Volume 4 no. 1 January 1977
Nucleic Acids Research
Levels of DNA polymerase -a and 0 in normal and xeroderma pigmentosum fibroblasts Umberto Bertazzoni, Miria Stefanini, Guido Pedrali-Noy, Fiorella Nuzzo and Arturo Falaschi Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Via S. Epifanio, 14-27100 Pavia, Italy
Received 8 October 1976 ABSTRACT We have determined the levels of DNA-polymerases - c and - A in fibroblasts obtained from normal subjects and from patients with Xeroderma Pigmentosum (XP) belonging to three different complementation groups and to the variant form. The assays have been performed in crude extracts and after fractionation on sucrose gradients. The levels of oC and /-polymerases in the different cases of XP were found to lie within the same range as the control values, and no correlation was found with the severity of the symptoms. The sedimentation coefficients of the two polymerases from all the pathological lines were identical to those of the normal fibroblasts.
INTRODUCTION The molecular basis for the deficiency in repair of DNA damage in
Xeroderma Pigmentosum cells (1, 2, 3), has escaped so far a clear description . The evidence from cell hybridization studies shows that up to 5 different complementation groups are involved in this genetic anomaly (4, 5), and that a sixth gene product is probably altered in the so called "variant
form" of the disease (6, 7). The evidence gathered from the studies on cultured cells points to a defect in some early step of the dimer excision process in the classical (as opposed to "variant") cases (2, 3, 8, 9), whereas the variant form is deficient in post-replication repair (10). The simplest hypothesis in the classical form involves a defect in an incision endonuclease; however, the high number of genes involved, the observation that in-
cision of UV damaged DNA may occur at a substantial level in XP cells (9), and the failure to obtain a direct evidence of enzyme deficiency, leave the possibility that the damage may lie at different steps. For the variant form, C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
141
Nucleic Acids Research a
particular DNA polymerase molecule could fit
thus
a
defect in this sort of A number of
enzyme
enzymes
seems
an
the simplest agent and
could explain the alteration.
of DNA metabolism have been assayed in
and normal cells; Brent (11), Bacchetti
(13) have assayed
as
et al. (12) and Duker and Teebor
endonuclease specific for
UV
irradiated DNA, which
present at normal levels in classical XP cell.
Cook et al. (14), in
classical XP fibroblasts, find normal levels of dimer excising activity DNA irradiated with
UV
and incised with
a
tivities, of the overall DNA polymerase activity and of DNA kinase in veral classical XP fibroblasts but have not observed a
on
bacterial UV-endonuclease. Pe-
drini et al. (15) have assayed the levels of DNA ligase, of two DNase
Sutherland et al. (16, 17) have reported
XP
any
ac-
se-
significant defect.
partial defect in photoreactivating
level in several fibroblast lines from patients with the classical
enzyme
and variant forms. Kuhnlein et al. (18) have observed
endonuclease specific for apurinic DNA in The reductions observed not "all
or
none", and
levels of those
no
enzymes
presence
a
are
reduction in
XP line (classical).
difficult to interpret, since they
available
an
on
are
the variability of the activity
in the normal population. Conversely, in the
in which "normal" levels
observed in
data
are
an
a
are
cases
reported (i. e., levels within the variability
number of normal controls) the results could be due to the
of different
enzymes
giving positive
response
with the
assay em-
ployed, but having different functions in vivo. This certainly applies to the DNase
assays,
and especially to the DNA polymerase one, since at least
three distinct molecules exist in human cells all responding to the
basic assay, and defined
as DNA
polymerase- X(,
and A -polymerases account for
activated DNA (20); the
-/3
over 95% of the
o( -enzyme is usually
and
same
(19). The
-
activity assayed with
prevalent, its level is posi-
tively correlated with DNA replication rate (21, 22) and it is the only polymerase
using RNA-primed natural DNA templates (23). The
A5
-enzyme
is usually less abundant ( from 1/5 to 1/10 of the total) and recent observations point to
a role in
repair type processes for this molecule (24).
In consideration of these uncertainties on the molecular basis of the defect, 142
we have
decided to check whether the o( - and
A
-polymerases
Nucleic Acids Research were present in in vitro cultured fibroblasts, and then to measure their
level in cells derived from XP patients either with the classical or the va-
riant form. MATERIALS AND METHODS
Fibroblasts from normal persons and XP patients were obtained from skin biopsies and cultured in vitro as already described (15). Cells were collected by trypsinization, washed with buffered saline and kept frozen at -80°C. The preparation of the cell extracts and the enzyme assays
were performed essentially as previously described for human lymphocytes (24). Homogenization was carried out in 3 volumes of 0. 2 5 M sucrose containing 50 mM Tris-HCl (pH 7. 6), 25 mM KC1,
5 mM MgCl2
with 75 strokes of a Teflon pestle in a glass homogenizer. After checking for cell breakage by microscopic examination, nuclei were disrupted by
the addition of potassium phosphate buffer (pH 7.2) to a final concentration of 0. 25 M; the suspension was then centrifuged for 1 hour at 40, 000 rpm in a Spinco rotor 50 at 2°C. The supernatants were used for crude extract assays and for sedimentation analysis; for this purpose 0. 25 ml aliquots
of the crude extract were dialyzed versus 0. 5 M NaCl, 25 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) and 1 mM 2-mercaptoethanol and layered onto a 5 ml gradient between 5 and 20% sucrose in the same buffer; after centrifugation at 0° - 2°C for 20 hours at 40, 000 rpm in a Spinco SW 50 L
rotor, 0. 2 5 ml fractions were collected by peristaltic pump aspiration from the bottom of tubes. DNA polymerase activities were measured by
the incorporation of H dTTP (specific activity 800-1200 cpm/pmol) into acid-insoluble material using activated DNA as template. One unit of enzyme activity is defined as 1 nmol of dTMP incorporated per hr. The dis-
tinction between the o( - and /3 -polymerase is based on the specific inhibition of o< -polymerase by N-ethylmaleimide (20); for the assay of the /3 -polymerase the extracts were preincubated at 0°C for 30 min with 5 mM N-ethylmaleimide. Protein concentration was measured by the Lowry
method.
143
Nucleic Acids Research RESULTS AND DISCUSSION Table 1 shows the results obtained in three normal and five XP strains. The values of C( and / -polymerases in the normal fibroblasts are of the order of 1 and 0. 2 U/mg respectively. These values are comparable to those observed in stimulated human lymphocytes (24) whereas the
heteroploid human cell lines (HeLa and EUE cultures, our unpublished data), show higher levels, namely between 6 and 9 U/mg for the oC -polymerase and between 0. 5 and 0. 9 U/mg for theft -enzyme. A variability within a factor of three of both enzymes is found for the control cells. This variability is not due to the assay, since its accuracy is within 10-20% in repeated experiments, and is probably related to the natural variability of the normal population more than to the state of the culture. The ratio of oC and /3 activities is about 5 to 1, not significantly
different from the value found in proliferative tissues (20, 24). The levels in the pathological cells are also variable, but they remain within the range
TABLE 1 in extracts of normal and XP Activity of DNA polymerases- oC and fibroblasts. Each value is the average of two determinations.
Cell line
Complementation group
Level of unscheduled DNA synthesis (26)
DNA polymerases (Units / mg Prot)
oc
A5
C XIII (Normal)
100%
1.20
0.29
C XIV (Normal)
100%
0.47
0.11
C XV (Normal)
100%
1.19
0.18
XP 25-RO
A
5%
0.69
0.15
XP4-RO XP5-PV XP 2-RO
C
10-15%
1.85
0.18
C
18%
0.63
0.11
E
40% 88%
0.48
0.08
0. 64
0. 13
XP 30-RO
144
variant
Nucleic Acids Research
4-
5
10
is
b
FRACTIONS
Fig. L Sucrose gradient fractionation of DNA polymerase- oc in normal and XP fibroblasts. For the procedure see Materials and Methods. Direction of sedimentation is from right to left. of the control cell; no correlation is observed with the severity of the disease. The ratio between the two enzymes is the same as in normal lines. We have measured the sedimentation rate of the two polymerases on all the extracts in order to check whether the molecular properties of
the enzymes were anomalous. The data for DNA polymerase- oC are reported in Fig. 1. The position of the main peak (the lower peak corresponds to the activity of the /3 enzyme which responds in part to the oc -assay) is the same in all cases and the quantitative differences observed correspond to those found in the crude extracts. Fig. 2 shows the fractionation of the
A -polymerase; the assay is absolutely specific for this enzyme and only one peak is observed. In no case the sedimentation coefficient seems alte145
Nucleic Acids Research
lii I--
0
a,
F
4
e7
5
15
X0
FRACTIONS
A
Fig. 2. Sucrose gradient fractionation of DNA polymerase - in normal and XP fibroblasts. For the procedure see Materials and Methods. Direction of sedimentation is from right to left. red and the quantitative variations are within the range of the controls. The -polymerases obtained for the Xesedimentation properties for o< and roderma Pigmentosum cells of the E group (not reported in the figures) -
were
also the
same as
A3
the normal cells.
These results lead
us
to conclude, in the first place, that normal
human fibroblasts and fibroblasts from all the cases of XP examined contain the two main DNA polymerases described in human cells. Second, the
levels of both enzymes in the "classical" XP lines are within the normal or range, and thus the repair defect is not due to a reduction in the oC the -enzyme. Third, the defect in post-replication repair observed in cx. XP variant is also not correlated with the alteration in DNA polymerase-
/3
146
Nucleic Acids Research or/S
. We cannot exclude that, whether in the classical or variant
form, the activities of other mammalian DNA polymerases may be affected, as for instance DNA polymerase-
g
(25), or that the alteration in the po-
lymerization step could be shown only when repair of an UV-damaged DNA is being made. We are at present investigating these possibilities.
ACKNOWLEDGMENT This work was partially supported by EURATOM (Contract 125-741-BIOI). U. B. is a Euratom scientific agent, and this publication is Contri-
bution n. 1381 of the Biology Division of the European Communities.
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