From the
A human apurinic/apyrimidinic endonuclease activity, called AP
endonuclease I, is missing from or altered specifically in cells
cultured from Xeroderma pigmentosum group-D individuals (XP-D cells)
(Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S.(1978) Nucleic
Acids Res. 5, 951-960). We have now observed that another
nuclease activity, UV endonuclease III, is similarly not detected in
XP-D cells and is inseparable from the AP endonuclease I activity. This
activity preferentially cleaves the phosphodiester backbone of heavily
ultraviolet-irradiated DNA at unknown lesions as well as at one of the
phosphodiester bonds within a cyclobutane pyrimidine dimer. The
nuclease activities have been purified from mouse cells to yield a
peptide of M
UV radiation is probably the most extensively used model system
for investigating the biological consequences of DNA damage. When DNA
is exposed to radiation at wavelengths near its absorption maximum of
260 nm, a large number of damages are formed, the most prevalent being
cyclobutane pyrimidine dimers (Loeber and Kittler, 1977). These dimers
are chemically stable and are corrected either by nucleotide excision
repair or in some cell types by enzymatic photo reversal (Boyce and
Howard-Flanders, 1964).
Xeroderma pigmentosum (XP)
Kuhnlein
et al.(1978) found that XP-D cells, and only cells from that
complementation group, lack or have altered an AP endonuclease,
designated AP endo I. This abnormality apparently manifests itself
through a higher than normal ability of XP-D cells to host
cell-reactivated depurinated SV40 DNA (Kudrna et al. 1979).
(As expected, on the other hand, in the same study, XP-D cells
displayed inefficient host cell reactivation of UV-irradiated SV40
DNA.) The abnormality of the AP endonuclease activity in XP-D cells is
peculiar, however, since XP-D cells are known to carry mutations in the
gene for a DNA helicase activity (Weber et al., 1990). We now
report that this AP endonuclease activity is inseparable from
activities acting upon UV-irradiated DNA, one of which cleaves a
phosphodiester bond within a cyclobutane pyrimidine dimer. Moreover,
these activities are apparently associated with ribosomal protein S3, a
protein located on the external surface of the 40 S ribosomal subunit,
which assembles onto preribosomal particles in the nucleus (Hadjiolov,
1985). Possible roles of this ribosomal protein in processing DNA
damage are discussed.
Antibodies S3 71/83 and S3 67/67 were isolated by immunoabsorption
from rabbit antisera prepared against purified rat liver ribosomal
protein S3. As a control, immunoabsorption-purified antibodies raised
in rabbits against rat ribosomal protein S26 (S26-67/8) were
used. These antibodies are described by Lutsch et al.(1990).
Escherichia coli endonuclease III was prepared and assayed
according to Gates and Linn(1977), T4 UV endonuclease was prepared
according to Friedberg and King(1971), and HeLa AP endonuclease II was
prepared according to Kane and Linn(1981). E. coli DNA
polymerase I was purchased from Boehringer Mannheim or from Life
Technologies, Inc. Uracil DNA glycosylase was prepared as described by
Gates and Linn(1977). The T4 endonuclease (Ser) construct was
originally obtained from Drs. J. K. de Riel and K. Valerie (VA
Commonwealth University). It has a W128S substitution due to
substitution of an AGT for a TGG codon, a substitution that also
generates a unique SpeI site in the ptac-denV plasmid
construct.
The average number of nicks introduced per PM2 genome
was calculated from the percentage of total PM2
[
The
supernatant was loaded onto a DE-52 DEAE-cellulose column (5
The dialysate was loaded onto a 1.2-liter phosphocellulose column,
which was equilibrated with buffer A at a flow rate of 50 ml/h. It was
washed with the same buffer, and flow-through fractions, which contain
UV endonuclease III, were collected. UV endonucleases I and II were
eluted from this column with a 4-liter linear gradient from 0 to 1.2
M KCl in buffer A (see Kim and Linn(1989)).
The supernatant was loaded
onto a DEAE-cellulose column (5
To the
dialysate (164 ml) was added 26.8 g of ammonium sulfate with constant
stirring on ice; then, after 10 min, it was centrifuged for 15 min at
20,000
The above material
was loaded onto a Sephacryl S-200 column (140 ml, bed volume), which
had been equilibrated with 25 mM potassium phosphate, 0.01%
Triton X-100, and 10 mM 2-mercaptoethanol. Activity separated
into 2 peaks, which eluted after 64 and 106 ml. The second peak was
dialyzed against 5 mM potassium phosphate (pH 8.1), 0.01%
Triton X-100, and 10 mM 2-mercaptoethanol (buffer C). It was
subsequently found that all of the activity elutes in the position of
the second peak if 0.7 M ammonium sulfate is included in the
elution buffer.
A 0.5-ml hydroxyapatite column was equilibrated with
buffer C, and the dialysate was applied to the column; then, the column
was washed with the same buffer and eluted with a linear gradient from
5 to 300 mM potassium phosphate (pH 8.1) in buffer C and
collected in 0.5-ml fractions. UV endonuclease III activity eluted at
70 mM potassium phosphate, and active fractions were pooled
and dialyzed against buffer C.
GM 3714 normal lymphoblast cells (5.6 liters of
culture, 6.7
10 liters of cells were grown to a density of
A
Since the
phosphocellulose flow-through fraction from XP-D cells was observed
specifically to lack AP endonuclease I activity (Kuhnlein et
al., 1978), phosphocellulose flow-through fractions from normal
and XP-D human fibroblasts and lymphoblasts and from normal rodent
MPC-11 cells were compared for their UV endonuclease III activity.
Indeed, this activity from XP-D cells was significantly lower or absent
compared with that from the corresponding normal cells ().
The content of UV endonucleases I and II activities, however, did not
differ greatly between the XP-D and the corresponding normal cells
(). This concurrent reduction suggested that the two
activities could be associated with the same protein.
Since this activity
appeared to be reduced in XP-D cells and since XP-D cells have a
reduced ability to process cyclobutane pyrimidine dimers, we wondered
whether the enzyme might have a more subtle interaction with pyrimidine
dimers. Weinfeld et al.(
(6) reported that excised
cyclobutane pyrimidine dimers isolated as dinucleoside monophosphates
from the growth medium of UV-irradiated human fibroblasts released free
thymidine and thymidine monophosphate after reversal of the
dimerization with high doses of UV light. This result was taken to
imply that the photo-liberated thymidine and thymidine monophosphate
had been attached solely by the cyclobutane ring of the dimer, i.e. that there had been a cleavage of an intradimer phosphodiester
linkage during the in vivo processing of DNA cyclobutane
pyrimidine dimers. To test whether UV endonuclease III might catalyze
such a phosphodiester bond cleavage within the pyrimidine dimer, the
consequence of sequential reactions of murine UV endonuclease III
followed by the pyrimidine-dimer glycosylase activity associated with
T4 endonuclease V was studied (see Fig. 3). In this experiment, a
mutant form (Nakabeppu et al., 1982) of the T4 endonuclease,
T4 endonuclease (ser), that lacks most of the AP endonuclease activity
of the enzyme but retains the DNA glycosylase activity was used.
The amount of
mutant T4 enzyme used in this experiment, on the other hand, while
producing no nicks, produced a number of alkali-labile sites equal to
roughly half of the pyrimidine dimers present (alkali-labile sites are
presumably sites at cyclobutane pyrimidine dimers, which have been
subject to the DNA glycosylase activity of the T4 enzyme but not to
phosphodiester bond cleavage) (see Fig. 3). The ordered reaction
of the mammalian enzyme followed by the phage DNA glycosylase activity,
however, resulted in the detection of a number of nicks equal to the
number of cyclobutane pyrimidine dimers present in the DNA substrate.
In addition, no alkali-labile sites were present in this DNA, which had
been treated with both enzymes. Hence, the combination of activities
ultimately generated one nick for each pyrimidine dimer present and
left no additional substrate for the T4 enzyme to convert to
alkali-labile sites. Notice that this experiment demonstrates that the
T4 enzyme acts more efficiently upon the UV-irradiated DNA, which had
been exposed to the UV endonuclease III than upon the DNA prior to
exposure to the mammalian protein. In other experiments utilizing more
of the enzyme, the T4 enzyme alone could convert all pyrimidine dimers
present to alkali-labile sites. In conclusion, the UV endonuclease III
appears to be able to cleave a phosphodiester bond between the
dimerized pyrimidine nucleoside residues, but these cleavages are
observable only after exposure to the T4 DNA glycosylase (see
Fig. 3
).
The above experiment utilized nitrocellulose filter
binding to monitor DNA nicking. To verify that the filter binding truly
reflected cleavage of the DNA backbone, unirradiated and irradiated PM2
DNAs were treated with the combination of enzymes and then sedimented
through denaturing, alkaline sucrose gradients. DNA, which was
irradiated and treated with both enzymes, was indeed converted from
form I to form II and smaller DNA species (Fig. 4).
Significantly, irradiated DNA treated with either one of the enzymes
alone did not exhibit any altered sedimendation pattern. Similar
results were obtained when DNA nicking was monitored by gel
electrophoresis (data not shown).
To confirm that nuclease activity
was indeed associated with the ribosomal protein, affinity-purified
antibody to rat ribosomal protein S3 and a cDNA clone for the rat
protein were utilized. In the first instance, two antibody preparations
were shown to be non-neutralizing for the UV endonuclease III activity
but to be able to immunodeplete it (). In addition, these
antibodies were able to bind the protein associated with the activity
onto protein A-Sepharose beads from which it could be extracted
(Fig. 5). Hence, the affinity-purified antibodies, which were
made against rat rpS3 isolated from ribosomes, recognized both the
protein and the nuclease activity of the murine UV endonuclease III
preparation.
Perhaps most significant,
however, was the fact that the rat rpS3 expressed in E. coli was able also to catalyze the apparent nicking of the
phosphodiester bond within cyclobutane pyrimidine dimers. The protein
expressed in E. coli produced little apparent nicking in
lightly irradiated DNA unless the T4 UV endonuclease (ser) was
subsequently added (, Experiment II). In this case, a
number of nicks was again observed that equaled the number of
cyclobutane pyrimidine dimers expected to have been present
(, Experiment II). Since this unique type of activity
observed with UV endonuclease III from MPC-11 cells (but neither
reported to be present in E. coli nor observed by us in
extracts from E. coli not containing the rpS3
plasmid
In summary, the results obtained with the
antibodies taken together with those using the rpS3 cDNA clone strongly
indicate that ribosomal protein S3 is indistinguishable from UV
endonuclease III in endonuclease specificity, antigenicity, and primary
structure. They are also consistent with the recent report of Wilson
et al. (1994) that ribosomal protein S3 from Drosophila also has AP lyase activity.
If rpS3 were to interact with DNA, it might
at some time be recruited back into the nucleus from the cytoplasm. Two
signals are possibly involved in the transport of rpS3 to the nucleus.
Both the peptide and the nuclease activity associated with UV
endonuclease III are strongly bound by concanavalin A affinity columns
(data not shown). Lectin blots confirmed that the protein interacts
with concanavalin A, as well as with Ulex europeus agglutinin
and soy bean agglutinin; however, it does not interact with wheat germ
agglutinin, Dolichos biflorus agglutinin, or ricin (data not
shown). This lectin binding pattern suggests a nuclear or nuclear
membrane localization (Hart et al., 1989).
Rat ribosomal
protein S3 also contains a potential nuclear localization signal motif
(Silver, 1991), KKRK, near the N terminus. Sequence comparison among
the rpS3 proteins from various species reveals that this sequence is
conserved among the eukaryotic rpS3 proteins but is absent from the
prokaryotic homologs (Fig. 7). Either this signal or the
glycosylation modifications could be utilized to mark the protein for
transport to the nucleus, and future study in this regard is warranted.
Kuhnlein et al.(1978) reported that AP endonuclease
activity from cultured human fibroblasts was resolved into two peaks,
AP endonucleases I and II, by phosphocellulose column chromatography
and that the activity corresponding to AP endonuclease I was missing
from extracts of XP group D cells. AP endonuclease I passed through
phosphocellulose, whereas AP endonuclease II (the major AP endonuclease
activity, which was not accompanied by UV endonuclease activity) was
retained by the resin. Subsequent study revealed that AP endonuclease I
cleaves on the 3`-side of the AP site by a
Kudrna et al.(1979) observed
that depurinated SV40 DNA transfects XP-D cells considerably more
efficiently than it does normal cells. Hence, there is a phenotypic
correlation with the absence of the AP endonuclease. These results were
unexpected since AP endonuclease activity was presumed to be involved
in the repair of AP sites. It may be, therefore, that at least in
mammalian cells, cleavage of DNA at AP sites by a
The
cleavage of the phosphodiester bond within a cyclobutane pyrimidine
dimer that was observed for rpS3 also calls for a novel function. The
activity could relax DNA distortions brought about by the dimer,
possibly then affecting the facility to replicate past the dimer as
suggested also by Galloway et al.(1994). Replication past
pyrimidine cyclobutane dimers is common in vivo in mammalian,
especially rodent, cells. Perhaps, for example, both the
On storage or during purification, UV
endonuclease III from mammalian cells or expressed in E. coli loses its preference for DNA damage, i.e. its activity
upon undamaged DNA increases and approaches that upon undamaged DNA. It
also behaves as though it becomes less anionic during ion-exchange
chromatography. Tycowski et al.(1993) reported that human U15A
RNA is coded for within intron I of the rpS3 gene. We tested the
possibility that U15A RNA (prepared by in vitro transcription)
could re-establish the specificity for UV-irradiated DNA. Approximately
half the nonspecific activity could be suppressed by addition of the
U15A RNA, suggesting that some RNA-like species, but probably not U15A
RNA, could be a specificity factor that suppresses activity upon
undamaged DNA.
Ribosomal protein S3 forms
part of the domain on the ribosome where the initiation of translation
occurs; it can be cross-linked to eukaryotic initiation factors eIF-2
(Westermann et al., 1979) and eIF-3 (Tolan et al.,
1983), and it appears to be directly involved in
ribosome-mRNA-aminoacyl tRNA interactions during translation (Bommer
et al., 1991). However, rpS3 has also been proposed to have
functions other than protein synthesis by several laboratories based
upon observations made in those laboratories. Mutants in the gene
encoding rpS3 in Drosophila have a recessive lethal and a
dominant Minute phenotype, which suggested a possible other
role (Andersson et al., 1994). As noted above, the small
nucleolar RNA, U15A, is processed from an intron of the human gene,
which suggested a relation between regulation of translation and RNA
splicing (Tycowski et al., 1993). Finally, rpS3 is
overexpressed in colorectal cancer cells, which suggested a role in
cell immortality (Pogue-Geile et al., 1991). The observations
in this paper now present a fourth such report.
RpS3 could be
utilized for DNA repair while in the nucleus prior to assembly into
ribosomes, or it might be recruited from functional ribosomes. However,
it finds its way to the genome, we would expect rpS3 to be involved in
DNA damage processing in some way by virtue of (1) the preferences of
the nuclease activity for DNA damage and
(2) by the recent
observation that transfection of normal or Fanconi's anemia human
cell lines with a rpS3 cDNA construct makes these cells considerably
more resistant to the DNA cross-linking agents, mitomycin C and
diepoxybutane.
DNA repair processes appear
at least in part to be coupled to transcription, presumably for
monitoring of damage and possibly for checkpoint control and stress
responses (Drapkin et al., 1994). Whether an involvement of
rpS3 in DNA damage processing would reflect a coupling of translation
to DNA repair is an intriguing possibility. SSL1, for example, has been
implicated in both of these functions in yeast (Yoon et al.,
1992). Finally, it is interesting to note that in E. coli,
rpS3 is a DNA binding protein, the H protein, that appears to be
associated with the nucleoid and is required for activity of several
DNA metabolic enzymes in vitro (Bruckner and Cox, 1989).
The ERCC2 gene corrects the phenotype of XP-D cells after gene
transfer (Flejter et al., 1992). ERCC2 shares 52% amino acid
sequence identity with yeast RAD3, and both genes encode a protein with
DNA helicase and ATPase activity (Weber et al., 1990; Sung
et al., 1993). Why then don't we detect the rpS3
nuclease activities in any XP-D cells? The rpS3 cDNA sequence is
unchanged in XP-D cells, and rpS3 is clearly present on ribosomes in
XP-D cells (GenBank Accession Nos. U14990, U14991, U14992(1994)).
Possibly, a subset of rpS3 molecules is differently subjected to
post-translational modification in XP-D cells, or possibly, rpS3 is
complexed differently to other macromolecules in XP-D cells, thus
changing its chromatographic or catalytic properties. Both of these
possibilities are currently being investigated.
Fibroblasts were harvested from T-150 flasks, 40 for the normal
cells and 15 for the XP-D cells. 10 mg of crude extract protein was
processed for the lymphoblasts. Processing of extracts and assay
procedures are described under ``Experimental Procedures.''
UV endonuclease III is taken as the activity passing through
phosphocellulose, whereas UV endonucleases I plus II are the activities
that bind to the resin.
After
removal of approximately 0.5 pmol (Experiment A) or 1 pmol (Experiment
B) of uracil from 0.35 nmol of [
Reaction mixtures contained 40 mM Tris
(pH 8.0), 50 mM KCl, 0.01% Triton X-100, 5 mM 2-mercaptoethanol, 4 mM EDTA, 20 µM PM2 DNA
(nucleotide residues), and 1.9 units of murine UV endonuclease III
hydroxylapatite fraction. Incubation was for 15 min at 37 °C.
In
Experiment I, reactions (0.2 ml) contained 40 mM Tris-HCl (pH
8.0), 50 mM KCl, 5 mM 2-mercaptoethanol, 0.01% Triton
X-100, 3 mM EDTA, 20 µM PM2 DNA (nucleotide
residues), UV-irradiated with 23 J/m
Immunodepletion
experiments were those described in Fig. 5. UV endonuclease activities
in the supernatants were assayed after exposing UV endonuclease III to
antibody, which had been attached to protein A-Sepharose beads and
centrifuging to remove the beads.
For supplying materials, we are indebted to Dr.
Jacques Laval (FAPY-DNA glycosylase and its substrate), Dr. Joan Steitz
(pS3
(31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) ),
Dr. Ira Wool (pGEM2S315), and Drs. Stephen Lloyd, Jon K. de Riel, and
K. Valerie for clones of T4 UV endonuclease V (Ser). We thank Dr. M. S.
Meyn (Yale University) for making his unpublished transfection data
available to us, Dr. D. G. Burbee (General Atomic, San Diego) for
making the unpublished yeast rpS3 sequence available to us, B. L.
Raether for providing unpublished human rpS3 sequences, and Ann Fisher
for expert tissue culture services.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 32,000, whose sequence
indicates identity with ribosomal protein S3. The nuclease activities
all cross-react with immunopurified antibody directed against authentic
rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the
activities was recovered and was indistinguishable from those of the
mammalian UV endonuclease III. Moreover, the protein expressed in
E. coli and its activities cross-react with the rat protein
antibody. Ribosomal protein S3 contains a potential nuclear
localization signal, and the protein isolated as a nuclease also has a
glycosylation pattern consistent with a nuclear localization as
determined by lectin binding. The unexpected role of a ribosomal
protein in DNA damage processing and the unexplained inability to
detect the nuclease activities in extracts from XP-D cells are
discussed.
(
)
is a rare, autosomal recessive disease that is clinically
characterized by hypersensitivity to ultraviolet radiation and a large
increase in the frequency of sunlight-induced skin carcinomas or
malignant melanomas (Kraemer et al., 1984) and in some cases
by UV-induced ocular lesions and progressive neurologic degeneration.
Cells cultured from XP patients exhibit enhanced sensitivity to killing
by UV light (Kraemer et al., 1976; Andrews et al.,
1978) and display reduced levels of UV-induced DNA repair synthesis
(Cleaver, 1968) as well as inefficient removal of a wide range of
chemically induced bulky DNA damages (Amacher et al.,1977; Galloway et al.,1994). Based upon
restoration of UV-induced repair synthesis following cell fusion, eight
complementation groups have been defined: seven classical groups
(A-G) that are abnormal in excision repair capability (de
Weerd-Kastelein et al., 1972; Hoeijmakers, 1993) and a variant
group that exhibits normal levels of excision repair but has impaired
post replication repair (Lehmann et al., 1975).
Materials
Murine plasmacytoma cell
strain MPC-11 was obtained from Dr. M. Koshland (University of
California, Berkeley). Human normal fibroblast strain F65 was obtained
from the Naval Biomedical Research Laboratory (Oakland, CA) and XP
fibroblast strain GM5424 (complementation group D) was from the
National Institute of General Medical Sciences Human Genetic Mutant
Cell Repository (Camden, NJ). Normal (GM 3714) and XP group D (GM
2485-A) human lymphoblasts were from Dr. T. Lindahl (Imperial Cancer
Research Fund, London, United Kingdom). Supercoiled PM2 phage
[thymidine-H]DNA (5000-16000
cpm/nmol) was purified as described (Kuhnlein et al., 1976).
U15A RNA was synthesized using SP6 RNA polymerase and plasmid
pS3
(31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) (Tycowski et al.,1993) linearized with
NsiI as described by Melton et al.(1984).
Phosphocellulose was from Sigma, DE-52 DEAE-cellulose was from Whatman,
and BA85S597 0.45-mm nitrocellulose membrane filters were from
Schleicher and Schuell.
[methyl-
H]Thymidine was from Amersham.
Cell Culture
MPC-11 cells were split 1:2
daily in suspension medium containing 50% Dulbecco's modified
Eagle's medium, 50% RPMI 1640 medium (Life Technologies, Inc.),
20% heat-inactivated horse serum, 2 mML-glutamine,
100 units/ml each of penicillin and streptomycin, 0.02 M Hepes
(pH 7.2), and 50 µM 2-mercaptoethanol. F-65 diploid human
fibroblasts were cultured in a humidified 5% CO incubator
at 37 °C in 150-cm
flasks. XP fibroblasts were grown
and maintained in Dulbecco's modified Eagle's medium
containing 20% fetal bovine serum. GM 3714 normal lymphoblasts and GM
2485A XP-D lymphoblasts were grown in RPMI 1640 medium using roller
bottles in the dark.
Preparation of Damaged DNAs
Depurination
of PM2 DNA was carried out at 70 °C in 10 mM sodium
citrate (pH 4.5), 100 mM NaCl for 10 min so as to generate 1.5
AP sites per DNA duplex. PM2 DNA was UV-irradiated in 10 mM
Tris-HCl (pH 7.5) and 0.02% glycerol at 2.5 J/m/s for the
times indicated with a Westinghouse germicidal lamp (G15T8). As a
comparison, a total dose of 525 J/m
produces approximately
2 sites sensitive to E. coli endonuclease III per PM2 DNA
duplex (Demple and Linn, 1982), whereas 23 J/m
produces
roughly 2 cyclobutane pyrimidine dimers per molecule (Snapka and Linn,
1981).
Endonuclease Assays
A filter-binding
assay with nitrocellulose filters and covalently closed, circular (form
I) PM2 DNA was used to monitor endonuclease activity. Standard reaction
mixtures (100 µl) for UV endonuclease activity contained 40
mM Tris-HCl (pH 8.0), 70 mM KCl, 0.01% Triton X-100,
3 mM EDTA, 10 mM 2-mercaptoethanol, and 20
µM (nucleotide residues) UV-irradiated PM2
[H]DNA. AP endonuclease assays were similar
except that depurinated PM2 [
H]DNA was substrate.
Reactions were incubated for the indicated times at 37 °C and then
terminated by the addition of 100 µl of 0.3 M
K
HPO
(pH 12.4), which partially denatures the
PM2 DNA. To detect total DNA by total denaturation, 100 µl of 0.3
M K
HPO
(pH 13.2) was added. A sample
of the reaction mixture was also spotted directly onto the filter for
determining the total amount of DNA present. After 5 min, the samples
were neutralized with 50 µl of 1 M KH
PO
(pH 4.0), and then 100 µl of 5 M NaCl was added.
This treatment results in the renaturation only of form I DNA. Each
sample was finally diluted to 3 ml with 50 mM Tris-HCl (pH
8.2), 1 M NaCl and filtered through a Schleicher and Schuell
nitrocellulose filter to selectively retain the denatured DNA. The
filters had been previously equilibrated with the same buffer. After
being washed with 5 ml of 50 mM Tris-HCl (pH 8.2), 1
M NaCl and then with 5 ml of 0.3 M NaCl, 0.02
M sodium citrate, the filters were dried and counted by liquid
scintillation.
H]DNA bound to each filter assuming a Poisson
distribution of endonucleolytic sites (Kuhnlein et al., 1976).
1 unit of rpS3 endonuclease activity specifically introduces 1 fmol of
nicks into depurinated or heavily UV-irradiated DNA per min at 37
°C. 1 unit of T4 UV endonuclease produces 1 fmol of alkali-labile
sites in DNA containing cyclobutane pyrimidine dimers as measured in
.
Separation of Three UV Endonucleases from MPC-11
Cells
96 liters of culture (1.1 10
cells) in logarithmic growth were harvested and resuspended in a
final volume of 200 ml of 10 mM Tris-HCl (pH 8.0), 0.7
M KCl, 1 mM EDTA, 5.0% sucrose, and 10 mM
2-mercaptoethanol, and then the suspension was immediately sonicated
five times for 15 s with a Branson sonifier and a large probe. After
adjustment to a final concentration of 0.3 M KCl with 20
mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM
2-mercaptoethanol, the extract was further sonicated four times for 15
s, then gently stirred for 2 h at 0 °C and centrifuged twice at
12,000 rpm for 20 min in a Sorvall GSA rotor at 4 °C.
16
cm), which had been equilibrated with 0.4 M NaCl, 20
mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 mM
2-mercaptoethanol to remove nucleic acids, and then the column was
eluted with the same buffer at a flow rate of 100 ml/h. Flow-through
fractions (400 ml), which had significant UV absorbance at 280 nm, were
pooled and dialyzed against 20 mM Tris-HCl (pH 8.0), 0.1
mM EDTA, and 10 mM 2-mercaptoethanol (buffer A).
Further Purification of UV Endonuclease III from
MPC-11 Cells
96 liters of culture (1.1 10
cells) in logarithmic growth were harvested 8 liters at a time,
washed once with PBS,
frozen as a pellet in liquid
nitrogen, and stored at -70 °C for up to 30 days. The total
collection of cells was thawed and resuspended in a final volume of 200
ml of 10 mM Tris-HCl (pH 8.0), 0.7 M KCl, 1
mM EDTA, 5.0% sucrose, and 10 mM 2-mercaptoethanol,
and then the suspension was immediately sonicated five times for 15 s
with a Branson sonifier and a large probe. After adjustment to a final
concentration of 0.3 M KCl with 20 mM Tris-HCl (pH
8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol (buffer B),
the extract was further sonicated four times for 15 s, then gently
stirred for 2 h at 0 °C and centrifuged twice at 12,000 rpm for 20
min with a Sorvall GSA rotor at 4 °C.
16 cm), which had been
equilibrated with 0.4 M NaCl, 20 mM Tris-HCl (pH
8.0), 0.1 mM EDTA, 10 mM 2-mercaptoethanol to remove
nucleic acids, and then the column was eluted with the same buffer at a
flow rate of 100 ml/h. Flow-through fractions, which had significant UV
absorbance at 280 nm, were dialyzed against buffer B and loaded at a
flow rate of 50 ml/h onto a 1.2-liter phosphocellulose column, which
was equilibrated with buffer B. The column was washed with the same
buffer, and fractions that contained UV endonuclease III were collected
(UV endonucleases I and II could be also eluted from this column with a
4-liter linear gradient from 0 to 1.2 M KCl in buffer B (see
Fig. 1
)).
Figure 1:
Phosphocellulose
chromatography of murine UV endonucleases. Preparation and
chromatography of the extract from MPC-11 cells are described under
``Experimental Procedures.'' The characterization of the
activities in peaks I and II was previously described by Kim and Linn
(1989).
The phosphocellulose fraction was loaded onto a
400-ml second DEAE-cellulose column, which had been equilibrated with
buffer B. The column was washed with buffer B and then eluted with a
1.2-liter linear gradient from 5 to 200 mM potassium phosphate
(pH 8.1) in 0.01% Triton X-100, 0.1 mM EDTA, and 10
mM 2-mercaptoethanol. Enzyme eluted at 90 mM
potassium phosphate, and active fractions were pooled.
g at 4 °C. To the supernatant was added 40
g of ammonium sulfate, and the precipitate was collected as above and
dissolved in buffer B to a final volume of 8 ml.
Quantitation of UV Endonuclease III Activity from
Normal and XP-D Human Cells
Log phase normal F65 fibroblast
cells (40 T-150 flasks) or XP-D fibroblast cells (15 T-150 flasks) were
harvested by scraping, rinsed with PBS, resuspended, sonicated, and
centrifuged as described for the purification of the murine enzymes
(Kim and Linn, 1989). The supernatant was collected and loaded onto a
4-ml DEAE-cellulose column equilibrated with 0.4 M NaCl, 40
mM Tris-HCl (pH 8.0), 10 mM 2-mercaptoethanol, 0.1
mM EDTA, and 0.005% Triton X-100 to remove nucleic acids.
Flow-through fractions (39 ml for normal and 15 ml for XP-D cells) from
the DEAE-cellulose column, which had significant UV absorbance at 280
nm, were pooled and dialyzed against 20 mM Tris-HCl (pH 8.0),
0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.005% Triton
X-100 (buffer B) and loaded onto a phosphocellulose column (15 ml for
normal and 8 ml for XP-D cells), which had been equilibrated with
buffer B and then washed with the same buffer. UV endonuclease III
activity flowed through the column. A steep gradient from 0 to 1.2
M KCl in buffer B was applied to elute UV endonucleases I plus
II for comparison.
10
cells), GM 2485A XP-D lymphoblast
cells (5.6 liters, 3.7
10
cells), or MPC-11 cells
(4.8 liters, 3.7
10
cells) were harvested, rinsed
with PBS, and then resuspended in 5 volumes of TE sucrose buffer (10
mM Tris (pH 7.5), 1.0 mM EDTA, and 0.25 M
sucrose). The cells were lysed by homogenization in a Waring blender
three times for 10 s at maximum power, and then the blender was rinsed
with an additional 5 ml of TE sucrose buffer. Nuclei and cell debris
were pelleted by centrifugation (Anderson and Friedberg, 1980), and
then the supernatant was collected by centrifugation in a Sorvall SS34
rotor at 12,500 rpm for 30 min to remove mitochondria and finally
dialyzed against 40 mM Tris (pH 8.0), 10 mM KCl,
0.05% Triton, and 10 mM 2-mercaptoethanol. Dialysates
containing about 10 mg of protein from normal and XP-D lymphoblast
cells were each loaded onto 5-ml phosphocellulose columns, which were
equilibrated with 40 mM Tris (pH 8.0), 10 mM KCl,
0.05% Triton, and 10 mM 2-mercaptoethanol. Flow-through
fractions were collected, and, as a reference, UV endonucleases I and
II were eluted from the columns with a buffer containing 1.2 M
KCl, 40 mM Tris (8.0), 10 mM KCl, 0.05% Triton, and
10 mM 2-mercaptoethanol.
Incision of PM2 DNA and DNA Synthesis
Reactions
Reactions for incision (100 µl) contained 40
mM Tris-HCl (pH 8.0), 3 mM EDTA, 0.01% Triton X-100,
70 mM KCl, 5 mM 2-mercaptoethanol, 40 µM
depurinated PM2 [H]DNA, and endonucleases as
indicated. Incubation was at 37 °C, and the reactions were stopped
by heating for 5 min at 70 °C. Duplicate aliquots of 10 µl were
removed to determine the extent of endonuclease incision. Reactions for
DNA synthesis (200 µl) contained 70 mM potassium phosphate
(pH 7.5), 9 mM 2-mercaptoethanol, 7 mM
MgCl
, 90 µM each of dATP, dGTP, and dCTP, 85
µCi of [
-
P]dTTP (500 cpm/pmol), 2.6
nmol of the incised PM2 [
H]DNA, and E. coli DNA polymerase I. Incubation was at 37 °C, and aliquots of 50
µl were removed at 10-min intervals, chilled on ice, and then mixed
with 200 µl of 0.1 M sodium pyrophosphate and 50 µl of
5 mg/ml bovine serum albumin prior to precipitation with 0.7 ml of 10%
trichloroacetic acid. The precipitates were filtered onto Whatman GF/C
filters, washed with 50 ml of 1 N HCl, 0.1 M sodium
pyrophosphate and then dried with 5 ml of 95% ethanol. Radioactivity
retained on the filter was measured by double isotope counting using
liquid scintillation with Betamax (West Chem) fluor.
Identification of the Product of Combined Reactions
with Class II AP
Endonucleases
[P,uracil-
H]Poly(dA-dT)
was prepared, and uracil DNA glycosylase was assayed as previously
described (Kim and Linn, 1988). 1 unit of the glycosylase releases 1
pmol of uracil per min from phage PBS2 DNA at 37 °C. AP
endonuclease activity was measured as described above; 1 unit of
activity produces 1 fmol of nicks specifically into AP DNA per min at
37 °C. Reaction mixtures containing 50 µM
[uracil-
H]poly(dA-dT) were incubated for
20 min at 37 °C to produce approximately 1 pmol of AP sites. After
enzyme inactivation for 5 min at 70 °C, the reactions were placed
on ice and brought to 40 mM Tris-HCl (pH 8.0), 70 mM
KCl, 0.01% Triton X-100, and 3 mM EDTA, and AP endonucleases
were added. After the enzyme reaction, the products were spotted onto
Whatman 3MM chromatographic paper (27
45 cm) and
chromatographed as previously described (Kim and Linn, 1988).
Subcloning, Overexpression, and Purification of Rat
Ribosomal Protein S3 from E. coli
NdeI and
BamHI restriction sites were introduced at the beginning and
the end of the rpS3 cDNA, respectively, by polymerase chain reaction
mutagenesis from pGEM2-S3-15 (Chan et al., 1990). The
polymerase chain reaction fragment was subcloned into the T7 RNA
polymerase expression vector, pT7-7 (Tabor and Richardson, 1985)
using the NdeI and BamHI restriction sites to produce
pT7-7S3. Then, pT7-7S3 was introduced into E. coli strain BL21plysS.
= 0.5 and then induced by the presence
of isopropyl-1-thio-
-D-galactopyranoside at a final
concentration of 1 mM for 4-5 h at 37 °C. The
bacteria were pelleted by centrifugation and resuspended in buffer C
(20 mM Tris-HCl (pH 7.5), 10% glycerol, and 1 mM
dithiothreitol) with 0.5 M KCl and protease inhibitors (1
mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1
µg/ml aprotinin), sonicated 6
30 s with a Branson sonicator
and the large probe on ice, and then centrifuged for 20 min at 12,000
g at 4 °C. The supernatant was loaded onto a
DEAE-cellulose column equilibrated with buffer C plus 0.5 M
KCl. The flow-through was dialyzed overnight against buffer C plus 0.1
M KCl and loaded onto an S-Sepharose column. Ribosomal protein
S3 was eluted with a gradient of 0.1-1 M KCl in buffer
C. The fractions containing rpS3 (0.50-0.55 M KCl) were
pooled, concentrated 20-fold using an Amicon apparatus, and loaded onto
a fast protein liquid chromatography-Superose 12 HR 10/30 column
equilibrated with buffer C plus 0.1 M KCl. The column was
eluted with the same buffer. Fractions of 0.3 ml each were collected,
and the activity eluted after 14 ml.
Purification of UV Endonuclease III Activity and
Its Absence or Chromatographic Abnormality in XP Group D Cells
When extracts from cultured murine or human cells (data not
shown) are passed through phosphocellulose, three peaks of activity,
which nick heavily UV-irradiated DNA, are observed (Fig. 1). The
two peaks of activity which bind to phosphocellulose, and UV
endonucleases I and II, were purified and characterized previously (Kim
and Linn, 1989). The flow-through activity, which we called UV
endonuclease III, is the subject of this report. We have now purified
UV endonuclease III by chromatography upon DEAE-cellulose,
phosphocellulose,sephacryl S-200, hydroxyapatite, heparin-agarose, and
S-Sepharose followed by sucrose gradient sedimentation. The final
preparation has a specific activity of roughly 33,000, a value that is
comparable with 38,000 and 29,000 obtained for UV endonucleases I and
II, respectively, from the same cells (Kim and Linn, 1989). Throughout
the purification procedure, the activity was inseparable from the AP
endonuclease activity present in the phosphocellulose flow-through
fraction,(
)
previously designated as AP
endonuclease I (Kuhnlein et al., 1976, 1978).
UV Endonuclease III Substrate Specificity
Characterization of the AP Endonuclease
Activity
To characterize the murine and human AP
endonuclease I activities, depurinated PM2 DNA was incised with either
the human or murine enzyme. Nicks were produced that did not support
DNA synthesis with E. coli DNA polymerase I (Fig. 2).
These results indicate that these enzymes leave a baseless sugar, a
sugar phosphate, or a phosphomonoester at the 3` terminus. To
distinguish among these possible cleavage products at the 3` terminus,
we examined the products formed by the combined action of the AP
endonuclease activity of human or murine UV endonuclease III and the
major class II AP endonuclease from HeLa cells (see Kim and Linn
(1988)). In both cases, the product was an unsaturated sugar residue,
indicating that UV endonuclease III, like UV endonucleases I and II,
mediates phosphodiester bond cleavage via a class I -elimination
process ().
Figure 2:
Mechanism of action of AP endonuclease
activities of human and murine UV endonuclease III. Reactions at 37
°C as described under ``Experimental Procedures''
utilized 5 units of E. coli endonuclease III for 15 min, 5
units of AP endonuclease II for 15 min, 1 unit of human UV endonuclease
III for 40 min, and 0.1 unit of DNA polymerase I (panelA) or 4 units of murine UV endonuclease I (a class I
-lyase enzyme) for 40 min, 5 units of AP endonuclease II for 40
min, 4 units of murine UV endonuclease III for 40 min, and 0.18 unit of
DNA polymerase I (panelB). The PM2 DNA was first
incised by the endonucleases and then used as substrate for DNA
synthesis with E. coli DNA polymerase I. The number of
incisions per duplex DNA molecule, which had been made by each
endonuclease, is given in parentheses. The UV endonuclease III used in
these experiments were hydroxyapatite
fractions.
UV Endonuclease III Interacts with Cyclobutane
Pyrimidine Dimers
Since UV endonuclease III activity
appeared to be weak or absent in fractions from XP-D cells and since
XP-D cells are defective in excising cyclobutane pyrimidine dimers
(Galloway et al., 1994), interaction of the enzyme with DNA
containing cyclobutane pyrimidine dimers was explored. To generate
approximately 4 cyclobutane pyrimidine dimers (and roughly 1
pyrimidine-pyrimidone (6-4) photo product) per covalently closed,
circular phage PM2 DNA molecule without generating a significant number
of other base or sugar damages (Demple and Linn, 1982), DNA was
irradiated at a dose of 46 J/m. This DNA was compared as a
substrate to PM2 DNA, which had received a dose of 525 J/m
so as to generate DNA molecules containing not only cyclobutane
pyrimidine dimers but also a significant frequency of hydrated
pyrimidines, AP sites, cross-links, and a number of other damages. When
the more lightly irradiated DNA was treated with UV endonuclease III,
no conversion of form I to form II DNA was detected; however, DNA
receiving 525 J/m
of irradiation was converted to form II
by the enzyme (I). Hence, the enzyme puts observable nicks
into DNA containing damages brought about by heavy irradiation but not
into DNA containing predominantly cyclobutane pyrimidine dimers and
some) photo products as the damage products.
Figure 3:
Proposed sequential reactions of UV
endonuclease III and the cyclobutane pyrimidine dimer glycosylase
activity of T4 UV endonuclease (Ser). If UV endonuclease III were to
cleave a phosphodiester bond within a cyclobutane dimer contained in
form I PM2 DNA, a nick would not be observed as it would be masked by
the pyrimidine-pyrimidine linkage. Breakage of the 5`-pyrimidine
glycosylic bond with phage T4 endonuclease V would expose the nick,
however, and give rise to form II DNA.
PM2
DNA was irradiated with a dose of 23 J/m to yield roughly 2
cyclobutane pyrimidine dimers per molecule, and then the sequential
reactions shown schematically in Fig. 3were carried out
(, Experiment I). The UV endonuclease III alone produced
neither observable nicks nor alkali-labile sites (in particular,
alkali-labile AP sites) in this lightly UV-irradiated DNA. Hence, UV
endonuclease III neither nicks DNA adjacent to pyrimidine dimers nor
does it form AP sites or other alkali-labile sites.
Figure 4:
Nicking of UV-irradiated PM2 DNA by
sequential reactions with UV endonuclease III and T4 UV endonuclease
(Ser) as demonstrated by alkaline sucrose gradient sedimentation.
Reactions were carried out as in Table IV with H-labeled
PM2 DNA irradiated with 23 J/m
where indicated and then
sedimented through 5-25% gradients of sucrose containing 0.25
M NaOH, 0.9 M NaCl, and 5 mM EDTA at 20
°C for 75 min at 43,000 rpm in a Beckman SW50.1 rotor. The
gradients were collected from the tube bottoms and radioactivity
determined. A, sequential enzyme reactions where indicated.
B, T4 enzyme only.
Another technique for monitoring a
phosphodiester cleavage within a cyclobutane pyrimidine dimer would be
to treat with photolyase, an enzyme that reverses the pyrimidine
dimerization to regenerate normal pyrimidine bases on the DNA. When
this scheme was tried, erratic results were obtained. The photolyase
did not act well on the product of the UV endonuclease III (in contrast
to the T4 endonuclease V, which preferred it). This weak
activity of the photolyase may have been due to the presence of the
nick within the dimer in a supercoiled substrate and/or the particular
phosphodiester cleavage (3` or 5`) within the dimer. In particular, the
photolyase appeared to act, albeit weakly, only when the DNA was
relaxed with topoisomerase subsequent to UV endonuclease III treatment.
Attempt to Identify Lesions Recognized in Heavily
Irradiated DNA
Certainly, UV endonuclease III recognizes
baseless sights formed by heavy UV irradiation, but it also appears to
act at some alkali-stable sites in this DNA. We have attempted without
success to characterize these lesions. Both the murine and the human
fibroblast UV endonuclease III act preferentially on supercoiled,
UV-irradiated DNA. When such DNA is relaxed with topoisomerase, the
rate of nicking of the substrate was 22 and 35% optimal for the murine
and human enzymes, respectively. Moreover, we rarely detected more than
two or three nicks per molecule, regardless of the UV dose. This
preference for closed, circular DNAs precludes easy identification of
the lesion(s) recognized by the enzyme without having more specific
substrates available. Using several such substrates, we have shown that
the mammalian preparations have no detectable uracil-,
5-hydroxylmethyluracil, or FAPY DNA glycosylase activity. When very
radioactive [thymidine-H]DNA was
prepared by nick translation and then irradiated, some release of
radioactivity was detected, but HPLC analysis of the label released did
not identify conclusively the several peaks to be any known base
damages, both because of limited material and because oxidized forms of
thymine are not well resolved by HPLC. Given the very limited activity
of this enzyme due to its preference for closed, circular DNA and its
apparent preference for uncommon lesions, baseless sites remain the
only characterized substrate brought about by the heavy irradiation.
Relationship between UV Endonuclease III and
Ribosomal Protein S3
UV Endonuclease III and AP Endonuclease I
Activities Are Associated with Ribosomal Protein S3
(rpS3)
UV endonuclease III from MPC-11 cells was purified
to yield a single visible protein band of M = 32,000 on denaturing gels. During the purification, both
UV and AP endonuclease activities copurified with this band. Three
peptides obtained from this protein by treatment with V8 protease were
purified and sequenced: KRFGFP, KVATRGLCAIAQAESL, and KGGKPEPPAMPQPV.
These have 100% identity with sequences found in rat ribosomal protein
S3 (Chan et al., 1990).
Figure 5:
Depletion with antibodies to ribosomal
protein S3 of the protein associated with the nuclease activity. 20
µl of suspension of protein A-Sepharose CL (Pharmacia Biotech Inc.)
suspended in 5% glycerol, 50 mM KCl, 25 mM Tris-HCl,
pH 8.0, and 0.005% Triton X-100 were mixed with antibody as indicated
and incubated for 90 min at 4 °C. The beads were then washed three
times by centrifugation with 2-fold concentrated suspension buffer, and
finally purified UV endonuclease III from MPC-11 cells was added and
incubated with the beads for 1 h at 4 °C. The beads were then
removed and washed by centrifugation. The initial supernatants
(leftgel) and material bound by the beads (rightgel) were then probed by immunoblotting with antibody
directed against rpS3. The supernatants were also assayed for enzyme
activity (Table V). The protein that had bound to the protein
A-Sepharose was released for the immunoblotting by boiling in the SDS
buffer used for loading sample onto the SDS-polyacrylamide gel
electrophoresis gel. Blots were developed with an alkaline phosphatase
immunoblot kit (Bio-Rad). Tracks are M, unprocessed UV
endonuclease III run on the gel; C, no enzyme added to the
beads; Ab1 is antibody 71/83; Ab2 is antibody 67/67;
CS is a control antibody, which had been directed against rat
ribosomal protein S26.
In the second approach to confirm the association of
the activities with rpS3, the cloned cDNA for rat ribosomal protein S3,
which was isolated by Chan et al.(1990), was sub-cloned into
the phage T7 expression vector, pT7-7 (Tabor and Richardson,
1985) to obtain pT7-7S3 (see ``Experimental
Procedures''). Expression of the protein corresponding to the
cloned cDNA was then induced in E. coli. The induced protein
had the expected apparent molecular weight of 32,000 and was purified
by chromatography upon DEAE-cellulose, S-Sepharose, and fast protein
liquid chromatography Superose 12. The purified peptide cross-reacted
with the antibodies to rpS3 as expected. It also had UV endonuclease
activity (Fig. 6). Preparations of the rat rpS3 expressed in
E. coli consistently have had UV and AP endonuclease
activities in approximately a 1:1 proportion, the same ratio as found
for UV endonuclease III isolated from mammalian cells.
Figure 6:
Overexpression of rat ribosomal protein S3
in E. coli and assay for nuclease activity. Rat ribosomal
protein S3 was overexpressed in E. coli, purified, assayed for
UV endonuclease activity (toppanel), and displayed
on a denaturing gel stained with silver (bottompanel) as described under ``Experimental
Procedures.'' The profile shown is that of the final purification
step, Superose 12 fast protein liquid chromatography. TracksM and L contain protein size
markers.
The
overexpressed rpS3 preparation purified from E. coli produced
nicks on depurinated DNA and heavily irradiated DNA linearly with time,
and these reactions were unaffected by the presence of MgCl or EDTA (data not shown). Only two endonuclease activities that
are able to act on depurinated DNA and UV-irradiated DNA in EDTA are
known to exist in E. coli: Fpg protein (FAPY DNA-glycosylase)
and endonuclease III (Linn and Deutscher 1993). FAPY DNA-glycosylase
activity was assayed for and was not present in the preparation of rat
rpS3 expressed in E. coli. To rule out contamination by E.
coli endonuclease III, immunoblotting of the purified,
overexpressed rpS3 preparation with antibody made against E. coli endonuclease III was performed. No E. coli endonuclease
III was detected when the recombinant rpS3 preparation was probed with
E. coli endonuclease III antibody, although sufficient
endonuclease activity was probed so that if E. coli endonuclease III were responsible for the activity in the
preparation, it would easily have been detected. Since the presence was
ruled out of the only two known E. coli nucleases that could
have been responsible for the nuclease activity in the rat rpS3
preparation obtained from E. coli, we conclude that the
nuclease activities on heavily UV-irradiated DNA and depurinated DNA
are associated with the overexpressed rat ribosomal protein S3 itself
(parenthetically, the presence of AP and UV endonuclease activity in
the overexpressed rpS3 preparation also supports the conclusion that
those same two activities found in the preparations from animal cells
also are associated with the ribosomal protein rather than with a
contaminant from the animal cells).
(
)
) is present in the purified rat rpS3
expressed in E. coli, we conclude that all of the activities
observed for mammalian UV endonuclease III are physically associated
with ribosomal protein S3.
Ribosomal Protein S3 Appears to be Located Both in
the Nucleus and in the Cytoplasm
In situ Confocal
immunolocalization using rpS3 antibodies revealed that rpS3 is present
both in the cytoplasm and the nucleus but not in the nucleolus (data
not shown). These results are in agreement with earlier studies on
ribosome assembly, which showed that rpS3 joins to preribosomal
particles after leaving the nucleolus but before transport to the
cytoplasm (Hadjiolov, 1985). After ribosomes are fully assembled in the
cytoplasm, rpS3 is apparently loosely bound to the 40 S subunit (Lutsch
et al. 1990; Bommer et al., 1991) and exchanges
freely among cytoplasmic ribosomes (Hadjiolov, 1985). The apparent
facility with which rpS3 detaches from the ribosome in vivo is
lost upon purification of ribosomes from cell extracts, however. We
find that with isolated ribosomes, rpS3 can only be released from
ribosomes under denaturing conditions, suggesting that some active
process might operate in vivo to facilitate release of the
protein. All of these data strongly indicate that rpS3 is normally
present in the nucleus.
Figure 7:
Peptide sequences of ribosomal proteins
S3. Sequences were obtained from GenBank.
-lyase mechanism to
produce a 3`-sugar terminus, which is not an efficient primer for
E. coli DNA polymerase I (this paper), whereas AP endonuclease
II (now called APE (Demple et al., 1991) or HAP1 (Robson
et al., 1992)) cleaves on the 5`-side of the AP site to
produce 3`-hydroxyl nucleotide and deoxyribose 5-phosphate termini
(Mosbaugh and Linn, 1980).
-lyase activity
is not a DNA repair event in the classical sense. Instead, an
alternative, novel function for this ubiquitous enzyme activity, such
as forestalling DNA repair, may be worthy of consideration.
-lyase
and the intradimer nicking activities serve to delay or modulate DNA
repair in an overstressed damage situation so as to avoid excessive
manipulation of the genome. These activities could in this way
contribute to regulating DNA repair processes rather than to
participate in them directly.
Thus, while the gene for U15A RNA is within
the rpS3 gene, there is no evidence for a functional interaction
between the respective gene products.
(
)
Table:
Comparison of UV endonuclease activity in
phosphocellulose flow-through fractions from normal and XP-D cells
Table:
Product of UV endonucleases acting upon AP
sites in DNA in concert with a class II AP endonuclease
P,
uracil-
H]poly(dA-dT) with uracil DNA
glycosylase, the glycosylase was inactivated for 5 min at 70 °C.
The DNA was then incubated with murine UV endonuclease III (20 units,
40 min), human UV endonuclease III (4.5 units, 120 min), or HeLa AP
endonuclease II (200 units, 10 min). These enzymes were inactivated for
5 min at 70 °C before a second incubation with enzyme where
indicated. Reaction mixtures were adjusted to 10 mM MgCl
for AP endonuclease II. The specific activity of the
[
P]dUMP residues was 6700 cpm/pmol for
Experiment I and 600 cpm/pmol for Experiment II. The sugar phosphate
products were separated and quantitated as described under
``Experimental Procedures.''
Table:
UV endonuclease III activity on
UV-irradiated DNAs
Table:
Effect of alkali and T4 UV endonuclease (Ser)
on cyclobutane pyrimidine dimers exposed to UV endonuclease III
, 4 units of UV
endonuclease III, and sufficient T4 endonuclease to convert half of the
pyrimidine dimers to alkali-labile sites. 20 µM PM2
DNA-nucleotide in 0.2 ml is equivalent to 212 fmol of DNA molecules,
and 23 J/m
produces roughly two cyclobutane pyrimidine
dimers per molecule. Thus, there were roughly 420 such dimers in the
reaction. After incubation for 15 min at 37 °C (1st incubation),
the reaction mixture was heated at 70 °C for 5 min to inactivate
the first enzyme; then, the reactions were incubated with the second
enzyme (added as indicated) for 10 min at 37 °C (2nd incubation).
Samples treated with alkali were mixed with 0.3 M potassium
phosphate (pH 12.4 with KOH), incubated for 2 h at room temperature,
and then neutralized with 1 M potassium phosphate (pH 4 with
HCl). Nicks were monitored by nitrocellulose filter binding. In
Experiment II, reactions (0.1 ml) contained the same buffer components,
except that dithiothreitol replaced 2-mercaptoethanol. 10 µM PM2 DNA, which had been UV-irradiated with 23 J/m
to
yield roughly 110 cyclobutane pyrimidine dimers per reaction and 0.15
unit of rat ribosomal protein S3 expressed in E. coli were
used. Both incubations were for 20 min with 1.9 units of the T4 enzyme
added in the second incubation where indicated. ND, not done.
Table:
Immunodepletion of endonuclease activity with
antibody directed against ribosomal protein S3
-Larssen, S., Lambertsson, A., Merriam, J., and Jacobs-Lorena, M.(1994) Genetics 137, 513-520
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.