The EMBO Journal Vol.2 No.5
Microinjection of human cell extracts corrects xeroderma
A.J.R. de JongeY,2, W. Vermeulent, B. Klein3 and
'Department of Cell Biology and Genetics, Erasmus Univerity, PO Box
1738, 3000 DR Rotterdam, 2Free University of Amsterdam, Medical Facul-
ty, Department of Medical Microbiology and Parasitology, v.d.
Boechorststraat 7, Amsterdam, 3Sylvius Laboratories, State University
Leiden, Department of Medical Biochemistry, 2333 AL Leiden, and
4Medical Biological Laboratory, TNO, PO Box 45, Rijswijk, The
Communicated by D. Bootsma
Received on 23 December 1982; revised on 21 February 1983
Cultured fibroblasts of patients with the DNA repair syn-
drome xeroderma pigmentosum (XP) were injected with
crude ceil extracts from various human cells. Injected fibro-
blasts were then assayed for unscheduled DNA synthesis
(UDS) to see whether the injected extract could complement
their deficiency in the removal of u.v.-induced thymidine
dimers from their DNA. Microinjection of extracts from
repair-proficient cells (such as HeLa, placenta) and from cells
belonging to XP complementation group C resulted in a tem-
porary correction of the DNA repair defect in XP-A cells but
not in cells from complementation groups C, D or F. Extracts
prepared from XP-A cells were unable to correct the XP-A
repair defect. The UDS of phenotypically corrected XP-A
cells is u.v.-specific and can reach the level of normal cells.
The XP-A correcting factor was found to be sensitive to the
action of proteinase K, suggesting that it is a protein. It is pre-
sent in normal cells in high amounts, it is stable on storage
and can still be detected in the injected cells 8 h after injec-
tion. The microinjection assay described in this paper pro-
vides a useful tool for the purification of the XP-A (and
possibly other) factor(s) involved in DNA repair.
Key words: xeroderma pigmentosum/microinjection/pheno-
typic correction/ DNA repair enzymes
Xeroderma pigmentosum (XP), an autosomal recessive
human disease, is characterized by an extreme sensitivity of
the skin to sunlight, a very high incidence of skin cancer and
frequently neurological abnormalities (for a review,
Kraemer, 1980). Cultured skin fibroblasts from most XP pa-
tients are deficient in the excision repair of u.v.-induced
pyrimidine dimers from their DNA and this is thought to be
the primary biochemical defect. As a consequence, excision-
deficient XP cells show a decreased rate of unscheduled DNA
synthesis (UDS), monitored as the incorporation of [3H]TdR
in cells in the GI and G2 phase of the cell cycle after u.v. ir-
radiation (Cleaver, 1968; Bootsma et al., 1970).
Using cell hybridization, seven complementation groups
have been identified so far within the XP syndrome (De
Weerd-Kastelein et al., 1972; Keijzer et al., 1979). This exten-
sive genetic heterogeneity indicates that the repair of u.v.-
induced DNA lesions in mammalian cells follows a complex
pathway. Efforts to unravel this pathwayand to characterize
*To whom reprint requests should be sent.
IRL Press Limited, Oxford, England.
the factors involved have been undertaken using essentially
non-viable systems: such as isolated nuclei (Smith and Hana-
walt, 1978), cell-free extracts obtained by osmotic disruption
(Ciarrocchi and Linn, 1978), sonication (Mortelmans et al.,
1976) or permeabilized cell systems (Roberts and Lieberman,
1979; Dresler et al., 1982). Up to now these studies have not
resulted in the identification of such factors. We have chosen
to use the living XP cell as a 'test tube' and to try to provide it
with the lacking factor using microinjection. Microinjection
into living cells via glass micro-needles has been applied suc-
cessfully to study cellular processes (Kreis et al., 1979; Bur-
ridge and Feramisco, 1980) and the activity of vatious bio-
logical macromolecules (Graessmann and Graessmann, 1976;
Graessmann et al., 1980b; Liu et al. 1979; Capecchi, 1980;
Anderson et al., 1980). In the cited studies, pure or purified
material was injected. We have injected crude extracts
prepared from human cells. Here we report the finding of an
activity in these extracts which corrects the repair defect in
cells belonging to XP complementation group A. This activi-
ty is abundantly present in normal cells, inactivated by the ac-
tion of proteinase K and specific for XP complementation
Correction of the XP-A defect by microinjection
Crude extracts prepared from repair-proficient cells were
injected into the cytoplasm of at least 3-days old XP25RO
homopolykaryons (XP complementation group A) using
standard microinjection procedures. Usually each extract was
injected into at least 50 homopolykaryons of which >70Vo
survived the injection. The ability of the surviving cells to per-
form excision repair was tested by determining their rate of
UDS. Briefly, this is carried out as follows: after injection,
the cells are irradiated with a saturating dose of u.v.; they are
then cultured in the presence of [3H]thymidine and the
radioactivity incorporated in the repair patches is visualized
A u.v.-exposed XP25RO homopolykaryon, injected with a
HeLa cell extract is shown together with two non-injected
monokaryons in Figure la, and repair-proficient control
fibroblasts in Figure lb (C5RO, non-injected). Quantitative
data on UDS
treatments are summarized in Table I. Although there is con-
siderable variation in the level ofUDS with different extracts,
the UDS of >907o of the surviving injected XP-A cells is
significantly above that found without injection (i.e., at least
2x the highest level found in non-injected cells in the same
preparation). With some extracts, the UDS of many injected
cells was close to the wild-type (C5RO) UDS level (compare,
for example, the cells of Figure la and b, see also some ex-
tracts in Table I and II). Even giant polykaryonswith >20
nuclei were found that displayed wild-typeUDS levels after a
single microinjection. We attribute the spreading of UDS
values with different HeLa extracts (see Table I), at least in
part, to uncontrolled variation in the qualityof extracts.
A number of control experimentsconfirmed that thegrains
observed above nuclei of injected cells were due to u.v.-
after injection of various
A.J.R. de Jonge et al.
Fig. 1. (a) Micrograph of a XP25RO homopolykaryon (containing three
nuclei) after microinjection of a HeLa extract, followed by assay for UDS.
See Materials and methods for experimental details. (1) Injected XP25RO
homotrikaryon. (2) Uninjected XP25RO monokaryons. Silver grains above
nuclei indicates UDS. (b) Micrograph of a control (C5RO) homopoly-
karyon after assay for UDS. (1) Homobikaryons showing UDS.
(2) Monokaryon showing S-phase labelling.
induced UDS and not to some artefact of the procedure.
(i) The increase in the level of nuclear labeling was dependent
on injection of the extract and on u.v. irradiation since no in-
crease was observed when the extract was replaced bybuffer
(not shown) or when the u.v. irradiation was omitted (Table
I). (ii) Neither the injection procedure itself nor the injected
HeLa extract noticeably affected the u.v.-induced UDS of
repair-proficient control polykaryons (Table I). (iii) A dif-
ference in UDS between injected monokaryons and injected
homopolykaryons was not observed (data not shown).
(iv) Finally, UDS was the same whether recipient cells were
u.v. irradiated before or after injection (Table I) ruling out
the possibility that the UDS was due to u.v. irradiation and
repair of any injected DNA or chromatin present in the ex-
Characterization of the correcting activity
The factor in the HeLa extract responsible for the restora-
tion of UDS is ubiquitous among human cells and acts
specificallyon the repair defect ofcomplementation groupA.
No significant stimulation of UDS was found when the ex-
tract was injected into cells belonging to complementation
groups C, D or F (Table I). On the other hand, HeLa factor
did correct the defect in XP2CA, a XP-A cell line unrelated
to XP25RO (Table I). Extracts prepared from an SV40-trans-
formed XP-C cell line (XP8CA C SV2) and an SV40-trans-
formed repair-proficient fibroblast (VH1O SV40) stimulated
the UDS of XP25RO to the same extent as HeLa extracts
(Table I). On the other hand, five extracts prepared from
three different SV40-transformed XP-A cell lines [three from
XP12RO SV40, one from XP25RO SV40 and one from
XP20S (SV)] were unable to induce UDS after injection into
XP25RO fibroblasts. The result with one extract is presented
in Table I. The absence of XP-A correction was not due to
any inhibitors of UDS present in the XP-A extracts, since in-
jection of these extracts in repair-proficient fibroblasts did
not influence their u.v.-induced UDS (see Table I). More-
over, a 1:1 mixture of XP-A and HeLa extracts stimulated
the UDS of XP25RO almost to the same extent as the HeLa
extract alone (data not shown). From the foregoing data, we
conclude that the correction observed is specific for XP com-
plementation group A. We have also found XP-A correcting
activity in extracts prepared from human placenta (Table I)
demonstrating that this property is not limited to transformed
or cultured cells.
The XP-A correcting factor is reasonably stable on storage.
We have not found considerable loss of activity after storage
of the extract for 7 weeks at 4°C or for longer periods at
-70°C (Table I). In the injected cell its activity can still be
detected 8 h after injection (Table I).
To determine whether the factor involved is a protein, the
extract was incubated with proteinase K covalently linked to
CNBr-activated Sepharose beads. After removal of the im-
mobilized protease by centrifugation and injection of the
supernatant into XP25RO polykaryons, the correcting activi-
ty was no longer detectable (see Table II). In contrast, activity
was retained in a control incubation with beads to which
bovine serum albumin (BSA) had been attached. Injection of
a 1:1 mixture of proteinase K-incubated extract and untreated
extract showed that the loss of UDS-correcting activity was
not due to inhibiting factors generated during the proteinase
K incubation. Furthermore, treated extract did not affect
UDS in normal (C5RO) homopolykaryons. The proteolytic
action of the proteinase K beads under these conditions was
confirmed by the substantial reduction of two enzymatic ac-
tivities present in HeLa extracts. The first was glucose-6-
phosphate dehydrogenase (G6PD) quantitatively determined
in an enzyme assay (see Table II). The second enzyme tested
assayed in a similar way to the XP-A factor, i.e., by micro-
injection into HPRT-deficient mouse cells. From the fore-
going data, we conclude that the XP-correcting factor con-
tains a protein moiety essential for its function.
The experiments presented here demonstrate that biologi-
cal activities in crude (cell) extracts can be assayed by micro-
needle injection into suitable recipient cells. Using this pro-
cedure, we have identified a protein in extracts of normal
human cells which specifically corrects the XP-A repair
defect. Although the identification of factors involved in
DNA repair is possible with the microinjection assay, there
are also limitations. One of these is that the activity to be
assayed must be present in sufficient amounts in the injected
extract. The XP-A correcting protein certainly fulfils this
criterion. A single microinjection is sufficient to restore the
UDS to the maximal level of repair-proficient cells. Even a
Human cell extracts correct XPrepairdefect
Table I. Levels of u.v.-induced UDS after microinjection of various human cells extracts into XP homopolykaryons
UDS (grains per nucleus)
07 of wild-type (
92 ± 7
5 ± le
83 ± 6
79 ± 5
28 ± 3
48 ± 4
33 ± 3
43 ± 3
36 + 3
5 ± le
47 ± 4
23 ± 3e
21 X 2
38 ±i 5
15 ± 2e
15 ± 3
u.v. before injection
UDS 8 h after injection
Extract >7 weeks at - 70°C
Extract 7 weeks at 40C
aBetween brackets the XP complementation group. For further details on the cell lines used see Table II.
bUnless indicated otherwise cells were u.v. irradiated immediately after injection.
'The UDS of injected cells is expressed as the 07 of the UDS of C5RO a repair-proficient control cell line, used as a standard in each experiment. This wild-
type UDS level differed between individual experiments but was always >50 grains/nucleus.
dExtract prepared from XP12RO SV40.
eResidual repair activity.
fExtract prepared from XP8CA C SV2.
gExtract prepared from VHI0 SV40.
3-fold diluted HeLa extract gave clearly detectable correction
(unpublished observations). This in itself is an interesting
observation, given the fact that the injected volume is very
small relative to the volume of the injected polykaryon (we
estimate < 100/). Furthermore, the concentration of the fac-
tor in the extract is considerably lower than in the cells from
which the extract was prepared (a factor of 2 to 3 is a minimal
estimate). Even ignoring possible loss or inactivation of the
XP-A factor during preparation of the extract, the activity of
the protein in normal (non-u.v.-irradiated) cells must be at
least 20- to 30-fold higher than necessary for maximal UDS
activity. This apparent excess of the XP-A correcting compo-
nent renders it unlikely that the factor is involved in a rate-
limiting step in the normal repair process. Our observation
that the UDS over the first 2 h after injection is already close
to that of control cells indicates that the correcting protein
can exert its function rapidly after introduction into the cell.
This extends results obtained in cell hybridization (Mat-
sukuma et al., 1981; Giannelli et al., 1982) and cybridization
experiments involving XP and control cells (Keijzer et al.,
1982). Also, our finding that the activity of the XP-A correc-
ting protein is still detectable 8 h after injection agrees well
with data from cybridization experimentsin whichcytoplasts
from normal cells were fused with XP-A fibroblasts(Keijzer
Phenotypiccorrection of the XP-Arepairdefect was also
obtainedby injectionof theprokaryotic enzymes (Micrococ-
cus luteus u.v.-endonuclease and T4 endonuclease V(A.J.R.
deJongeetal.,inpreparation).In these cases correction was
not specific: all XPcomplementation groupswerecorrected,
in agreement with results reported by others who used a
Hayakawaetal., 1981). Itappearsthat theseprokaryoticen-
zymes cause a complete by-passof all therepairdefects in
XP. This is not the case for theproteinfactor described in
thispapersince the correction was found to be XP-Aspecific.
However, a by-passof somestepsin therepair process,in-
cluding the stepaffected in XP-Acells, is not ruled out. In
that case thecorrecting proteinshould bemissingin XP-A as
a direct or indirectconsequenceof the XP-A-deficientstep.It
is evenpossiblethat theinjectedextractprovidedtwo or more
(protein)factorsresponsiblefor the XP-A correction. If so all
these factors should bepresentin 20- to 30-fold excess in nor-
mal cells and all of them should be deficient in XP-A cells.
cell system (Tanaka
A.J.R. de Jonge et al.
We favour, therefore, the more simple interpretation that the
correction is due to just one component: the gene product
deficient in XP complementation group A.
With the microinjection repair assay described here, we are
now trying to further characterize and purify this protein.
Materials and methods
Cell lines and culture conditions
Relevant data on the cell lines used in this study are listed in Table III. All
cells were cultured in Ham's FIO medium (Flow supplemented with 7.5%o
fetal and 7.5%o newborncalf serum and penicillin and streptomycin
(100Ag/ml).Cells to be microinjected were cultured on 0.6 x 0.8 cm pieces of
a microscope slide with a 2 mm grid.
Table H. The effect of treatment with proteinase K on the XP-A correcting
activity in HeLa extracts.
Treatment of extract
No of wild-
(i) No incubation
47 + 4
(ii) Proteinase K-beadsa
3 + 0.3
40 + 2
1:1 mixture (i) and (ii)
16 + 2
Sepharose beads with covalently attached proteinase K or BSA were in-
cubated with aliquots of a HeLa extract. The beads were removed by cen-
trifugation. The supernatant was microinjected into XP25RO polykaryons
and UDS was assayed. The enzymatic activity of HPRT was assayed by
microinjection into HPRT-deficient mouse LTH-1 cells as described in
Materials and methods. The enzymatic activity of G6PD was assayed ac-
cording to Jongkind (1967). The activity in the untreated aliquot was set at
aThe proteinase K-treated extract, injected into control cells resulted in 50
i 3 grains/nucleus, versus 51 j 2 grains/nucleus observed in non-injected
n.d. not determined.
Preparation of cell extracts and microinjection
Cultures of HeLa S3 cells (8-20 x 107 cells) in log phase were harvested by
trypsinization or scraping (using a rubber policeman) and washed twice in Na-
K reversed phosphate buffered saline (RPBS: 4.05 mM Na2HPO4;
KHPO4; 140 mM KCI; pH 7.2). After the second wash the supernatant was
removed and the 'dry' pellet (with a volume of0.2 to >1 ml) was subjected to
sonication (six pulses of 10 s with 10 s intervals, at 0°C, using the microtip of
a MSE sonicator operating at maximum output). The sonicate was centrifug-
ed for 40 min at 130 000 g, at 4°C in a type 50 fixed angle rotor, using a
L5-65 Beckman ultracentrifuge. Aliquots of the supernatant were either used
directly for microinjection or rapidly frozen in liquid nitrogen and stored at
- 70°C until use. Under these conditions, extracts can be stored for > 7 mon-
ths without notable loss of XP-A correcting activity. The activity of the ex-
tract is sensitive to repeated cycles of freezing and thawing. Crude extracts
from other cells were prepared in the same way as described above. Placenta
extract was made by sonication of finely cut fresh placental tissue and ultra-
centrifugation as specified for the HeLa extract.
The crude cell extracts obtained above were microinjected into the
cytoplasm of homopolykaryons (see below) via glass micro-needles using the
procedure described by Graessmann et al. (1980a). Relevant data on the injec-
tion and the injected cells were recorded during microinjection with the aid of
a tape recorder.
Cellfusion and assay of repair activity
Only homopolykaryons were used for microinjection. These were obtained
by fusion of cells of an (XP) cell strain in suspension using inactivated Sendai
virus at a concentration of 200 HAU/ml as described by De Weerd-Kastelein
et al. (1972). The fused cell population was cultured for at least 3 days after
seeding to allow completion of DNA replication (S-phase) in the homopoly-
karyons (Jaspers et al., 1981, and unpublished observations). This eliminates
possible confusion of radioactive labeling due to a short period of normal
DNA replication with that due to UDS. Moreover, the unique morphology of
each polykaryon facilitates registration and subsequent re-identification of in-
jected cells. Usually 50-100 homopolykaryons each containing 2 to >20
nuclei were injected with the same cell extract. The percentage of cells which
died from the injection varied between 5 and 30. The ability of the surviving
injected cells to perform UDS was assayed as follows. After injection, cells
were irradiated with a saturating dose of u.v. (15 J/m2; Jaspers and Bootsma,
1982), cultured for 2 h with 10 1sCi/ml [3H]thymidine (sp. act. 20 Ci/mmol)
and washed, fixed and processed for autoradiography according to Zelle and
Bootsma (1980). Exposure time was 1 week. After development, fixation and
staining with Giemsa's solution the slides were mounted, the injected cells
were relocated and the average number of grains per nucleus ( kSEM) was
determined to calculate the level of UDS. All data on UDS of injected cells
refer to the fraction of cells which survived the microinjection treatment. In
some experiments the u.v. irradiation was carried out prior to the microinjec-
Table HI. Relevant information on the cell lines used
Kraemer et al. (1975)
Kramer et al. (1975)a
Takebe et al. (1974)
De Weerd-Kastelein et al. (1972)b
Hashem et al. (1980)
Kleijer et al. (1973)
XP8CA C SV2
Hashem et al. (1980)a
de Weerd-Kastelein et al. (1976)
Arase et al. (1979)
Repair-proficient primary fibroblast
Repair-proficient, SV40-transformed fibroblasta
Mouse cell line, HPRT-
de Jonge et al. (1982)
'These cell lines were transformed with a SV40 ori- fragment (6-17, Gluzman et al., 1980, and unpublished results).
bSV40 transformation of this cell line was carried out by G. Veldhuizen (Medical Biological Laboratory, Rijswijk).
Human ceU extracts correct XP repair defect
Studies using proteinase K
Proteinase K (pretreated for 2 h at 37°C to destroy any contaminating
DNase or RNase activity) or BSA was covalently linked to CNBr-activated
Sepharose beads and after extensive washing with ice-cold RPBS to remove
unattached protein, the beads were incubated at 37°C with aliquots of HeLa
cell extracts. After 30 min, beads were removed by centrifugation, the super-
natant was microinjected with XP25RO homopolykaryons and UDS was
assayed as described. The effect of proteinase K on the enzymatic activity of
HPRT in the cell extracts was studied by microinjection of the treated extracts
into HPRT-deficient mouse LTH-l cells (de Jonge et al., 1982), followed by
culturing in the presence of 10ACi/ml[3H]hypoxanthine (sp. act. 1 Ci/mmol)
for 24 h. Further processing was as described above for injected XP cells. The
enzymatic activity of G6PD in the treated extracts was quantitatively deter-
mined according to Jongkind (1967). The G6PD activity in the untreated all-
quot was set at 10007o.
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