvrC
DNA complex production of 3`
incision by UvrB leads to a conformational change in the complex
enabling UvrC to make the 5` incision (Lin et al., 1992). The
inhibition of both 3` and 5` incisions by
-ERCC1 and the
detectability of uncoupled 5` incision in our experiments led us to
suspect that in human excinuclease also there was an order of incision
with 5` preceding the 3` incision. However, further experiments with
the defined system revealed the presence of uncoupled 3` incision (Fig. 9). Thus it is possible that in humans as well 3` incision
precedes 5` incision, but that in the presence of
-XPG the enzyme
system is induced to make 5` incision only. Whether uncoupled 5`
incision occurs at all in the absence of antibodies remains to be
determined.