[CANCER RESEARCH 34, 3318—3325,December 1974]
Two Forms of Repair in the DNA of Human Cells Damaged
by Chemical Carcinogens and Mutagens'
JamesD. Reganand R. B. Setlow2
Carcinogenesis Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
sequence of repair events in human cells is believed to be
similar to that well established for such microorganisms as
DNA repair in human cells, as assayed by the photolysis of Escherichia coli (23, 26). Initial strand breakage for the repair
5-bromodeoxyuridine incorporated during repair, takes one of of these lesions is made by a specific endonuclease. An
two limiting forms, depending on the nature of the original exonuclease then causes a rather extensive excision of
insult to the DNA. Damage from ionizing radiation is repaired nucleotides (including the dimers) from that portion of the
by the insertion of three or four nucleotides during a brief DNA (16, 22), the resulting gap is filled with undamaged
period (‘@‘.‘60
mm) after the insult, but in the case of ultraviolet
nucleotides by polymerase action, and a ligase completes the
radiation there is extensive excision of bases (@‘l00)during a repair (26). This UV or “long
patch―type of repair continues
protracted period (1 8 to 20 hr). Similarly, chemicals that for at least 20 hr after radiation (21).
To measure DNA repair we use an assay (Chart 2) that
damage DNA fall into two categories, those that result in the
ionizing or “short―
type of DNA repair (ethyl methanesulfo
involves the photolysis of dBrUrd3 incorporated into labeled
nate, methyl methanesulfonate,
propane sultone) and those parental DNA during repair (20, 21). Briefly, cells whose DNA
that result in the ultraviolet or “long―
type of repair has been labeled with dThd-3 H are exposed to radiation or
(N-acetoxy-2-acetylaminofluorene,
2-methoxy-6-chloro-9-[3chemical insult and then immediately placed in medium
(ethyl-2-chloroethyl)aminopropylamino]
acridine dihydrochlo
containing i04
M dBrUrd, an analog to dThd. As repair
ride). In addition, some chemicals (4-nitroquinoline
1- proceeds, dBrUrd is inserted in place of excised dThd residues.
oxide) cause damage that apparently is repaired by both The cells are then exposed to 3 13 nm of radiation, which
mechanisms. When agents that induce long repair in normal causes the dBrUrd-containing regions to become sensitive to
cells are used to treat cells from patients with the genetic alkali. When the cells are lysed and their DNA is sedimented in
disease xeroderma pigmentosum (characterized by extreme alkali, breaks appear at the sites at which dBrUrd has been
sensitivity to ultraviolet radiation), defective repair is seen. inserted, providing an estimate of the number of repaired
Thus it is possible that long repair involves the action of an regions and their size. This assay is rapid and sensitive (1 repair
endonuclease (thought to be lacking in xeroderma cells) on a event in 108 daltons of DNA can be detected), and since it
distortion or intercalation in the DNA. Short repair may indicates both the average number and size of the repaired
involve the simple excision and replacement of a few bases at regions it can easily distinguish between the long (UV) and
short (ionizing) types of repair. Thus it is better suited for our
the site of a single-strand break.
experiments than are other methods currently used for
measuring DNA repair (2, 6, 9, 17).
INTRODUCTION
We have previously reported briefly the differential response
of XP cells versus normal cells to N-acetoxy-AAF as examined
When the DNA of human cells is damaged by ionizing or
by dBrUrd photolysis (24). Stich et al. (27—31) and Cleaver
UV radiation (for review, see Ref. 26), a specific pattern of
(5)
havereportedon the reducedrepairseenin XPcells,
repair (18) is initiated, the nature and time course of which
compared with normal cells, after treatment with a variety of
depend on the kind of insult (ionizing or UV) and, hence, it
carcinogens. Cleaver (5) also found normal repair in XP cells
would seem, on the nature of the lesions in the DNA (Chart
with 2 agents we classify as short-repair inducing agents in
1). Ionizing radiation, either directly or indirectly, causes
normal cells, i.e., ionizing radiation and MMS.
many single-strand breaks in DNA, which are quickly closed
We have examined the repair of damage caused by 6
by DNA polymerase and ligase, with the incorporation of only
chemical carcinogens and mutagens: EMS, MMS, propane
a few nucleotides (17). This ionizing or “short
patch―type of
sultone, N-acetoxy-AAF, ICR-170 (7), and 4-NQO. On the
repair is completed 30 to 60 mm after the original insult (1 1,
basis of our results, these chemicals fall into 2 categories, 1
13, 16). In the case of UV radiation, however, dimers are
resulting in short repair and the 2nd in long repag. Moreover,
formed between adjacent pyrimidine residues, and the
SUMMARY
1 Research
sponsored
jointly
by
National
Cancer
Institute
Grant
Y01-CP-40013 and by the United States Atomic Energy Commission
under contract with the Union Carbide Corporation.
2 Present
address:
Biology
Department,
tory, Upton, N. Y. 11973.
Received November 12, l973;accepted
3318
Brookhaven
National
Labora
3The abbreviations used are: dBrUrd, bromodeoxyuridine; dThd,
deoxythymidine; XP, xeroderma pigmentosum; N-acetoxy-AAF, Nacetoxy-2-acetylaminofluorene; MMS, methyl methanesulfonate; EMS,
ethyl methanesulfonate; ICR-i 70, 2-methoxy.6-chloro-9-[3-(ethyl-2-
chloroethyl)aminopropylaminoj
August 28, 1974.
acridine dihydrochloride; 4-NQO,
4-nitroquinoline 1-oxide; BrUra, bromouradil.
CANCER RESEARCH VOL. 34
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Two Forms oJDNA Repair
cells from patients with the hereditary disease xeroderma
pigmentosum, characterized by extreme sensitivity to UV
radiation, are deficient not only in the repair of UV-induced
pyriinidine dimers but also in the repair of lesions caused by
chemical carcinogens that in normal human cells result in long
repair patches.
MATERIALS AND METHODS
@
@
@
Cell Culture. The normal human cells used were diploid
fibroblasts derived from fetal skin. The XP cells were also
diploid fibroblasts, derived from skin biopsies of patients, as
previously described (25). In certain experiments, an aneu
ploid tumor cell line was used that was derived from an
amelanotic melanoma of a xeroderma patient (J. D. Regan, M.
J. Snodgrass, R. B. Setlow, and E. Klein, manuscript in
LONG
(UV-TYPE)
REPAIR
SHORT
(IONIZING-TYPE)
REPAIR
DNA WITH
UV-INDUCED LESION
I
@
1
UV-ENDONUCLEASE NICKS
DNA NEAR LESION
DNA WITH
y-RAY INDUCED BREAK
EXONUCLEASE
EXCISES
LESION AND MANY BASES
EXONUCLEASE EXCISES
A FEW BASES
@
POLYMERASE INSERTS
80 —
100 NEW BASES
preparation).
Cells were routinely cultured by standard
procedures in Eagle's medium (8) with 15% fetal calf serum.
For experiments, cells were planted in 50-mm plastic Petri
dishes (Permanox; Lux Corp., Thousand Oaks, Calif.) at 1 to 4
x l0'@cells/dish.
Labeling. The DNA of the experimental cultures was labeled
by incubation of the cells for 16 to 18 hr in medium
containing dThd-3 H (1 .9 Ci/mmole, Schwarz BioResearch,
Orangeburg, N. Y.) at 1 to 5 j.tCi/ml plus 10% calf serum or in
medium containing
2P04 (Schwarz BioResearch) plus 3%
calf serum.
Delivery of the Insult and Analysis of DNA. Chart 3 is a
diagrammatic representation of our experimental protocol.
After the labeling period, the radioactive medium was replaced
with growth medium containing 10% fetal calf serum. After
about 2 hr, hydroxyurea at 2 X 10 M and unlabeled dBrUrd
or dThd at 10
M were added to the H- and 2P-labeled
cultures, respectively. The chemical or physical insult was then
delivered, and the cells were incubated for several hr to
overnight. The monolayer of cells was washed free of any
floating cells with an EDTA solution (25). Only attached cells
were assayed. The labeled cells were harvested, mixed together
at 2 X i0@ cells/ml in the EDTA solution, and exposed to 313
nm of light from a large quartz-prism Hilger monocromator.
Ten thousand cells were lysed on top of, and their DNA was
spun through, an alkaline sucrose gradient (5 to 20% sucrose, 2
M NaCl at 30,000 rpm for 180 min in 4-ml tubes in a SW 56
rotor of a Beckman Model L centrifuge. Drops were collected
from a hole punched in the bottom of each tube onto strips of
filter paper, and the acid-insoluble radioactivity in H and 2p
was measured in a toluene-based scintillator in a Packard
scintillation counter. The distributions of radioactivity were
converted to weight-average molecular weights by a computer
program based on the distances sedimented by phage DNA's of
known molecular weights: T4 DNA, 55 X 106; DNA, 15 X
POLYMERASE INSERTS
A FEW BASES
Label
Insult
Incubation
T
dlhd-3H
C dBrUrd —‘-—@ 313 nm
Irradiation
32
@
Insult
Calculate
values of
l/M@
Compute A (1/Ma) : (I/MW)3H
LABELED
DNA
PYRIMIDINE
DIMER
@
/
uv IRRADIATE
REPAIR
(l/M@)32p
Chart 3. Protocol for dBrUrd photolysis assay for DNA repair.
DIMERS EXCISED
dBrUrd
RESIDUES INSERTED
V
INNONRADIOACTIVE
MEDIUM CONTAINING
dBrUrd
Count
x dlhd
LIGASE CLOSES STRAND
Chart 1. Diagram of the 2 classes of DNA repair in human cells.
Sediment
in alkali
Unlabeled
P04
LIGASE CLOSES STRAND
Mix
Unlabeled
IRRADIATED WITH
313nm
LYSE CELLS
IN ALKALI
SEDIMENT
>1
IN
ALKALI
I'
Chart 2. Principleof the dBrUrd assay for DNArepair.
DECEMBER 1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
3319
J.D. Regan andR. B.Setlow
106 ; and aXl 74 DNA, 1.7 X 106 (10). Details of the method
have been described previously (20, 21).
Ionizing radiation was delivered from a 60 Co source at
about 3500 rad/min. UV radiation was delivered from a
germicidal lamp at a fluence rate of “-7.4
ergs/sq mm sec. Data
on concentrations and treatment times for chemical insults are
given in the figure legends.
The method of analysis of data we use, to be described
more fully elsewhere (R. B. Setlow and J. D. Regan, in
preparation), assumes that per i0@ daltons there are N
repaired regions each of which contain n BrUra residues. From
data (not shown) on fully substituted DNA we know that the
cross-section, a, for strand breaks by 3 13-nm radiation is 0.76
x l0@
sq mm/erg.
Hence a fluence Fmakes
an average of aF
breaks/residue (for example; if F
106 ergs/sq mm, oF
0.076), and the probability of no breaks in a repaired region is
e'―'@'@
Thus 1 —e naF is the probability of 313-nm radiation
making a break in a repaired region. Since one or more breaks
in a given repaired region change the sedimentation pattern of
the repaired DNA by the same amount, very large fluences
have little additional effect on the sedimentation pattern and
the number of breaks, B, approach a plateau at the value N.
The number of breaks per 108 daltons, B, is given by
B=N(1
(A)
_e@0F').
if n is sufficiently large so that naF > 0.5 the measurement of
B as a function of F gives enough data to evaluate both N and
n. If n is small, e―@
1 —naP', and only the product Nn
may be found from initial slopes of the B versus F curve
dB/dF = Nna.
(B)
We compute the weight-average molecular weights, M@, of
cells incubated in dThd and in dBrUrd for different fluences
and calculate
fl\
@
@
@‘\
M@ )
1
1
dBrUrd
M
dThd
(C)
and takeB
2i@(1/M@).
If we could get to high enough fluences, we could break
every repaired region, but for practical reasons we have limited
the 3 13-nm radiation to <10 mm which, for a fluence rate of
1.5 X l0@ ergs/(sq mm mm), means F
1.5 X 106 ergs/sq
mm. Hence for n > 5 , the graph of B versus F will show
curvature and permit the evaluation of n whereas, for n < 5 , B
versus F will be a straight line of slope Nn and an estimate of
probably because of small differences in growth characteristics
from experiment to experiment and because of errors in the
physical and, especially, chemical dosimetry. Hence, for
example, i@(l/M@) is a curvilinear function of F for normal
cells treated with UV (Chart Sb ; Ref. 19) and with
N-acetoxy-AAF (Chart 8). A curve also fits the ICR-l70 data
(Chart 7b) better than does a straight line, but the difference
between the 2 functions, at the relatively small values of
@(l/M@) observed, is close to the resolution of our
techniques.
RESULTS
Ionizing Radiation. When normal human diploid cells were
exposed to 10 krad of y-radiation and then assayed for DNA
repair as described above, the alkaline sucrose gradient profiles
(Chart 4@i)of the DNA from cells incubated in dBrUrd did not
differ greatly from those for the controls incubated in dThd.
Incubation for longer than 60 mm did not increase the
sensitivity of the DNA to 3 13-nm radiation, hence repair was
essentially complete at 60 mm. The 3 13-nm radiation caused
only a small relative decrease in the molecular weight of the
1st group, suggesting that only a few dBrUrd residues were
incorporated during the repair process. The number of breaks
in the DNA can be estimated from the observed value of
@(l/M@)by use of Equation C. Values of @(l/M@)for the
DNA from v-irradiated cells are given in Chart 4b. The
differences between the dBrUrd and control groups (“-0.1
break in 108 daltons) are close to the level of the background
noise of the experiment and would not have been measurable
without the double-label technique.
For 10 krads we observe 5 single-strand breaks per l0@
daltons immediately after radiation, Le., N 5 in Equation B.
A fluence of 106 ergs/sq mm gives @@(l/M@)0.15, so that
Equation B reads B 0.3 Sn(0.76 X l07)106 when n 0.8
BrUra residues are inserted per 7-ray-induced single-strand
break. If nucleotides were chosen randomly during strand
break repair, we would expect an average of 2 .7 nucleotides
inserted since BrUra makes up 0.3 of the bases. Thus we
estimate that about 3 nucleotides are inserted per average
single-strand break in the DNA of these cells after 10 krad
7-radiation. This figure agrees well with the data of Painter and
Young (1 7), who used repair replication methods to arrive at
an estimate of about 3 nucleotides inserted in each repaired
region of HeLa cells after 5 krad of'y-radiation.
Uv Radiation.Whennormalcellsweregiven200ergs/sq
mm of 254-nm radiation rather than 10 krad of 7-rays, the
time to complete repair was 20 hr, and 3 13-nm irradiation
at 20 hr caused a marked decrease in the molecular weight of
the DNA of the cells incubated in dBrUrd after the UV insult
(Chart 5a). Qualitatively similar results (not shown) were
obtained at earlier times. These results suggest that the dBrUrd
target for the 3 13-nm radiation was considerably larger than in
the case of ionizing radiation damage. Chart Sb shows that the
changes in l/M@ are much larger after UV than after ionizing
radiation, and there is a clear indication that z@(l/M@)
approaches a limit at high fluences of 3 13 nm (see also Ref.
19). Thus, the probability that a repaired region will be
N isneeded
toestimate
n.Theformer
procedure
isfollowed
for UV damage, the latter for 7-ray damage for which N is
taken as the number of single-strand breaks for 108 daltons.
Since the 2 treated cell samples in Chart 3 were mixed
together before they were irradiated with 3 13 nm, lysed,
centrifuged, collected, and counted, systematic errors cancel
out. In any one determination of @(1/M@),the precision is
±0.05
X lO_8 for small z@(1/M@) and ±5% for large
@(1/M@)
(R. B. Setlow and J. D. Regan, in preparation); the differences
between different experiments are usually twice these values
3320
CANCER RESEARCH VOL. 34
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@
0@
@
‘
\
Two Forms ofDNA
@
ib8
I
ib@ ib6
313nm
/Oergs/mm
>-1O
6x1O5
51\\I
4
Chart 4. a, Alkaline sucrose gradients of DNA
from normal human cells radiated with 10 krad
6 °Co
7-rays
and
nonradioactive
then
incubated
for
60
mm
in
dBrUrd or dThd; dBrUrd-incubated
cells were previously labeled with dThd-3; dThd-in
@
313nm
ii
I \/\
•@_\\@
I
I
I
‘
\
x@
3 11
3@3nm
I
‘\,-‘
I'
>
@
ergs/mm2
i@6
+
12 xlO5ergs/mm2
—
i'@@
io@
doltons
Repair
‘@
I,!
II
0
(3
<4
cubated cells were previously labeled with 2P; left,
0 or 6 X 10' ergs/sq mm 313-nm radiation;right, 12
x i0@ergs/sq
mm313-nmradiation
before
sedi
mentation in alkaline sucrose; b, differences between
a
the reciprocals of the weight-average molecular
weights of DNA from cells incubated in dBrUrd and
OiA
06
@r;@ 02
DISThNCE
in dThd [i@(l/M@)] during short (ionizing type)
repair as a function of increasing fluence of 3 13-nm
radiation. 0, S 0, data from different experiments.
Incubation for times longer than 60 min after
d8
SEDIMENTED
x108
0.15@
d6
04
02
0
radiation did not increase the sensitivity of the DNA
to 313-nm radiation.
@+Q.O5‘1-O.O5@
o.15Q
b
@
@
‘@
110
313 nm FLUENCE
1'2
1:4
(ergs/mm2)
b
>-
I-
6- xlO@
>
IC.-)
5.
0
Lx
4-
0
w
0
3.
2
w
C.-)
/0
a:
w
2-
0@
1-
@
0.8
0.6
04
02
08
DISTANCE SEDIMENTED
0.6
04
0.2
/
4
I'OI
l@4
x1O@
313nm FLUENCE Iergs/mm2)
Chart 5. a, Alkaline sucrose gradients of DNA from normal human cells radiated with 200 ergs/sq mm 254-nm UV radiation and then incubated
for 20 hr in nonradioactive dBrUrd or dThd; dBrUrd-incubated cells were previouslylabeled with dThd-3H; dThd-incubated cellswere previously
labeled with 32p; left, no 31 3-nm radiation; right, 3 x 10' ergs/sq mm 31 3-nm radiation before sedimentation in alkaline sucrose. b,
during long (UV-type) repair as a function of increasing fluence of 31 3-nm radiation.
@(1/M@)
DECEMBER 1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
3321
J. D. Regan and R. B. Setlow
photolysed apparently approaches unity, indicating that many
dBrUrd residues are incorporated per repaired region. Our
calculations from these and other experiments (20, 21)
indicate that “‘-25
residues (—-100bases) are incorporated into
the typical repaired region after UV damage.
Chemical Carcinogens and Mutagens. When DNA repair
after chemical insult was examined in the assay system
described above, 3 agents, i.e., EMS, MMS, and propane
sultone, were found to induce short (ionizing) repair. For
EMS (Chart 6), as for the other two,
@(1/M@)was
proportional
to the 3 13-nm fluence, indicating that the
repaired regions were small. The change in 1/M@ after a 2-hr
treatment with 10 2 M EMS was “‘3
times that observed after
10 krad of 7-rays. Comparable results were found with MMS
and propane sultone. The change in 1/Me after a 1-hr
treatment with 5 X l0@ M MMS was equivalent to that after
a 7-ray dose of ““40
krad, and the change after treatment with
2 X l0@ propane sultone for 2 hr was equivalent to that after
a 7-ray dose of 10 krad.
In contrast, 2 carcinogens, ICR-l 70 and N-acetoxy-AAF,
resulted in long (UV) repair events. The alkaline sucrose
gradient profiles (Chart 7a) and 3 13-nm sensitivity data (Chart
7b) for DNA
from normal
human
cells after treatment
Since both N-acetoxy-AAF and ICR-l 70 induced long
repair in normal human cells, we investigated the ability of
xeroderma cells to repair lesions induced by these carcinogens.
Patients with xeroderma pigmentosum are highly sensitive to
Uv radiationand frequentlydevelopactiniccarcinoma
(3).
Cells from most patients with this desease are incapable or are
only minimally capable of repairing UV-induced damage to
their DNA, although exceptions have recently been reported
(1 , 3, 25). The ability of such cells to repair the DNA damage
induced by N-acetoxy-AAF or ICR-170 was much lower than
in normal cells (Chart 9), and dBrUrd incorporation during the
repair assay was greatly reduced. (A brief report of the data
for xeroderma cells treated with N-acetoxy-AAF appeared
earlier.) These results indicate that xeroderma cells are
with
10 -6 M ICR-l70 for 1 hr suggest a pattern of repair similar to
that seen after UV irradiation (Chart 4, a and b), with “‘-10
dBrUrd substitutions in each repaired region. This value is
close to the resolution limit of our techniques (see “Materials
and Methods―). ICR-l70 at l06 M for 1 hr is equivalent to
-“‘25ergs/sq
mm
of
254-nm
radiation.
Comparison
of
@(l/M@) after UV radiation and after treatment with
N-acetoxy-AAF
(Chart 8) indicates that 7 X 10 6 M
N-acetoxy-AAF for 1 hr is equivalent to “-‘50
ergs/sq mm of
254-nm radiation.
0
4
8
12x1O5
313 nm FLUENCE (ergs/mm2)
Chart 6.
@(1/M@)as a function of increasing fluence of 313-nm
radiation in normal human cellsafter treatment with 10 2 M EMSfor 2
hr and subsequent incubation in dBrUrd or dThd for 18 hr. Note the
similarity to the graph for ionizing-type repair (Chart 4b); o, ., data
from different experiments.
I—
>
0
0
0
a:
Lx
0
LU
0
I2
LU
0
a:
LU
0@
DISTANCE SEDIMENTED
313nm FLUENCE (ergs/mm2)
Chart 7. a, Alkaline sucrose gradients of DNA from normal human cells treated with 10 6 M ICR-i 70 and then incubated for 18 hr in
nonradioactive dBrUrd or dThd ; dBrUrd-incubated cells were previously labeled with dThd-3 H; dThd-incubated cells were previously labeled with
3 2 P;
3 1 3-nm
radiation
(right)
induced
a
marked
shift
in
the
molecular
weight
of
the
DNA.
b,
@(1/M@)
as
a
function
of
increasing
fluence
of
3 13-nm radiation after ICR-i 70 treatment and repair. Note the similarity to UV-type repair (Chart 5b); o, ., normal cells in the different
experiments.
3322
CANCER RESEARCH VOL. 34
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Two For,ns ofDNA Repair
cells than of normal cells. Since 18 hr after treatment the
number of breaks induced by 3 13-nm radiation in normal cells
was close to that in untreated controls, there seems to have
,‘7x105M
been little or no 4-NQO left in their DNA, as opposed to the
I
appreciable amount remaining in xeroderma cells. In the
3
I
absence of 3 13-nm radiation, DNA from xeroderma cells
showed more breaks than normal DNA; these breaks disap
a
I
•
7x1O6M
peared with incubation.
When DNA strand breaks in 4-NQO-treated cells incubated
.11
in dBrUrd for 90 ruin were compared to those in dThd-incu
bated cells (Chart 11), both normal and xeroderma cells
showed some increase in sensitivity to 3 13-nm radiation after
the dBrUrd incubation, suggesting that repair was almost
complete by the end of the 90-mm treatment. (In these data
the photodynamic effect of the 313-nm radiation on 4-NQO
bound to the DNA is approximately subtracted out.) The
Tx,O5
difference between the 2 cell types is not striking. The effect
313 nm FLUENCE (ergs/mm2)
of 4-NQO on the DNA of both normal and xeroderma cells is
Chart 8. @(1/M@)
as a function of increasing fluence of 313-nm
radiation after treatment with the indicated doses of N-acetoxy-AAF complex and may represent damage and repair corresponding
to both of the classes we have described here.
and incubation for 18 hr and repair in normal human cells. The shape
NORMAL FIBROBLASTS
4.
@
x i08
N—ACETOXY-AAF
of the curve is typical of long (UV-type) repair (Chart Sb); o, •,
normal
cells in the different experiments.
xP CELLS
0.80 N-
040j!
DISCUSSION
A summary of the results of our experiments and a
classification of the chemical agents tested are presented in
ACETOXY
AAF
O@
5-
@“‘@@0
K 108
@
II
I
00
0
ICR -170
@
0
4
313nm
é
FLUENCE
(ergs/mm2)
2-
Chart 9. Repair in xeroderma cells It@(lIM@)l as a function of
increasing fluence of 313-nm radiation] after treatment with N-ace
/
//
//
/
NORMAL
CELLS
NO
1—
toxy-AAF or ICR-hO. The changes in 1/Me are ‘—10-fold
smallerthan
in normal cells. o, •,
XP fibroblasts; a, amelanotic melanoma from an
xP patient.
0
@
_O
defective in the repair of chemical lesions of this class, as in
the repair of UV-induced lesions.
Repair synthesis in human cells after treatment with the
carcinogen 4-NQO has been investigated rather extensively
(27—29) and has recently been shown to be reduced in
xeroderma cells compared to normal cells (28, 30). 4-NQO
binds to DNA (12, 14) and upon illumination causes the
photodynamic photolysis of unsubstituted DNA (1 5), provid
ing an index of the amount of carcinogen bound to the DNA
at various times after treatment. Chart lOa shows results for
cells incubated in dThd after 4-NQO treatment. Three points
are apparent: (a) there were more alkali-labile bonds or breaks
in the DNA of xeroderma than normal cells immediately and
18 hr after treatment; (b) 3 13-nm illumination made more
breaks in xeroderma DNA than in normal DNA; and (c) after
18 hr of incubation of XP cells, 313-nm radiation is able to
make 85% of the breaks it makes at time zero. The
corresponding percentage for normal cells is 53. Thus it
appears that more 4-NQO is bound to the DNA of xeroderma
313nm
7
3S
12
313nm
7
4-
0
NO
I@ 7.5x1O5ergs/mm2@
XP CELLS
I“—
:@;0.20-
@
I@
18
TIME AFTER
TREATMENT
18
(hr)
Chart 10. l/M@ for xeroderma cells and normal human cells after
treatment with 5 X 10 -@ M 4-NQO for 1.5 hr. Cellular DNA was
analyzed immediately after treatment (0 time) and 18 hr after
treatment. Cells were incubated in dThd only; no dBrUrd was used.
3 x 108N0313nmXPCELLS
313nmNORMAL
@@71
/
vi
@75xlO5ergs/mm2
CELLS
1,
0
0
18
TIME AFTER
0
TREATMENT
18
(hr)
Chart 11. @(1/M@)
for xeroderma cells and normal human cells after
treatment for I .5 hr with 5 X 10
M 4-NQO determined by the
dBrUrd repair assay.
DECEMBER 1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
3323
J. D. Regan and R. B. Setlow
Table 1. The 2 classes of DNA repair observed can be
conveniently described as short and long repair. In short
repair, the number of nucleotides involved in repairing the
lesion is small, and the repair process is completed relatively
quickly
(“—60
mm). In long repair, there is an extensive
run of
nucleotide excision and replacement, and repair continues for
a long time (“‘-20
hr). The model insult that results in typical
short repair is ionizing radiation, and long repair is typified by
the process that follows damage by UV radiation.
Three of the chemical agents tested in our assay system, the
mutagens EMS and MMS and the carcinogen propane sultone,
resulted in short repair. We suggest that these agents, either
directly or indirectly, cause single-strand breaks in DNA which
are then filled in by a small number of nucleotides.
N-acetoxy-AAF and ICR-l 70 both induced long repair in
normal cells, and the experimental evidence indicates that the
initial DNA lesion induced resulted in extensive base excision
and replacement. The amount of base excision differed with
the insult; 200 ergs/sq mm of 254-nm UV radiation resulted in
the excision and replacement of 100 nucleotides per average
repaired region; N-acetoxy-AAF treatment, “‘-140
nucleotides;
and ICR-l70 treatment, ‘—‘-40
nucleotides. These differences
are important and indicative of some mechanism that controls
the extent of base excision during long repair; but the major
distinction between short and long repair is that the former
involves excision of only a few nucleotides, whereas the latter
involves many (40 to 140) nucleotides per average repaired
region.
At present we interpret this dichotomy as having the
following molecular basis. Agents that induce short repair
cause
a single-strand
break
in the
DNA.
The
break
around UV-induced dimers). Thus they present a DNA lesion
that is recognizable as the substrate for the UV-endonuclease
that initiates the repair of UV damage. A single-strand break is
induced by this endonuclease, and extensive base excision
follows. In the case of UV-induced dimers, it is known that in
E. coli they are excised as parts of small oligonucleotides;
hence, excision initially makes a large gap that quickly gets
larger before polymerase and ligase close it. It is conceivable
that extensive exonuclease action can occur only where there
is a region of altered base stacking or denaturation of the
DNA. Long repair occurs over an extended time period ; this
fact may suggest that the number of UV-endonuclease
molecules per cell is the rate-limiting step in the process.
If, as present interpretations
suggest (3—5, 25) the
molecular basis for xeroderma pigmentosum is in fact the lack
of a functional UV-endonuclease, then xeroderma cells should
exhibit defective repair after damage by chemical agents that
induce long repair in normal cells. Our results are consistent
with this expectation.
ACKNOWLEDGMENTS
We are grateful to F. M. Faulcon and W. H. Lee for superb technical
assistance, to Dr. James Miller, University of Wisconsin, for the gift of
N-acetoxy-AAF, and to Dr. Hugh Creech, Institute of Cancer Research,
for the gift of ICR-I 70.
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and
subsequent exonuclease action may briefly involve excision of
a few bases, but these are quickly replaced by DNA
polymerase, and the strand is closed by polynucleotide ligase.
These agents cause no local denaturation or distortion of base
stacking. Agents that induce long repair do not initially cause
single-strand breaks but do intercalate in the DNA or
otherwise cause regions of local distortion (such as those
Xeroderma Pigmentosum and DNA Repair. Lancet, 1: 601, 1971.
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ClassificationofDNA-damaging treatments accordingto the type ofrepair induced
ofstrand
No.
breaks!108
daltonsafter
dBrUrdincubation
anddBrUrd106
mmnucleotidesTypeDose
ergs/sq
orof
313-nminserted!ofTreatmentconcentrationDurationradiation.lesionrepairUV
(254 nm)
ergs/sq mm
60Co
‘y-rays200
10 krad10
Short1'N-acetoxy-AAF7
mm4—‘40Long―4-NQO5X 10 6 M60
x 10 M90
short―EMS10
-2 M1
.0ShortMMS5
mm“-0.4ShortPropane
X 10 MS
hr‘—0.4ShortICR-l70106
X 10 @‘
M2
sultone2
@
@
0.625
mm‘—2Long
in normal
b Repair
equal
C See text
3324
for
cells
in normal
1 0-fold
greater
and xeroderma
and
20 mm‘—1
hr‘-‘1--10Long―
M1
a Repair
1Long°
than
in xeroderma
cells.
cells.
details.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
3325
Two Forms of Repair in the DNA of Human Cells Damaged by
Chemical Carcinogens and Mutagens
James D. Regan and R. B. Setlow
Cancer Res 1974;34:3318-3325.
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