Cipl inhibits DNA replication but not
PCNA-dependent nucleotide excision-repair
Mahmud K.K. Shivji, Sara . Grey, Ulrich P.Strausfeld,
Richard D. Wood and J.Julian Blow
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Background: 1)NA that is damaged by ultraviolet (UV)
light is repaired predominantly by nucleotide excision-repair, a process requiring the DNA polymerase
auxiliary factor PCNA. UV-irradiation also induces the
production of Cipl protein via activation of p53. Cipl is
an inhibitor of the cyclin-dependent kinases, which are
required for the cell cycle to proceed through the Gl/Sphase transition and initiate DNA replication. Inhibition
by Cipl probably causes the block to initiation of DNA
replication that is seen in irradiated cells. Cipl also
directly inhibits the function of PCNA during DNA
synthesis. As nucleotide excision-repair requires PCNA,
the physiological relevance of PCNA inhibition by Cipl
is currently unclear.
Results: We show that nucleotide excision-repair of
UV-damaged DNA occurs in extracts of Xenopus eggs,
and that this reaction is PCNA-dependent. The repair
reaction is not inhibited by Cipl, even when the level of
PCNA is reduced 100-fold so that it becomes limiting for
DNA repair. By contrast, CipI strongly suppresses the
function of PCNA in replicative DNA synthesis under
these conditions.
Conclusions: Cipl can potentially inhibit DNA replication in Xenopus egg extracts by inhibiting the cyclindependent kinase function required for the initiation of
replication forks, and also by inhibiting PCNA function.
The inhibition of PCNA is selective for its function in
DNA replication, however, as Cipl does not affect
PCNA function in nucleotide excision-repair. The
induction of Cipl in response to DNA damage,
therefore, allows repair to continue in the genome under
conditions in which replication is severely inhibited.
Current Biology 1994, 4:1062-1068
Background
likely to be interplay between the repair and cell-cycle
checkpoint pathways at the gap-filling step.
Dividing cells usually respond to DNA damage in two
distinct ways. Firstly, they must try to repair the damaged
DNA with minimal loss of genetic information.
Secondly, cell-cycle checkpoint controls are activated to
block cell-cycle events, such as DNA replication or
mitosis, which may be difficult to perform with damaged
DNA. The recently identified Cipl protein seems to
play an important part in the coordination of these
different pathways.
In higher eukaryotes, most UV-induced DNA lesions are
removed from the DNA by nucleotide excision-repair
(reviewed in [1]). This is a versatile repair mechanism
capable of removing a broad spectrum of DNA lesions,
including many types of chemically induced adducts, as
well as those produced by UV-irradiation. In the
nucleotide excision-repair reaction, damaged DNA is
recognized and nicked at two sites, 27-32 base pairs (bp)
apart, on either side of the lesion. The oligonucleotide
containing the lesion is then removed, and the gap is
filled in using the undamaged strand as a template. The
gap-filling synthesis is performed by DNA polymerase 8
or , and also requires the activity of PCNA (proliferating
cell nuclear antigen) [2,3], an auxiliary factor for these
DNA polymerases [4-7]. DNA polymerases 8 and and
PCNA are all also required for chromosomal DNA replication (reviewed in [8-10]), which means that there is
Following UV-irradiation, higher eukaryotic cells
activate the tumour suppressor p53; this activation is
required for cells to delay DNA replication if their DNA
has been damaged by ionizing radiation [11,12]. The p53
protein has transcription-factor activity and induces the
expression of Cipl ( also known as Wafl) [13,14], a
21 kD protein which can bind to and inactivate cyclindependent kinases (Cdks) [15-18]. Cdks are required for
a number of key transitions during the eukaryotic cell
cycle, in particular for chromosome replication and progression from the GI phase of the cell cycle into S phase
(reviewed in [19-211). Cell-free extracts of Xenopus eggs
that support chromosome replication in vitro require Cdk
activity to initiate DNA replication [22-25]. We have
recently shown that addition of bacterially expressed
Cipl to Xenopus egg extracts specifically blocks the initiation of DNA replication by inhibiting the Cdks
required for this process [25]. These results suggest that
the p53-dependent G1 checkpoint responsive to DNA
damage is activated by the Cipl-mediated inhibition of
the Cdks required for entry into S phase.
An additional function of Cipl may be to inhibit PCNA,
the DNA polymerase auxiliary factor. Cipl inhibits both
replication of the SV40 viral DNA in vitro as well as
DNA synthesis catalyzed by DNA polymerase 8, and
Correspondence to: . Julian Blow.
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Cipl, DNA replication and DNA repair Shivji et al.
RESEARCH PAPER
inhibition of both processes could be relieved by addition
of excess PCNA [26,27]. These results suggest that Cipl
might be able to inhibit chromosome replication by
inhibiting PCNA and blocking replication-fork elongation, as well as by a direct effect on Cdks.
As nucleotide excision-repair also requires PCNA, the
physiological relevance of the inhibition of PCNA by
Cipl is currently unclear. It would be surprising if Cipl,
having been induced by DNA damage, then functions to
block nucleotide excision-repair. We have therefore
investigated the effect of Cipl on the function of PCNA
in Xenopus extracts that can support both DNA synthesis
and nucleotide excision-repair.
Results
Nucleotide excision-repair in Xenopus egg extracts
When low-speed supernatants of Xenopus egg extracts are
centrifuged at > 100 000 x g, nuclear envelope precursors
are pelleted, leaving the resultant high-speed supernatants
unable to assemble interphase nuclei [28]. As the ability
of Xenopus extracts to initiate DNA replication on
double-stranded DNA is dependent on the template
DNA being assembled into interphase nuclei, high-speed
supernatants are therefore also unable to initiate DNA
replication [29-31], but do retain the soluble proteins
required for DNA synthesis [32,33]. The ability of
Xenopus high-speed supernatant to support nucleotide
excision-repair of UV-irradiated DNA was therefore
compared with human cell extracts (Fig. la). Mixtures of
irradiated and non-irradiated DNA were incubated with
[ac-3 2 P]dATP in extracts as described previously [34].
Like human cell extracts and Xenopus oocyte extracts
[35,36], Xenopus egg extracts preferentially incorporated
label into UV-irradiated DNA (Figs la,b). Repair of UVdamaged DNA was insensitive to the addition of Cipl at
concentrations of up to 15 pxg ml-1 (Fig. c). These levels
of Cipl are ample to abolish the replication of chromosomal DNA in Xenopus low-speed supernatants by
inhibiting Cdks [25].
In order to confirm that the reaction with Xenopus
extract did indeed represent nucleotide excision-repair,
DNA synthesis was analyzed using a DNA molecule
with a single d(GpTpG) adduct of cisplatin at a defined
position (Fig. 2) [37]. Repair synthesis in this DNA
construct took place predominantly in a 33 bp fragment
flanking the lesion (Fig. 2, lane 1), consistent with the
size of the excision fragment produced by nucleotide
excision-repair in Xenopus oocytes [38]. As in the experiment shown in Figure 1, Cipl did not significantly
inhibit repair synthesis in this assay (lane 2, Fig. 2).
Inhibition of PCNA function in DNA synthesis by Cipl
Using an assay system based on SV40 DNA replication,
the p53-inducible Cdk inhibitor Cipl has been shown to
inhibit PCNA-dependent DNA synthesis directly
[26,27]. No effect on Cdks was observed in this system,
Fig. 1. Repair DNA synthesis by extracts of Xenopus eggs and
human cells. (a) Cell extracts from the normal lymphoblastoid
line GM1953 (lane 1)or high-speed supernatants of Xenopus egg
extracts (lanes 2-6) were assayed at the indicated concentrations
for nucleotide excision-repair activity with a mixture of UVirradiated (+UV) and unirradiated (-UV) DNA in a buffer
including [- 32P]dATP. DNA was isolated, linearized with
BamHl and separated by agarose gel electrophoresis. The upper
panel shows an ethidium stain of total DNA and the lower panel
an autoradiograph of the dried gel. (b) Quantification of the
nucleotide excision-repair activity in the Xenopus egg extracts
shown in (a), normalized for DNA recovery. (c) Effect of different
concentrations of histidine-tagged human Cipl on the nucleotide
excision-repair activity in 6 mg ml- 1 Xenopus egg extract.
because the SV40 replication reaction reconstituted with
purified proteins does not require Cdk function [39,40].
In contrast, when Cipl was added to Xenopus extracts, it
efficiently blocked the initiation of chromosomal DNA
replication by inhibiting the Cdks required for this
process [25]. This is likely to be because the quantity of
Cdks (2 .g Cdk2 per ml) in unfractionated Xenopus egg
extract is much lower than the amount of PCNA
(200 Ig ml-1) [25]. Thus, the inability of 15 ig ml-1
Cipl to inhibit PCNA-dependent nucleotide excisionrepair in unfractionated extracts (Figs 1,2) might be due
to the high concentration of PCNA present in the assay.
Xenopus egg extract was therefore fractionated, in order
to control the level of PCNA. When human cell extract
is applied to a DEAE column, the flow-through fraction
contains all the proteins required for nucleotide
excision-repair except for PCNA, which is found in the
1 M salt eluate [3]. We fractionated Xenopus extract
similarly; quantitative immunoblotting with anti-PCNA
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Current Biology 1994, Vol 4 No 12
as a supplement (data not shown), suggesting that PCNA
is the only component lacking from XD1 that is required
for this reaction. Cipl strongly inhibited replicative DNA
synthesis in the presence of 0.4 jig ml-1 PCNA
(lanes 3-6, Fig. 3a), blocking both total DNA synthesis
(Fig. 3b) and the appearance of closed circular doublestranded DNA (Fig. 3c). This inhibition was relieved by
the addition of further PCNA to 1.4 or 2.4 fig ml -1
(lanes 7-16, Fig. 3a; Figs 3b,c). This suggests that the
inhibition of complementary strand synthesis by Cipl in
PCNA-depleted Xenopus egg extracts was mediated by
inhibition of PCNA.
Insensitivity of DNA repair to Cipl
Fraction XD1 was then used for the nucleotide
excision-repair reaction. XD1 on its own supported only
low levels of repair of UV-irradiated DNA (Fig. 4a).
Fig. 2. Nucleotide excision-repair of a defined lesion by Xenopus
egg extracts and lack of inhibition by Cipl. A single 1,3-intrastrand d(GpTpG) platinum cross-link in a closed circular duplex
molecule was used as the substrate for repair. (a) Substrates with
(lanes 1 and 2) or without (lanes 3 and 4) an adduct were
incubated in the presence (lanes 2 and 4) or absence (lanes 1 and
3) of 15 JLg ml-1 histidine-tagged Cipl. An autoradiograph of a
gel after BstNI-cleavage of the DNA is shown. (b) The DNA
construct, with the position of the platinum cross-link and the
size of BstNI restriction fragments indicated.
antibody PC10 [411 showed that more than 99% of the
PCNA was removed from the flow-through fraction
(XD1), and was recovered in the 1 M salt-wash fraction
(XD2) (data not shown).
To test the fractionated system, we made use of the fact
that high-speed supernatants of Xenopus egg extracts
support efficient complementary strand synthesis on
single-stranded DNA (Fig. 3) [32,33]. The PCNAdepleted fraction, XD1, supported only very low levels
of DNA synthesis (lane 1, Fig. 3a; asterisks in Figs. 3b,c).
When supplemented with 0.4 fig ml-1 human PCNA,
however, DNA synthesis in the XD1 fraction was
strongly stimulated (Fig. 3a, lane 2). As well as increasing
the total amount of DNA synthesis (Fig. 3b), addition of
PCNA also led to the production of fully replicated
closed circular DNA molecules (Fig. 3c). Maximum
yields of closed circular double-stranded DNA were
achieved with PCNA concentrations of 5-20 j.g ml - 1
(Fig. 3 and data not shown). Similar results were obtained
using the 1M salt-wash fraction, XD2, instead of PCNA
Fig. 3. Inhibition by Cipl of PCNA-dependent complementary
strand synthesis in fractionated Xenopus extracts. Xenopus highspeed supernatant was fractionated on a DEAE column and
the flow-through fraction (XD1) assayed for complementary
strand synthesis on M13 single-stranded DNA. XD1 (1 mg ml-1 )
was incubated with 4 ng Il-1' M13 single-stranded DNA,
[ot- 32 P]dATP and various concentrations of human PCNA and
human histidine-tagged Cipl. Reaction products were separated
by agarose gel electrophoresis. (a) Autoradiograph of 32p incorporation. Lane 1: no added PCNA; lanes 2-6: 0.4 jig ml-1 PCNA;
lanes 7-11: 1.4 jig ml- PCNA; lanes 12-16: 2.4 Ig ml-' PCNA.
Lanes 1, 2, 7 and 12: ro Cipl; lanes 3, 8 and 13: 1.9 g ml-1
Cipl; lanes 4, 9 and 14: 3.8 g ml-' Cipl; lanes 5, 10 and 15:
7.5 jig ml- Cipl; lanes 6, 11 and 16: 15 jLg ml-1 Cipl. The
migration of double-stranded open circular M13 (DS-oc), doublestranded closed circular M13 (DS-cc), single-stranded circular
M1 3 (SS-c) and single-stranded linear M1 3 (SS-I) is indicated. (b)
Quantification of 32p incorporation into total DNA or (c) doublestranded closed circular M1 3 DNA. Asterisk: no added PCNA;
blue circles: 0.4 I.g ml-1 added PCNA; green triangles: 1.4 jig
ml-1 added PCNA; red squares: 2.4 Jug ml-' added PCNA.
Cipl, DNA replication and DNA repair Shivji et al.
Repair was strongly stimulated by the addition of fraction
XD2 (not shown) or human PCNA, reaching a maximum
at about 400 ng ml-' added PCNA (Fig. 4a, open squares).
Thus, as in human cell extracts, nucleotide excision-repair
in Xenopus egg extracts is dependent on PCNA. Maximal
stimulation of the repair reaction occurred, however, at
lower PCNA concentrations than was seen for the replication reaction (Figs 3,4 and data not shown). Cipl had
virtually no effect on nucleotide excision-repair in fraction
XD1 supplemented with concentrations of PCNA at or
below those required for maximal stimulation: under these
conditions, PCNA is limiting for repair (Fig. 4a, closed
squares). This is a marked contrast to the severe inhibition
of replicative DNA synthesis by Cipl (Fig. 3). We also
tested the effect of Cipl on DNA repair in a human cell
extract system in which PCNA was limiting. As previously
reported [2], DNA repair in the human system was
strongly dependent on PCNA, but, as with the Xenopus
XDI fraction, repair was not inhibited by an excess of
Cipl (Fig. 4b). Cipl can therefore inhibit the function of
PCNA involved in DNA replication without inhibiting
the PCNA involved in DNA repair.
Discussion
PCNA-dependent DNA repair inXenopus egg extracts
Previous work has demonstrated the functional repair of
UV-damaged DNA molecules when introduced into
Xenopus oocytes [35,36,38]. We have investigated a
related reaction mediated by high-speed supernatants of
Xenopus egg extracts. Preferential repair synthesis in
either UV-damaged or cisplatin-damaged DNA was
observed, and the size of the repair patch was consistent
with the excision fragment of approximately 30 nucleotides produced by nucleotide excision-repair in Xenopus
and mammalian cells [38].
Studies of human cell extracts have shown that
nucleotide excision-repair requires the polymerase
auxiliary factor, PCNA [2,3]. To establish whether the
same is true in Xenopus egg extracts, we fractionated the
extract to remove PCNA whilst leaving all the other
components required for nucleotide excision-repair. The
PCNA-depleted fraction, XD1, supported repair only
poorly, and the reaction was strongly stimulated by complementation with human PCNA. Thus, as in
mammalian cells, nucleotide excision-repair in Xenopus
eggs seems to require PCNA activity. The Xenopus repair
reaction was saturated with approximately 0.4 Bug ml-I
PCNA. This is well below the level of PCNA (approximately 200 g ml-1) present in unfractionated Xenopus
eggs [25,42]. PCNA therefore appears to be present in
the Xenopus egg at concentrations well above those
required for efficient nucleotide excision-repair.
Inhibition of DNA synthesis by Cipi
Low-speed supernatants of Xenopus egg extracts support
the semi-conservative replication of added DNA
molecules following their assembly into interphase nuclei
RESEARCH PAPER
Fig. 4. Effect of Cipl on PCNA-dependent nucleotide
excision-repair in Xenopus and human cell extracts. (a) Repair
reactions contained 1 mg ml- 1 XD1, various concentrations of
PCNA, either with (filled symbols) or without (open symbols)
15 .Lg ml- 1 histidine-tagged Cipl. AMP incorporation into either
damaged (squares) or undamaged (circles) DNA was quantified.
(b) Repair reactions contained 1 mg ml- 1 human CFII and
0.5 mg ml-1 CFIA, various concentrations of PCNA, either with
(filled symbols) or without (open symbols) 40 j.g ml-1 bacterially
expressed GST-tagged human Cipl (greater than four-fold more
than the Cipl concentration required to inhibit DNA replication
in Xenopus egg extract 125]). AMP incorporation into either
damaged (squares) or undamaged (circles) DNA was quantified.
[29,31,33,43]. The initiation of DNA replication in this
system is dependent on the activity of a Cdk which
probably consists of a complex between Cdk2 and an Aor E-type cyclin [22,23,25]. When Cipl (around
4 pug ml-1) is added to this system, the initiation of DNA
replication is specifically blocked by inhibition of
Cdk2/cyclin function [25,44] (Fig. 5, red line). The
quantity of Cipl required to inhibit replication corresponds closely to the quantity of Cdk2 in the extract
(approximately 2 Bug ml-1), and no effect of Cipl could
be detected on PCNA function in replication-fork elongation [25]. The high levels of PCNA (approximately
200 Fag ml-') in unfractionated Xenopus extract would,
however, make it very hard to detect an effect of Cipl on
PCNA function.
In contrast, in the extracts used here, complementary
strand synthesis on single-stranded DNA does not require
Cdk/cyclin function, and is strongly dependent on
PCNA. The PCNA-depleted fraction, XD1, supported
complementary strand synthesis only poorly, and was
strongly stimulated by complementation with human
PCNA. This suggests that a PCNA-dependent DNA
polymerase (either 8 or E) is required to perform most of
the DNA synthesis in this reaction. DNA polymerase or,
with its associated primase subunit, is also likely to be
required to prime synthesis of nascent strands (data not
shown).
Using the PCNA-depleted XD1 fraction, we could
manipulate the PCNA concentration to levels that were
100-1 000 times lower than in the unfractionated extract;
at these levels efficient inhibition of complementary
strand DNA synthesis by Cipl could be observed (orange
line, Fig. 5). This is consistent with recent studies using
human cell extracts, which showed that Cipl is capable
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Current Biology 1994, Vol 4 No 12
during DNA replication. After our studies were
completed, Li et al. [50] reported that PCNA-dependent
nucleotide excision-repair by human cell extracts is
refractory to inhibition by Cipl; our results are consistent
with these observations. Both Li et al. [50] and FloresRozas et al. [26] presented evidence suggesting that the
synthesis of shorter tracts of DNA by polymerases 8 and
E is less sensitive to Cipl inhibition than is the synthesis
Fig. 5. The effects of Cipl expression in Xenopus extracts. The
major effect (red line) isto block the initiation of DNA replication by inhibiting a cyclin-dependent kinase, probably consisting
of Cdk2 plus cyclin A or cyclin E [251. At higher Cipl concentrations, PCNA function in DNA synthesis is inhibited (orange line),
but Cipl does not inhibit the function of PCNA in nucleotide
excision-repair under these conditions (dashed green line).
of inhibiting PCNA during DNA synthesis in the SV40
cell-free system, as well as DNA synthesis catalyzed by
purified polymerase 8 [26,27].
Differential effect of Cipl on repair and replication
With Xenopus XDI fraction supplemented with up to
0.4 Ig ml - ' PCNA, nucleotide excision-repair was
virtually unaffected by the addition of 15 Rg ml-1 Cipl.
Complementary strand synthesis under these conditions
was, however, strongly suppressed. A similar insensitivity
of nucleotide excision-repair to Cipl inhibition was
observed in human cell extracts.
These results go some way towards explaining the
function of Cipl following DNA damage (Fig. 5). Two
of the responses of dividing cells to DNA damage are to
repair the damage and to block cell-cycle events, such as
DNA replication, that it would be dangerous to
complete were the damage still present. There is good
evidence that Cipl is involved in this latter checkpoint
control, as it is induced when DNA damage occurs
[11-13,45-49] and can block the initiation of DNA
replication by inhibiting the Cdks required at the G1/Sphase cell-cycle transition (Fig. 5) [14,25]. Cipl also
seems to have a direct effect on DNA replication by
specifically inhibiting PCNA function in this process
(Fig. 5,orange line). As Xenopus eggs contain much less
Cdk2 (2 jig ml-1) than PCNA (200 jig ml-1) [25], this
inhibition is only revealed when PCNA levels are
lowered by fractionation. Under these conditions,
however, Cip does not inhibit the function of PCNA in
nucleotide excision-repair (Fig. 5, green line).
It is reasonable to find that Cipl can discriminate
between the roles of PCNA in repair and replication, and
only blocks the latter. The precise mechanism of this differential effect is currently unclear. One possibility is that
Cipl might be able to distinguish the structure of the 30nucleotide gap in DNA generated during the repair
process from the primer-template structures that occur
of longer tracts. It will be interesting to determine the
mechanism of this apparent discrimination, and to test
whether it occurs during filling of a gap produced by
nucleotide excision-repair. Alternatively, the specificity
of Cipl inhibition might be modulated by interaction
with excision-repair proteins - such as the XP and
ERCC repair factors that are associated with human
disease - at the site of DNA damage. As most of the
repair factors have been isolated, it should soon be
practical to explore these possible mechanisms.
Conclusions
Xenopus egg extracts support the nucleotide
excision-repair of UV-damaged DNA, and, as in
mammalian cells, this reaction is dependent on the DNA
polymerase auxiliary factor, PCNA. Cipl, the production of which is induced in response to DNA damage,
can potentially inhibit DNA replication in Xenopus
extracts by inhibition of the Cdk function required for
the initiation of replication forks, and, at higher concentrations, by inhibition of PCNA function. The inhibition
of PCNA in DNA replication is, however, selective:
Cipl does not affect the function of PCNA in nucleotide
excision-repair. The induction of Cipl in response to
DNA damage, therefore, allows DNA repair to continue
under conditions in which DNA replication is severely
inhibited.
Materials and methods
Preparation of extracts and proteins
High-speed supernatant was prepared from activated Xenopus
eggs by diluting interphase low-speed supernatant prepared as
described [51] with an equal volume of Buffer A (20 mM
Hepes-KOH, 40 mM potassium phosphate, 10 % sucrose,
2.2 mM MgC1, 2 200 pM EDTA, 2 mM dithiothreitol (DTT),
I [ig each of leupeptin, pepstatin and aprotinin, pH 7.8) and
centrifuging at 100000 x g for 30 min at 4 C in a swingingbucket rotor. Harder centrifugation (250000 x g) yielded
supernatants with reduced repair activity. The supernatant
(final protein concentration 25 mg ml-1) was stored in 20 p1
aliquots in liquid nitrogen.
Fractions XD1 and XD2 were prepared as follows: 1 ml
Xenopus high-speed supernatant prepared as described above
was applied to a 2 ml DEAE Biogel column (Bio-Rad) in
Buffer A containing 0.1 M KCI. The flow-through fraction,
containing 4 mg ml-1 protein was designated XD1. Bound
protein was eluted (2 mg ml-' protein) with Buffer A
containing 1M KCI and was designated XD2.
Cipl, DNA replication and DNA repair Shivji et al.
Whole-cell extract from GM 1953, a normal lymphoblastoid cell
line, was prepared as described [341 and had a protein concentration of 14 mg ml-1. CFII and CFIA fractions were prepared
[2] by loading HeLa whole-cell extract on a column of phosphocellulose in Buffer B (25 mM Hepes-KOH pH 7.8, 1 mM
EDTA, 0.01i % NP40, 10 % glycerol, 1 mM DTT) containing
0.1 M KCI. Flow-through fractions were collected and bound
protein eluted with Buffer B containing I M KCI. Pooled peak
fractions from the flow-through (CFI) and the M KCI elution
(CFII) were dialyzed against 25 mM Hepes-KOH (pH 7.9),
1 mM EDTA, 17 % glycerol, 1 mM DTT, 12 mM MgCI 2 and
0.1 M KCI. CFI was then loaded onto a DEAE Biogel column
equilibrated in Buffer B containing 0.15 M KC1. Peak fractions
from the flow-through (CFIA) were dialyzed as above. CFIA
and CFII contain all proteins essential for nucleotide
excision-repair except for PCNA. In experiments with these
fractions, reaction mixtures included I mg ml -1 CFI protein,
0.5 mg ml 1' of CFIA protein and the indicated amount of
PCNA (or a pre-mixture of PCNA and Cipl). Incubation was
for 3 hours at 30 °C before isolation of DNA.
Recombinant histidine-tagged human Cipl [27] and human
PCNA [52] were produced in Escherichia coli. Bacterially
expressed glutathione-S transferase (GST)-tagged human Cipl
was the same preparation as described previously [25].
Reaction conditions
Standard repair assay reaction mixture contained 250 ng each
of UV-irradiated plasmid pBluescript KS+ and non-damaged
plasmid pHM14 in a buffer including [c- 32 P]dATP as
described [34]. Reactions were performed for 90 min at 30 C,
and DNA was then isolated, linearized with BamHI and
separated by agarose gel electrophoresis. Nucleotide
excision-repair was quantified by scanning the autoradiograph
of 32 p incorporation and normalizing this figure to the DNA
recovery as determined by scanning the ethidium signal [2].
For the defined lesion experiments, a single 1,3-intra-strand
d(GpTpG) cisplatin cross-link in the oligonucleotide
5'-TCTTCTTCTGTGCACTCTTCTTCT-3' was incorporated into closed-circular duplex M13mp1 8GTG DNA with T4
DNA polymerase and ligase following described methods [37].
A control substrate was synthesized without the adduct. The
M13mpl 8GTG vector was derived by replacing the EcoRI-Sall
fragment of M13mp18 with a 42 bp fragment containing a
sequence complementary to the adduct-containing oligonucleotide. Reaction mixtures containing 100 ng substrate
DNA were incubated with 6 mg ml -1 Xenopus high-speed
supernatant with [- 32 P]dTTP and [- 32 P]dCTP as labels. After
the reaction, DNA was purified, digested with BstNI and
separated by electrophoresis on an 8 % polyacrylamide gel.
For complementary strand synthesis assays, Xenopus XDI1
fractions (1 mg ml-1) were incubated in the same buffer as used
for DNA repair reactions [34] with 4 ng i.1-1 M13 singlestranded DNA, [- 32 P]dATP and various concentrations of
human PCNA and human histidine-tagged Cipl. Reactions
were performed for 90 min at 30 °C, and products were then
separated by agarose gel electrophoresis. Gels were dried and
the label quantified with the aid of a Phosphorimager
(Molecular Dynamics).
Acknowledgements: We thank M. Howell for providing the Cipl
clones, B. Stillman for providing the PCNA clone, K. Yarema,
J. Essigmann, S. Lippard, D. Szymkowski and J. Moggs for
RESEARCH PAPER
the platinated substrate, and T. Hunt and T. Lindahl for helpful
discussions. J.J.B. is a Lister Institute Jenner Research Fellow.
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Received: 7 September 1994.
Accepted: 7 October 1994.