ABSTRACT
Mammalian nucleotide excision repair is the primary enzymatic pathway for
removing bulky lesions from DNA. The repair reaction involves three main steps:
(i) dual incisions on both sides of the lesion; (ii) excision of the damaged
base in an oligonucleotide 24-31 nt in length; (iii) filling in of the post-excision gap and ligation. We have developed assays that probe the
individual steps of the reaction. Using these methods (assays for incision,
excision and repair patch synthesis), we demonstrate that the mammalian
excision nuclease system removes bulky lesions by incising mainly at the 22nd-25th phosphodiester bonds 5
'
and the 3rd-5th phosphodiester bonds 3
'
of the lesion, thus releasing oligonucleotides primarily 26-29 nt in length. The resulting excision gap is filled in by DNA
polymerases
[delta]
and
[epsilon]
as revealed by the `phosphorothioate repair patch assay'. When these assays
were employed with cell-free extracts from the moderately UV-sensitive rodent mutants in complementation groups 6-10, we found that these mutants are essentially normal in all
three steps of the repair reaction. This leads us to conclude that these cell
lines have normal
in vitro
repair activities and that the defects in these mutants are most likely in
genes controlling cellular functions not directly involved in general excision
repair.
Nucleotide excision repair (excision repair) is the primary mechanism for
removing DNA damage caused by UV light and by agents which make bulky base
adducts (
1
-
3
). Lack of excision repair causes xeroderma pigmentosum (
4
,
5
), which may result from mutations in seven genes (XPA, XPB, XPC, XPD, XPE, XPF
and XPG). In addition, mutations in two genes, CSA and CSB, abolish
transcription-coupled repair and cause Cockayne's syndrome (
6
,
7
). To supplement these naturally occurring human mutations, rodent cell lines
sensitive to UV and UV mimetic agents have been isolated and characterized.
These fall into 11 complementation groups, some of which are the same as those
groups defined by XP and CS mutations: group 2 (XPD), group 3 (XPB), groups 4
and 11 (XPF), group 5 (XPG), group 6 (CSB) and group 8 (CSA) (
8
). It has also been shown that the protein defective in group 1, ERCC1, is in a
complex with XPF (
9
,
10
), which constitutes the 5' nuclease (
11
) of the human dual incision-excision nuclease system (
12
).
The excision repair nuclease has recently been reconstituted with proteins
purified to near homogeneity (
13
). This study revealed that six repair factors consisting of XPA, RPA, TFIIH
(XPB, XPD, p62, p44, p34), XPC, XPF-ERCC1 and XPG are necessary and sufficient for the initial steps of
excision repair resulting in high efficiency damage removal. Similar results
have been obtained with the structurally and functionally homologous
Saccharomyces cerevisiae
proteins (
14
). These studies raised questions as to the direct participation in general
excision repair of the proteins defective in rodent complementation groups 7, 9
and 10. The present study was undertaken to directly address these questions,
as well as to determine the possible effects of the CSA and CSB proteins
(groups 8 and 6) on basal excision repair.
Since excision repair encompasses dual incisions, excision of 24-31 nt oligomers and repair synthesis to fill in the excision gap, cell
lines representative of groups 6-10 were tested for each of these functions. Cell-free extracts were tested for 5' incision (both the location and level of activity), for the
level of excision as well as the size(s) of the excision products and for the
precise boundaries of the repair synthesis patch using the high resolution
phosphorothioate repair patch assay (
15
). Our results demonstrate excellent correlation between the size of the
excision product and the repair patch and show that representatives of
complementation groups 6-10 incise at the normal sites, excise damage at a normal rate and
generate a resynthesis patch indistinguishable from the wild-type repair patch. We conclude that these mutants are not defective in the
basal steps of general excision repair.
The oligodeoxyribonucleotides used for substrate preparation were from either
Operon Technologies (Alameda, CA) or Midland Certified Reagent Co. (Midland,
TX). A 20mer containing a cyclobutane thymine dimer (T<>T) was a kind gift from J.-S.Taylor (Washington University, St Louis, MO). T4 polynucleotide kinase
and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA) and [[gamma]-
32
P]ATP was from ICN (Irvine, CA). Nucleotide triphosphates (dNTP) were from
Pharmacia (Piscataway, NJ) and nucleotide thiotriphosphates (dNTP[alpha]S) were from Amersham Life Science (Arlington Heights, IL).
Repair-proficient HeLa and Chinese hamster ovary (CHO) cell lines AA8 and K1 and
the UV-sensitive mutant derivatives listed below were used in this study: UV41
(group 4), UV135 (group 5), UV61 (group 6), V-B11 (group 7), US31 (group 8), CHO7PV (group 9), CHO4PV (group 10) and
UVS1 (group 11). Cells grown either as a suspension or adherent culture in
Eagle's MEM supplemented with 10% fetal bovine serum were harvested and cell-free extracts (CFE) prepared as described (
16
) and stored at -80oC in 25 mM HEPES, pH 7.9, 100 mM KCl, 12 mM MgCl
2
, 0.5 mM EDTA, 2 mM dithiothreitol and 12.5% glycerol (v/v) buffer. The CFE
concentrations were 10-25 mg/ml and the extracts were stable for at least two cycles of thawing
and refreezing.
Substrates containing either a centrally located cholesterol residue (in place
of T at position 70) or a T<>T adduct at nt 74-75 were prepared as described (
17
) by phosphorylating with T4 polynucleotide kinase, annealing and ligation of
six overlapping oligonucleotides to generate a 140 bp duplex. Four types of
substrates were used: 5'-end-labeled DNA, either with no damage or with the cholesterol
lesion, was used for the incision and resynthesis (repair patch) assays and
internally labeled DNA, with either the cholesterol residue or the T<>T photoproduct, was used for the excision assay.
This assay measures cleavage 5' of the lesion using 5'-end-labeled DNA as the substrate.
The reaction mixtures contained 3-15 fmol radiolabeled DNA with a cholesterol residue, 50 [mu]g CFE and 12.6 fmol pBR322 in 25 [mu]l reaction buffer (35 mM HEPES, pH 7.9, 10 mM Tris pH 7.5, 60 mM
KCl, 40 mM NaCl, 5.6 mM MgCl
2
, 0.4 mM EDTA, 0.8 mM dithiothreitol, 2 mM ATP and 3.2% glycerol with bovine
serum albumin at 0.2 mg/ml). The reaction was carried out at 25oC for 60 min or at 30oC for 45 min. DNA was deproteinized with proteinase K (0.2 mg/ml for
15 min at 37oC), followed by phenol, phenol/chloroform and ether extractions and then
precipitated with ethanol in the presence of 20 [mu]g oyster glycogen. Recovered DNA was resuspended in formamide/dye mixture
and resolved on 12% denaturing polyacrylamide gels. Following autoradiography
to visualize the repair products, the level of 5' incision (oligomers in the 42-53 nt range) was quantified by scanning the dried gel with an
AMBIS systems scanner.
Using internally labeled DNA substrate (at the 6th phosphodiester bond 5' of the cholesterol residue or at the 13th bond 5' of T<>T), this assay detects the excised fragment resulting from both
5' and 3' incisions. The 25 [mu]l repair reaction was supplemented with 20 [mu]M each of dATP, dCTP, dGTP and TTP. The reaction was
initiated by addition of substrate DNA (2-6 fmol cholesterol-containing 140mer or 16 fmol T<>T substrate) and 50 [mu]g CFE and was incubated at 30oC for 45-60 min. Following resolution on 10% sequencing
gels and autoradiography to visualize the repair products, the level of
excision (oligomers in the 24-31 nt range) was quantified by scanning dried gels with an AMBIS systems
scanner.
This assay measures the repair patch at single nucleotide resolution. Terminally
labeled DNA (10-20 fmol) was incubated with 40-70 [mu]g CFE in 25 [mu]l reaction buffer containing 80 [mu]M each of three dNTPs and 80 [mu]M of one dNTP[alpha]S and the reaction was at 30oC for 90 min. To obtain the precise
patch sequence with wild-type CFE, four separate reactions were carried out with a different dNTP[alpha]S in each reaction. Only the dGTP[alpha]S- and TTP[alpha]S-containing reactions were performed for
analysis of repair patches made by mutant CFEs. Following the repair reaction,
deproteinized DNA was resolved on 8% denaturing polyacrylamide gels and full-length (140 nt) DNA was located by autoradiography and recovered by
electroelution. Following ethanol precipitation of the eluted DNA, samples were
resuspended in 9 [mu]l 20 mM HEPES, pH 7.5, heated for 3 min at 70oC and cooled to 25oC. Iodine was added to 0.5 mM and the mixture was incubated at 25oC for 1 min (
18
). The reaction was quenched by addition of sodium acetate, pH 5.2, to 0.25 M
and DNA was precipitated with ethanol, resuspended in formamide/dye mixture and
resolved on 12% sequencing gels without heating prior to loading onto the gel.
The repair patch was visualized by autoradiography.
We wished to examine the repair activity of mutant cell lines in the moderately
UV-sensitive complementation groups 6-11, whose representative cell lines are less sensitive than the
original mutants belonging to complementation groups 1-5 (
8
,
19
,
20
). To this end we employed assays that probe the three steps of basal excision
repair: incision, excision and repair synthesis and ligation. Although the
incision and excision assays have been used in earlier studies of excision
repair with CFEs (
9
,
17
), the high resolution repair patch assay has not previously been used in a
mammalian
in vitro
system.
Each of the repair assays was performed with normal, repair-proficient CFEs (HeLa, CHO K1 or CHO AA8) prior to use with mutant CFEs.
All three cell lines gave similar results and the data obtained with the CHO K1
extract are shown in Figure
1
. The incision assay, which utilizes a 5'-end-labeled substrate, detects incision sites 5' of the lesion and also 3' incision sites when this incision is not
accompanied by the 5' incision (3' uncoupled incision). The CHO K1 CFE primarily incises at the 22nd-25th bonds 5' of the lesion (Fig.
1
); this agrees with an earlier study using this substrate and HeLa CFE which
generates a strong incision site the 25th phosphodiester bond 5' of the cholesterol residue and weaker incisions at the 22nd-24th phosphodiester bonds (
17
). Also consistent with earlier work, the range of 5' incision sites covers about a full turn of the helix (
13
,
17
). At a low frequency, some 3' uncoupled incision may be observed at the 3rd phosphodiester bond 3' of the cholesterol residue; however, since a band at this
position is also observed with mutant extracts known to be defective in
incision (Fig.
2
, lane 4, and data not shown), this band cannot definitively be ascribed to
uncoupled 3' incision. This 73 nt fragment, which is particularly strong in rodent
cell lines compared with human lines, is most likely caused by both 3' uncoupled incisions and a strong, non-specific 3' -> 5' exonuclease which is blocked by the
cholesterol residue (
17
). When 5'-end-labeled, unmodified DNA was used for the incision assay (data
not shown), distinct incisions were not observed; instead we detected a DNA
ladder, indicating low levels of nicking at random positions.
The 5' incision site was localized using CFE and 5'-end-labeled cholesterol substrate. With this system and
HeLa extract, bands corresponding to incisions at the 18th-29th phosphodiester bonds 5' of the lesion are detected, with the major band being at the 25th
phosphodiester bond (
17
). When the incision assay was conducted with CFEs from complementation groups 6-10, the mutant extracts gave incision patterns similar to those observed
with wild-type CHO cell lines (Fig.
2
), whereas there were no bands above background in CFEs from groups 1-5 (lane 4, and data not shown). With this assay the background caused by
non-specific nucleases is relatively high, particularly with the group 8 CFE
(lane 7), which results in an apparent shift of the specific incision bands to
smaller fragments with the distal incision at nt 35 compared with nt 42 for
other rodent extracts. Quantitative analysis of Figure
2
shows that 1.6% of the radioactivity is in the 42-53 nt region for the excision-defective group 5 (XPG) CFE and the values for the wild-types (lanes 2 and 3) are 5-6% compared with 5-8% for groups 6-10 (lanes 5-9). Thus, within the limitations
of this assay, we conclude that complementation groups 6-10, with the possible exception of group 8, have normal 5' incision activity.
CFEs from rodent complementation groups 5-11 were tested for excision activity using internally labeled substrates
containing either a cholesterol residue or a T<>T photoproduct. Figure
3
A shows that, with the exception of CFEs from groups 5 and 11, all tested
extracts have similar excision activity with the cholesterol substrate.
Complementation group 5 is known to be mutated in XPG (
22
) and group 11 has recently been found to be the equivalent of group 4, which is
mutated in the XPF gene (
10
,
23
-
25
). Thus, it is not surprising that no or greatly reduced excision activity is
observed with these extracts. Quantitative analyses of several excision assays,
such as that shown in Figure
3
A, are summarized in Figure
3
B. With this substrate CFEs from groups 9 and 10 exhibited reduced levels of
excision compared with groups 6-8, but clearly were in the range of normal excision activity as defined
by the levels of excision observed with the wild-type cell lines K1 and AA8. We also conducted kinetic assays in which the
extract concentrations ranged from 0.5 to 2.5 mg/ml or the times of incubation
varied from 15 to 120 min (data not shown), but no clear differences were
observed.
The data presented thus far are consistent with normal incision and excision
activities for the representative cell lines of groups 6-10. We wished to know if these cell lines have a detectable defect in the
repair synthesis and ligation steps of excision repair. For this purpose we
conducted phosphorothioate repair patch assays (
12
,
15
) with the mutant CFEs using only two nucleotides in the dNTP[alpha]S form. The results are shown in Figure
5
. A normal sequence pattern is observed for the wild-type CHO K1 (lanes 1 and 2) but no patch is seen with XPF/group 4 (lanes 3
and 4) or XPG/group 5 (lanes 5 and 6) extracts. The patches for groups 6-10 (lanes 7-16) are qualitatively similar to those observed for the wild-type, although group 8 exhibits a patch enlarged by ~4 nt at the 5' boundary (lane 12), consistent with the
observed shift in 5' incision sites observed with this extract (see Fig.
2
, lane 7). Thus, we conclude that complementation groups 6-10, with the possible exception of group 8, have normal repair synthesis
and ligation activities.
Our data show that rodent UV-sensitive mutant cell lines in complementation groups 6-10 are normal in the incision, excision and resynthesis and
ligation steps of nucleotide excision repair when assayed
in vitro
. These findings support the recent reconstitution experiments with purified
proteins which concluded that XPA, RPA, XPC, TFIIH, XPF/ERCC1 and XPG were
necessary and sufficient for a high efficiency excision reaction (
13
,
26
,
27
). This leads us to consider other explanations for the UV-sensitive phenotypes of complementation groups 6-10.
The prototypic representative of complementation group 6 is UV61 (
28
,
29
), which was reported to remove (6-4) photoproducts but not cyclobutane dimers (
30
). Cloning of the gene (
ERCC6
,
CSB
) and related analyses demonstrated that this cell line is deficient in
transcription-repair coupling (
31
). Consistent with our previous data showing normal excision of T<>T from a semisynthetic covalently closed plasmid substrate (
9
), we here demonstrate that UV61 extracts have normal incision and excision
activities on another bulky lesion as well as on a T<>T photoproduct contained within a linear duplex molecule. Thus, our data
support earlier conclusions that the apparent lack of excision of Pyr<>Pyr
in vivo
is due to a defect in strand-specific repair (
6
,
31
,
32
). Furthermore, the normal
in vitro
incision, excision and repair synthesis observed for UV61 extracts suggest that
the CSB protein does not have an accessory role in the basal repair reaction.
The V-B11 cell line (complementation group 7) was isolated as a moderately UV-sensitive cell line (
33
) with normal unscheduled DNA synthesis (UDS) but a 70% reduction in incision
activity. It was also reported that it removes (6-4) photoproducts at a normal rate from the genome overall, but excises T<>T from a transcriptionally active locus at 20% of the rate of the
parental line (
34
). Superficially, these phenoytpic characteristics are similar to those of
Cockayne's syndrome cells, but somatic hybrids of V-B11 with other rodent cell lines demonstrate that this cell line
complements both groups 6 and 8 (
35
,
36
). Although a subtle defect in transcription-repair coupling cannot be eliminated, our
in vitro
results of normal incision, excision and repair synthesis do not reveal any
defect in basal/general excision repair in this mutant. Clearly, more work is
needed to explain the UV sensitivity of this cell line.
The sole representative of complementation group 8 is the UV-sensitive mouse lymphoma cell line US31 (
36
,
37
). This cell line is also moderately UV sensitive and removes (6-4) photoproducts, but not Pyr<>Pyr, at a normal rate
in vivo
. Recently it was demonstrated that a derivative of this mutant cell line did
not complement a human CSA
-
cell line and that the cloned
CSA
gene restored UV survival to normal rates (
38
). CSA mutants, like CSB (group 6) mutants, are defective in transcription-coupled repair and our observations of normal excision and repair
synthesis activities with a linear substrate are consistent with this
phenotype. In our incision and repair patch assays we observed a 4-6 nt shift at the 5' incision site with this mutant and it is conceivable that this
may be a direct effect of the lack of CSA protein in the mutant CFE. However,
the CFE from this cell line contains a potent non-specific nuclease which degrades input DNA (see lanes 7 of Figs
2
,
3
A and
4
A). Hence, we consider this mutant to be normal in all aspects of basal excision
repair. This conclusion is consistent with the finding that in a reconstituted
excision nuclease system devoid of CSA the 5' incision site corresponds to the 5' incision site we detect with all other cell lines (
13
).
The sole members of complementation groups 9 and 10, CHO7PV and CHO4PV
respectively, were isolated from the CHO K1 cell line and characterized as
mutants with reduced UDS after UV irradiation (
39
). Detailed analyses revealed a 2- to 3-fold increased UV sensitivity for both cell lines with reduced
excision of both (6-4) and Pyr<>Pyr photoproducts. With our
in vitro
assays we observe excision of the cholesterol residue and the T<>T photoproduct within the normal range.
The high efficiency reconstitution of the human excision nuclease with six well-defined factors (
13
) makes it unlikely that other proteins contribute to the efficiency of excision
repair in our model system. We considered the possibility that groups 7, 9 and
10 might be leaky alleles of human repair proteins not previously correlated
with rodent mutations (e.g. XPA, XPC-HHR23B or TFIIH subunits). To test these possibilities, mutant CFEs were
supplemented with purified XPA, XPC-HHR23B or TFIIH (p89/XPD, p80/XPB, p62, p44 and p34) and tested for
excision of the T<>T photolesion; no effect on excision activity was observed (data not shown).
These results suggest, but do not definitively confirm, the possibility that
groups 7, 9 and 10 are not leaky mutants of known excision repair genes.
Since our
in vitro
assays, which do not reveal repair defects in these cell lines, were conducted
with naked DNA and not chromatin, it is quite possible that the proteins
defective in groups 7, 9 and 10 are responsible for the enhanced accessibility
of DNA (i.e. chromatin factors) to the other repair factors known to have an
essential role in excision repair. Cloning these genes and characterization of
the encoded proteins is likely to provide useful information on the repair of
lesions in chromatin.
We thank Dr M.Z.Zdzienicka (Leiden, The Netherlands), Dr M.Stefanini (Pavia,
Italy) and Dr A.Yasui (Sendai, Japan) for the V-B11, CHO7PV, CHO4PV and UVS1 cell lines, representative of groups 7, 9, 10
and 11(
4
). We also thank Dr C.P.Selby (Chapel Hill, NC) for the CFE prepared from US31,
Drs D.Mu and M.Wakasugi for critical comments on this manuscript and Dr
T.Matsunaga for inspiration. This work was supported by National Institutes of
Health grant GM32833 (A.S.).
REFERENCES
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