One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a singlestrand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

In the past 20 years or so, DNA repair has developed from a vague, almost unacceptable, concept into a series of well-studied phenomena about which a tremendous amount of information has accumulated. It is probably only natural, then, that many investigators have attributed the whole of the cell’s ability to withstand DNA damage to DNA repair alone. And yet, if the study of science has taught us anything, it is that simple concepts are often flawed just because they are too simple.

Attempts to correlate extent of DNA repair in mammalian cells with survival parameters have, in general, failed. The principal exception has been demonstrated by cells from patients with xeroderma pigmentosum (XP): cells from those patients with the most severe deficiencies in excision repair are in general the most sensitive to ultraviolet light (u.v.) (Andrews et al. 1978). But similar correlations do not exist for other comparisons between mammalian cells. Cultured mouse cells, which exhibit almost no excision repair of u.v.-induced DNA damage (Klimek, 1965), survive u.v. irradiation almost exactly as do excision-competent human cells (Rauth & Whitmore, 1966). The repair of single-strand breaks (ssbs) and double-strand breaks seems to be almost complete in cells irradiated with ionizing radiation at doses causing up to 99% killing (Hariharan et al. 1981; Lehmann & Stevens, 1977), and ionizing radiation-induced base damage seems to be rapidly and completely repaired in all normal mammalian cells (Remsen & Cerutti, 1977). Cell lines from many, but not all, patients with ataxia telangiectasia (AT) are defective in the excision of such base damage; yet survival curves of cells from all AT patients tested, whether or not defective in base damage repair, are virtually identical (Paterson & Smith, 1979).

One of the cellular responses that have been cited as candidates for cellular protection is the inhibition of DNA synthesis that is induced by DNA-damaging agents. It has been proposed that this slowing down of DNA synthesis increases the time for repair before replication can fix the damage (Tolmach et al. 1980). One of the components of DNA synthesis inhibition is DNA damage-induced delay of replicon initiation. This ‘low-dose’ effect is exhibited by many, perhaps all, DNA- damaging agents. The inhibition of replicon initiation by ionizing radiation, which induces large numbers of ssbs in cellular DNA, can be mimicked by exposing cells to 313 nm light after their DNA thymine has been partially replaced with bromouracil. Because of this, Povirk & Painter (1976) proposed that the lesion that causes the block to replicon initiation is the ssb.

Kaufmann et al. (1980) proposed that the u.v.-induced inhibition of replicon initiation in HeLa cells and normal human fibroblasts was mediated by ssbs formed during excision repair of pyrimidine dimers. However, when this hypothesis was tested, Kaufmann & Cleaver (1981) found that replicon initiation in XP cells of complementation group A, which exhibit virtually no excision repair of pyrimidine dimers (Kleijer et al. 1973), was inhibited by u.v. just as well as in excisionproficient normal cells. To determine if this phenomenon holds true for other mammalian cells, we examined the effects of u.v. on DNA synthesis in excisiondeficient and excision-proficient Chinese hamster cells.

Chinese hamster ovary (CHO) cell line AA8 was a gift from Lloyd Fuller, and CHO 43-3B, which is u.v.-sensitive and excision-deficient (Wood & Burki, 1982), was donated by Regine Goth- Goldstein. Both cell lines were grown in modified Eagle’s medium with 10% bovine serum. Inhibition of DNA synthesis after u.v. or ionizing radiation was measured by preincubation of cells with [1C]thymidine for 24 h and incubation of cells with [1H]thymidine for 15 min after irradiation (Painter, 1977). Alkaline sucrose gradient analysis was performed as described previously (Painter & Young, 1976). Cells were exposed to u.v. under saline at a fluence rate of 1· 3 J m-2s-1; cells were X-irradiated (250 kVp) at a dose rate of 1 gray (100 rad) per min.

After exposure to either 0 · 3 J m-2 or 1 · 0 J m-2, the initial decrease in rate of DNA synthesis was about the same for both the radiosensitive 43-3 B cell line and the AA8 line; the AA8 cells then began to recover and reached virtually normal rates of DNA synthesis by 4h after exposure, whereas the 43-3B cells continued at depressed rates of synthesis until at least 7h after exposure (Fig. 1). Similar results were obtained with UV-5 cells, which are excision-defective cells derived from AA8 (data not shown). Rudé & Friedberg (1977) observed a similar lack of recovery in repairdeficient human cells compared with normal human cells.

Fig. 1.

Inhibition of DNA synthesis in CHO AA8 (○) and CHO 43-3B (•) cells as a function of time after exposure to 0·3 J m-2 (A) or 1·0 J m 1 (B) of u.v.

Fig. 1.

Inhibition of DNA synthesis in CHO AA8 (○) and CHO 43-3B (•) cells as a function of time after exposure to 0·3 J m-2 (A) or 1·0 J m 1 (B) of u.v.

To determine the basis for the deficiency in recovery of DNA synthesis, we used alkaline sucrose gradient analysis to determine the status of nascent strands of DNA in the two cell lines at various times after exposure to u.v. At 30 min after irradiation, the inhibition of replicon initiation was about the same in both cell lines (Fig. 2), as indicated by a fluence-dependent decrease in radioactive DNA in the low molecular weight regions (fractions 5-13) of the gradients. In no case did the low fluences we used have a significant effect on chain elongation, as evidenced by virtually the same radioactivity in DNA of high molecular weight (fractions 14-24) from all cells. Thus, the early drop in DNA synthesis rate in both cell lines is due exclusively to inhibition of replicon initiation.

Fig. 2.

Alkaline sucrose gradient profiles of nascent DNA labelled by incubating CHO AA8 cells (A) or CHO 43-3B cells (B) for 10 min with [1H]thymidine at 30min after exposure to 0·3 J m-2 (O), 1·0 J m-2 (▿), or 0 (•) u.v. Sedimentation is from left to right.

Fig. 2.

Alkaline sucrose gradient profiles of nascent DNA labelled by incubating CHO AA8 cells (A) or CHO 43-3B cells (B) for 10 min with [1H]thymidine at 30min after exposure to 0·3 J m-2 (O), 1·0 J m-2 (▿), or 0 (•) u.v. Sedimentation is from left to right.

At 60 min after u.v. irradiation, a difference between the two lines could be distinguished (Fig. 3). Although the status of nascent strands in cells of both lines exposed to 0-3 J m-1 was about the same, a continuing depression of replicon initiation was apparent in 43-3B cells, but not in AA8 cells. And at 90 min after irradiation, there was a marked difference between the two cell lines (Fig. 4). After both fluences, the AA8 cells had completely recovered from the inhibition of replicon initiation by this time, whereas in 43-3B cells, the inhibition of replicon initiation was greater than at earlier times. (The rate of recovery in experiments using alkaline sucrose gradient analysis was faster than in the experiments of Fig. 1, but the trend was the same.) Inhibition of DNA chain elongation in 43-3B cells was also evident at 90 min after exposure to u.v. When AA8 cells and 43-3B cells were exposed to X-rays, there was no difference between the two lines in the dose response for inhibition of DNA synthesis (data not shown).

Fig. 3.

Same as Fig. 2, but incubation with [1H]thymidine began 60min after exposure to u.v.

Fig. 3.

Same as Fig. 2, but incubation with [1H]thymidine began 60min after exposure to u.v.

Fig. 4.

Same as Fig. 2, but incubation with [1H]thymidine began 90 min after exposure to u.v.

Fig. 4.

Same as Fig. 2, but incubation with [1H]thymidine began 90 min after exposure to u.v.

The initial u.v.-induced inhibition of replicon initiation was the same in both excision-proficient and excision-deficient CHO cells. Kaufmann & Cleaver (1981) observed the same results for excision-proficient and excision-deficient human cells. It therefore seems certain that neither the ssb nor any other event associated with nucleotide excision repair is responsible for the inhibition of replicon initiation observed in u.v.-irradiated cells. However, the recovery of overall DNA synthesis was greatly delayed in excision-defective cells. The simplest interpretation of these results is that pyrimidine dimers are responsible for the original inhibition of replicon initiation in both cell types and for the delayed recovery of overall DNA synthesis in excision-defective cells. Again, however, the simplest explanation may not be the correct one.

Consider the case of the effects of u.v. on inhibition of DNA synthesis in human AT cells. Although it is generally true that AT cells are normal in their response to u.v., the inhibition of replicon initiation after exposure to u.v. is slightly but significantly less in AT cells than in normal human cells (Painter, 1985). If u.v.- induced lesions themselves were responsible for inhibition of replicon initiation, there would be no such difference between AT and normal cells. Therefore, some response of AT cells to u.v.-induced lesions must be abnormal. It is generally believed that the reduced inhibition of replicon initiation observed in X-irradiated AT cells is due to a defective effector, which in its normal state is activated by an ssb and causes the inhibition of replicon initiation. This is probably also the basis for the reduced inhibition of replicon initiation in u.v.-irradiated AT cells. Because there is no difference in inhibition of replicon initiation by u.v. between excision-proficient and excision-deficient cells, the effector must not be stimulated by ssbs formed during repair of pyrimidine dimers or the 6-4 photoproduct, neither of which is reparable by u.v.-sensitive cells (Mitchell et al. 1985). The lesion that causes the inhibition of replicon initiation in u.v.-irradiated cells may be a relatively rare photoproduct. Perhaps, like thymine glycol, it is repaired by the base-excision repair pathway in both normal and u.v.-sensitive cells. During this kind of repair, the endonucleolytic attack that follows glycosylase action causes an ssb. This ssb would be the stimulus for inhibition of replicon initiation in u.v.-irradiated normal cells but not in u.v.-irradiated AT cells, because of the abnormal effectors in AT. Such an interpretation is supported by results with both chick cells (Lehmann & Stevens, 1975) and cells of the fat-tailed marsupial mouse, Sminthopsis crassicaudata (Wilkins, 1983), in which photoreactivation of pyrimidine dimers failed to reverse the u.v.-induced inhibition of DNA synthesis.

In u.v.-irradiated cells, it is possible that the delay in initiation of replicons gives the cell extra time to repair DNA lesions before they are fixed by replication. The result of this would be complementary to the effect reported by Bohr et al. (1985) that CHO cells repair u.v.-induced pyrimidine dimers preferentially in transcribed genes such as that for dihydrofolate reductase. Removing damage from crucial DNA before it replicates seems to be a cellular priority.

This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy (contract no. DE-AC03-76-SF01012).

Andrews
,
A. D.
,
Barrett
,
S. F.
&
Robbins
,
J. H.
(
1978
).
Xeroderma pigmentosum neurological abnormalities correlate with colony-forming ability after ultraviolet radiation
.
Proc. natn. Acad. Sci. U.S.A.
75
,
1984
1988
.
Bohr
,
V. A.
,
Smith
,
C. A.
,
Okumoto
,
D. S.
&
Hanawalt
,
P. C.
(
1985
).
DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall
.
Cell
40
,
359
369
.
Hariharan
,
P. V.
,
Eleczko
,
S.
,
Smith
,
B. P.
&
Paterson
,
M. C.
(
1981
).
Normal rejoining of DNA strand breaks in ataxia telangiectasia fibroblast lines after low X-ray exposure
.
Radiat. Res.
86
,
589
597
.
Kaufmann
,
W. K.
&
Cleaver
,
J. E.
(
1981
).
Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts. J’, molec. Biol.
149
,
171
187
.
Kaufmann
,
W. K.
,
Cleaver
,
J. E.
&
Painter
,
R. B.
(
1980
).
Ultraviolet radiation inhibits replicon initiation in S phase human cells
.
Biochim. biophys. Acta
608
,
191
195
.
Kleijer
,
W. J.
,
De Weerd-Kastelein
,
E. A.
,
Sluyter
,
M. L.
,
Keijzer
,
W.
, De
Wit
,
J.
&
Bootsma
,
D.
(
1973
).
UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes
.
Mutat. Res.
20
,
417
428
.
KlÍmek
,
M.
(
1965
).
Formation but no excision of thymine dimers in mammalian cells after UV- irradiation
.
Neoplasma
12
,
459
460
.
Lehmann
,
A. R.
&
Stevens
,
S.
(
1975
).
Postreplication repair of DNA in chick cells: Studies using photoreactivation
.
Biochim. biophys. Acta
402
,
179
187
.
Lehmann
,
A. R.
&
Stevens
,
S.
(
1977
).
The production and repair of double strand breaks in cells from normal humans and from patients with ataxia telangiectasia
.
Biochim. biophys. Acta
474
,
49
60
.
Mitchell
,
D. L.
,
Haipek
,
C. A.
&
Clarkson
,
J. M.
(
1985
).
(6-4) Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane dimers
.
Mutat. Res.
143
,
112
115
.
Painter
,
R. B.
(
1977
).
Rapid test to detect agents that damage human DNA
.
Nature, Land.
265
,
650
651
.
Painter
,
R. B.
(
1985
).
Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light
.
Mutat. Res.
145
,
63
69
.
Painter
,
R. B.
&
Young
,
B. R.
(
1976
).
Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells
.
Biochim. biophys. Acta
418
,
146
153
.
Paterson
,
M. C.
&
Smith
,
P. J.
(
1979
).
Ataxia-telangiectasia: An inherited human disorder involving hypersensitivity to ionizing radiation and related DNA-damaging chemicals. A
.
Rev. Genet.
13
,
291
318
.
Povirk
,
L. F.
&
Painter
,
R. B.
(
1976
).
The effect of 313 nanometer light on initiation of replicons in mammalian cell DNA containing bromodeoxyuridine
.
Biochim. biophys. Acta
432
,
267
272
.
Rauth
,
A. M.
&
Whitmore
,
G. F.
(
1966
).
The survival of synchronized L cells after ultraviolet irradiation
.
Radiat. Res.
28
,
84
95
.
Remsen
,
J. F.
&
Cerutti
,
P. A.
(
1977
).
Excision of gamma-ray induced thymine lesions by preparations from ataxia telangiectasia fibroblasts
.
Mutat. Res.
43
,
139
146
.
Rudé, J. M. &
Friedberg
,
E. C.
(
1977
).
Semi-conservative deoxyribonucleic acid synthesis in unirradiated and ultraviolet-irradiated xeroderma pigmentosum and normal human skin fibroblasts
.
Mutat. Res.
42
,
433
—442.
Tolmach
,
L. J.
,
Hawkins
,
R. B.
&
Busse
,
P. M.
(
1980
). The relation between depressed synthesis of DNA and killing in X-irradiated HeLa cells. In
Radiation Biology in Cancer Research
(ed. R. E. Meyn &
H. R.
Withers
), pp.
125
142
.
New York
:
Raven Press
.
Wilkins
,
R. J.
(
1983
).
Photoreactivation of UV damage in Sminthopsis crassicaudata.
Mutat. Res. Ill, 263-276
.
Wood
,
R. D.
&
Burki
,
H. J.
(
1982
).
Repair capability and the cellular age response for killing and mutation induction after UV
.
Mutat. Res.
95
,
505
—514.