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Postreplication repair of DNA in ultraviolet-irradiated mammalian cells
J. Mol. Biol. (1972) 66, 319-337
Postreplication
Repair of DNA in Ultraviolet-irradiated Mammalian Cells A. R. LEHMMN~
Biology Division, Oak Ridge National Laboratory Oak Ridge, Tenn. 37830, U.X.A. (Received 21 April 1971, and in revised form 9 December 1971) DNA synthesized in ultraviolet-irradiated L5178Y mouse lymphoma cells is smaller than that made in u&radiated cells, ae shown by pulse-labeling with tritiated thymidine, followed by sedimentation of the DNA in alkaline sucrose gradients. Hence, DNA synthesized on ultraviolet-irradiated DNA templates contains discontinuities. The estimated distance between these discontinuities is comparable to the average spacing between pyrimidine dimers in the parental strands, suggesting that the replication machinery leaves gaps opposite the dimers. On subsequent incubation these discontinuities are Clled in by a process which is inhibited by hydroxyurea. In order to determine whether the gaps are filled in by a recombinational mechanism or by de 7aoe)osynthesis, irradiated cells were pulse-labeled with radioactive thymidine, followed by incubation in bromodeoxyuridine and exposure to light of wavelength 313 nm. Bromuracil-containing sections of DNA are selectively disrupted by light of this wavelength. Thus if the gaps in the radioactively labeled daughter strands are filled in by exchange with thymidine-containing DNA from the parental strands (recombinational exchange), they will not contain bromuracil, and the molecular weight distributions of the radioactive DNA from initially irradiated and unirradiated cells will be similar after exposure to 313-nm light. On the other hand, if the gaps are filled in by bromuracil-substituted DNA (arising from de nOvo synthesis), the small pieces of radioactive DNA synthesized in the initially irradiated cells during the pulse-labeling will be reformed on exposure to 313-nm light, and the molecular weight distribution from those cells will have a lower average molecular weight than that from similarly treated unirradiated cells. The results are in accordance with the second alternative, indicating that the gaps in the daughter strands opposite the pyrimidine dimers are filled in with newly synthesized DNA. Furthermore parental DNA does not seem to be involved in the gap-filling process. This excludes many of the recombinational models which have been put forward to account for similar gap-Clling phenomena observed in bacteria. The results give an approximate value of ,800 nucleotides for the size of the filled-in gaps.
1. Introduction A surprising aspect of the effect of ultraviolet light on mammalian cells is that, despite the fact that mammalian DNA offers a target about 1000 times as big M the DNA in bacteria, the U.P. sensitivities of mammalian cells and bacteria are very similar. This is in contrast to the situation with X-rays, to which mammalian cells are in general 10 to 100 times more sensitive. A typical U.V. survival curve for mammalian cells has a shoulder and a D, value both of about 50 to 100 ergs/mm2 (see review by t Present address: Biology England. 21
Building,
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A. R. LEHMANN
Rauth, 1970). In this dose range there are about 10e pyrimidine dimers per cell (assuming a total mass of DNA per cell of lo-l1 g and a percentage of pyrimidines as dimers of 0.02 to 0.04). This clearly implies that mammalian cells are capable of tolerating the production in their DNA of a large number of such alterations without being killed. In bacteria there are three known types of repair processes for removing or circumventing pyrimidine dimers: (1) photoreactivation (see review by J. K. Setlow, 1967). This process has been detected in marsupials but in no other mammals (see review by Cook, 1970). (2) Excision-repair of dimers, whereby a section of the DNA containing the dimers is excised by the cell’s repair machinery (see review by R. B. Setlow, 1968). The resulting gap is then f&d in by a process termed repair-replication (see review by Hanawalt et al., 1968). Human and other primate cell lines excise ditners efficiently from their DNA (Regan, Trosko & Carrier, 1968) but very little dimer excision (Trosko, Chu & Carrier, 1966; Steward & Humphrey, 1966; Klimek, 1966; Cleaver & Trosko, 1969) and only very low levels of repair-replication (Painter & Cleaver, 1969 ; Cleaver, 1970) have been detected in rodent cells. (3) Postreplication repair (“recoli which was unable to excise combinational repair”) ; using a strain of Edwichia thymine dimers yet could still survive a U.V. dose producing about 50 dimers per cell, Rupp & Howard-Flanders (1968) showed that the DNA synthesized in u.v.-irradiated cells had a lower molecular weight (as measured in alkaline sucrose gradients) than that from unirradiated cells, suggesting that this newly synthesized DNA contained gaps. Subsequently the gaps were filled in, as demonstrated by an increase in size of the newly synthesized material during further incubation. In later work Rupp and co-workers presented data which suggested that the gaps in the new strands were opposite pyrimidine dimers in the parental strands (Howard-Flanders, Rupp, Wilkins & Cole, 1968) and estimated that the size of the gaps was about 1000 to 1600 nucleotides in length (Iyer & Rupp, 1971). They have recently presented evidence (Rupp et al., 1970; Rupp, Wilde, Reno & Howard-Flanders, 1971) for end-to-end association of daughter and parental DNA after U.V. irradiation, supporting their hypothesis that recombinational exchanges are involved in the filling-in of the gaps in E. coli. The process of postreplication repair has been detected in all strains of E. coli tested, exoept for those carrying the recA mutation (see review by Smith, 1971). The existence of similar processes in Chinese hamster cells and mouse lymphoma L5178Y cells has been briefly reported by Cleaver t Thomas (1969) and Rupp et al. (1970). This paper describes the process in detail in mouse lymphoma cells (L5178Y) and presents evidence that in these cells postreplication repair does not involve recombinational exchanges.
2. Materials and Methods The cell line used in all experiments was the mouse lymphoma line L6178Y (generously provided by Dr G. A. Fischer), which was grown in suspension culture at 37°C in Fischer’s medium (Fischer & Sartorelli, 1904) supplemented with 10% foetal calf serum. The doubling time was 11 to 12 hr.
(a) Ultraviolet
irradiation
Cells were spun down, washed in PO, saline (phosphate buffered saline: 8.0 g NaCl, 0.2 g KCl, 1.15 g Na,HPO,, 0.2 g KH2P0,, 0.1 g CaCl,, 0.1 g MgCl, . 6H,O/l.) and resuspended in 5 to 10 ml. PO4 saline at a cell concentration of about 3 x 105/ml. The cell suspension was stirred in a Falcon Petri dish and irradiated with U.V. light (from a mercury germicidal lamp) at 264 nm with an incident U.V. exposure of 110 or 220 ergs/mm2.
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(b) Radioache lxzb&ng After irradiation the cells were resuspended in Fischer’s medium, and Biter 8 certain interval of time, ,25 or 50 &i [naet~~Z-3H]thymidin+nl. (lSCi/m-mole, Schwarz BioResearch, Inc.) were added and the cells incubated usually for O-5 hr. (c) Ckz8e eampe9immt8 In ch8sse experiments, the cells were spun down after radioactive labeling, washed, and resuspended in medium containing 25 M deoxycytidine/ml. 8nd either 2-5 pg thymidine/ml. or 5 pg BrdUrd/ml., and incubsted for various periods of time. (d) Degmdatkn wing 313~nm light In these experiments, after incubation with BrdUrd the cells were spun down, washed in PO4 saline, and resuspended in this buffer at 8 concentrstion of 2 to 3 x lo6 cells/ml. The suspension ~8s then exposed in 8n ice-cooled l-cm-path quertz microcuvette to light of wavelength 313 nm from 8 18rge, qu8rtzprism Hilger monochrom8tor illuminated with 8 Philips 1000 W, high-pressure, water-cooled mercury arc. A thin Myl8r film W&Binserted between the monochromator and the sample to eliminate 8ny scattered shortw8velength light. The exposure rate 8s measured by a calibrated thermopile ~8s about 1.3 x lo5 ergs/mmz/min, end the incident exposure was between 0 and 6 x IO5 ergs/mma. Cells were incubated for 4 to 5 hr with 20 PCi [3H]BrdUrd/ml. at total concentration of 5 pg/ml. end 2.5 CcQdeoxycytidine/ml. and then treated exactly as in the shove section, but irrsdiated at 313 mn in the exposure range of 0 to IO4 ergs/mma. The exposure r&e w&8 5 to 10 x lo3 ergs/mma/min. A 3.9~ml. gradient of 5 to 20% sucrose, O-1 M-N&~, O-1 M-N~OH wss formed in a 4ml. polyallomer tube. One-tenth ml. of 2% sodium dodeoyl sulfate, 0.02 as-EDTA wae layered over this. In all experiments, immediately before lysis and centrifugation, the cells, resuspended in PO4 saline, were irmdi8ted with 2 kred of X-rsdietion (see Results, se&ion (8)) delivered from a 250 kVpf30 mA, G. E. k&xitron X-ray msohine (+-mm Al filter), and a volume containing 5 to 15 x lo3 cells (i.e. about 0.1 ccgof DNA) WBBthen 18yered into the sodium dodecyl sulf8te on top of the gradients. These were centrifuged immediately et 20°C in the SW56 rotor in 8 Beckman model L ultracentrifuge, usually at 40,000 rev./min for 75 min. After centrifug8tion the bottoms of the tubes were pierced with 8 Buchler tube piercer, and 26 to 29 &drop fractions were collected on whatman no. 17 peper strips (1 cm width), 8 modification of the filter-paper disk technique 8s described by Carrier & Setlow (19718). The strips were washed in 5% trichloroacetic &d, twice in ethanol, and fhmlly in 8cetone. When dry, they were cut up into scintillation vi&~ and the radioactivity ~8s measured in 8 Packard liquid-scintillation spectrometer, with toluene containing 4 g 2,5-bis-2-(S-tert.-butylb enzoxazolyl)-thiophene/l. 8s ecintilletor. Calculations of moleoulsr weights and percentage counts for each fraction were performed by a computer program which used the empiric&y determined relationship between molecular weight, M, and the mtio of the distanoe sedimented to the length of the liquid column, D: M = 5-02 x lo3 (loll D/wat)a’6, where w is the rotor speed (rev./mm) and t is the centrifug&ion time (mm). In experiments in which accurate molecular weights were required, marker DNA from h phsge ~8s used, aesuming a single-strend molecular weight of 16 x lo6 d&ons (Caro, 1965).
3. Results (B) Problems and interpretations of sedimentation pro$les of DNA from mammalian cell8 Presently available data suggest that 8 replicating DNA molecule in 8 mammalian cell hes the structure shown in Figure 1(e) (Huberman & Riggs, 1968 ; Lehmann, 1970 ; Lehmann t Ormerod, manuscript in prep8ration). Replicating units (replicons) are
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FIU. 1. Sedimentation profiles of DNA from unirmdiated celk labeled for various periods of time and the DNA struotures they represent. The DNA of the cells was labeled with radioactive thymidine, after which the cells were X-irradiated 8nd lysed in detergent on top of mlkmline sucrose gradients. These were then centrifuged for 75 min 8t 40,000 rev./min. (8) Cells labeled with 0.5 pi [‘*C]thymidine/ml. for IS hr; (b) and (c) cells lsbeled with 25 PCi [3H]thymidine/mJ. for 30 min and 16 min, respectively. Total cts/min: (8) 4225, (b) 9661, (c) 3779. (d) Replicated DNA, (e) replicating DNA mt different stages of the S-period. The discontinuous verticel lines delineate separate replicons. Sediment&ion in all Figs is from right to left.
joined end-to-end and are approximately lo* daltons in length (Huberman & Riggs, 1968; Lehmann & Ormerod, manuscript in preparation). In a pulse-labeling experiment onIy the growing DNA strands (the inside strands in Fig. l(e)) become radioactively labeled. However, the much longer parental strands, though unlabeled, are unfortunately present, and in alkaline sucrose they entangle with the growing strands, rendering any interpretation of the profiles meaningless (Lehman, 1970; LehnmM & Ormerod, manuscript in preparation). This can be circumvented by degrading the DNA to a small extent, so that the long parental strands are somewhat broken down but the shorter, growing strands are hardly affected. Although this can be done chemically, with strong alkali (Regan, Setlow & Ley, 1971; Elkind & Kamper, 1970; Lett, Klucis t Sun, 1970), a low dose (1 to 3 krads) of X-rays (Lehmann BEOrmerod, manuscript in preparation; Lehmann, 1970; Elkind & Kamper, 1970) has been found to be much more reproducible, and can be more accurately controlled. It also takes less time, and is therefore more convenient. Hence at the end of all treatments cells were X-irradiated for 5 seconds (2 brads)---a dose that produces approximately one single-strand break per 200x lo6 daltons (Lehmann & Ormerod, 1970). The growing
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strands, being mostly shorter than this, were hardly affected. It should be emphasized that the sole purpose of this X-irradiation was to fragment the parental DNA strands and thus prevent entanglement. The cells were always lysed immediately (i.e. within seconds) after this irradiation. All the experiments described below utilized mouse lymphoma L5178Y cells, which do not excise pyrimidine dimers from their DNA after U.V. irradiation (results to be published elsewhere). Figure 1 shows sedimentation profiles of DNA from unirradiated cells under different circumstances together with the structures of the DNA they are presumed to represent. The first (Fig. l(a)), which will be referred to as type I, represents “joined-up DNA” (as shown in Fig. l(d)) and is typical of a profile from DNA labeled for several hours. Its main characteristics are a peak with a sharp front at &f = 150 to 200 x lo6 daltons and a low molecular weight tail. As discussed in some detail in an earlier paper (Ormerod & Lehman, 1971) and as reported by McBurney, Graham & Whitmore (1971) and Elkind (1971), this sharp front is caused by a sedimentation anomaly and is seen in all profiles of DNA with molecular weights greater than 2 x 10s daltons, whether from bacteria or mammalian cells, if the DNA is centrifuged at high rotor speeds (20,000 rev./min or more) (e.g. McGrath & Williams, 1966; Rupp & Howard-Flanders, 1968; Lett et al., 1970; Elkind & Kamper, 1970; Ormerod & Lehmann, 1971; Regan et al., 1971). The material at this peak represents an accumulation of DNA of molecular weight between 15 and 10 x 10s daltons. The second and third types (type IIA in Fig. l(b) and IIB in Fig. 1(c)) represent “growing DNA” (Fig. l(e)). They are characterized by broad, flat-topped profiles stretching across the middle of the gradient. The distribution arises because in an asynchronous population there are growing strands of all sizes between zero and the size of the replicating unit (about 1O8) such as those shown in Fig. l(e). After a l&minute pulse (Fig. I(c), type IIB), a small peak is seen at fractions 15 to 17 (M w 4 x 107) ; this arises from a population of relatively small replicating units and is less marked in the 30-minute pulse (Fig. l(b)), type IIA), when much of the DNA in the small units has elongated and joined up to the DNA in adjacent units. These profiles have also been discussed in detail elsewhere (Lehmann, 1970; Lehmann BE Ormerod, manuscript in preparation). Prolonged incubation after a short pulse label would be expected to convert type II prollles to type I, corresponding to elongation of the growing strands and joining up of adjacent replicons after synthesis. (b) Formation of gaps after ultraviolet irradiation Pulse-labeling cells after U.V. irradiation produces completely different profiles as shown in Figure 2. These proflles are referred to as type III. There is a pronounced peak near the top of the gradient at a molecular weight of 15 to 25 x lo6 daltons after 110 ergs/mm2 and 5 to 15 x lo6 daltons after 220 ergs/mm2, showing that the DNA synthesized after U.V. irradiation is indeed shorter than that synthesized without prior U.V. exposure. This confirms the results of Cleaver & Thomas (1969) and a preliminary report of Zipser & Rupp (1970, Biophys. Sot. Ann. Meeting, Abstr. 10, p. 261a). In order to test whether the gaps in the DNA synthesized after U.V. exposure were opposite pyrimidine dimers, cells were labeled for 16 hours with O-5pCi [14C]thymidine/ ml. (50mCi/m-mole) and U.V. irradiated. Half of the irradiated cells were examined for dimer content as described by Carrier & Setlow (1971b). The rest were incubated and pulse-labeled with [3H]thymidine, and a sedimentation experiment was carried
A. R. LEHMANN
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FIG. 2. Sedimentation profiles from u.v.-irrediated oells. cells were incubated (b) 220 ergs/mm a. After irradiation, 30 min with (a) 25 pCi [sH]thymidine/ml. or (b) 60 pCi carried out et 40,000 rev./mm for (a) 76 min or (b) 86 min.
Exposures were (a) 110 ergs/mms and for 46 min and then pulse-labeled for [3H]thymidine/ml. Centrifugation was Total ots/min: (a) 4259, (b) 2398.
out as described in Materials and Methods. In Table 1 the number-average molecular weights for the low molecular weight pieces of the newly synthesized 3H-labeled DNA (calculated from the sedimentation experiment) are compared with the interdimer spacings in the parental strands (calculated from the fraction of l*C radioactivity in pyrimidine dimers). Good agreement was obtained, suggesting that the gaps might indeed be opposite the dimers. The replication mechanism envisaged by Rupp & Howard-Flanders (1968) for u.v.-irradiated E. coli was as follows: in an irradiated cell the replication machinery continues to elongate the growing DNA strand until it reaches a dimer. It then leaves a gap and synthesis recommences at some point beyond the dimer. In my experiments, as shown above, the average interdimer distance is about lo7 daltons after 110 ergs/mnP. As shown in earlier work (Lehmann t Ormerod, 1970), the replication rate in these cells is about O-8x lo6 daltons/minute. Hence it should take on the average 1245 minutes for the replication machinery to proceed from one dimer to the
TABLE
1
Convpari8on of dimer spacing and size of piece8 synthesized in irradiuted cell8 U.V. exposure @f3d=*)
Percentage of W radioactivity as dimers
Calculated interdimer spacing? (108 daltons)
110
0.019
9.5
220
0.032
54
Measured ikf,, of3 low molecular weight pieces ( x 10s) 12.0 6.6
t Based on assumptions: (1) ratio of dimers in E. COGDNA, T-T/T-C/Cc = 6:4: 1 (Setlow & Cerrier, 1966); (2) thymine content of mouse DNA = 30% henae; (3) ratio of dimers in mouse DNA, T-T/T-C/C-C= 6*2:3.3:05. t. Calculated from the slope of a plot of log (number of molecules in eaoh fraction) against moleoular weight (Lehmann & Ormerod, 1970).
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next. Thus it might be expected that during the first few minutes immediately after irradiation, in some replicons the replicating machinery would not yet have reached a dimer, so that the labeled DNA would not be in short pieces. This prediction is borne out by the experiment shown in Figure 3, in which cells were pulse-labeled for 16 minutes at 0, 10, and 20 minutes after irradiation. With increasing times before labeling, the amount of low molecular weight material increased. However, after 20 minutes there was still a high molecular weight tail, and this remained even if the cells were labeled 30 to 60 minutes after irradiation. Possible explanations for this are given in the Discussion. In subsequent experiments, in order to obtain maximum amounts of low molecular weight DNA, the cells were always incubated for 30 to 60 minutes between U.V. irradiation and pulse-labeling.
Pulse after
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FIG. 3. Sedimentation profiles from cella pulse-labeled at various times efter irradiation. Cells were exposed to U.V. light of 110 er&nma, incubated for 0, 10, or 20 min and then pulselabeled for 16 min with 26 $i [3H]thymidine/ml. Total ctsjmin: 3080 (-), 4898 (---), 2571 (. . .).
(c) Filling-in
of gaps
Figure 4 shows results from pulse-chase experiments. With unirradiakl cells, a 0*5-hour pulse (Pig. 4(a)) gave, as expected, a typical type II pro6le (seePig. l(b) and discussion thereof). A subsequent 4 hour incubation in unlabeled thymidine converted this to a type I pro& (Pig. 4(b)), indicating elongation of the growing strands and joining of the strands in adjacent replicons. This process was prevented by 10e3 Yof DNA synthesis (Pfeiifer & Tolmach, hydroxyurea (Pig. 4(c)), an inhibitor 1967). With u.v.-irradiated cells the straight pulse gave a type III profile (pig. 4(d)). Pilling-in of the gaps should be manifested by a conversion of a type III to a type II profile and subsequent elongation and joining up by conversion of type II to type I. Both processes occurred during a 4-hour incubation in unlabeled thymidine, and a type I profile was obtained (Pig. 4(e)). In the presence of hydroxyurea (Pig. 4(f)) a type III profile remained, demonstrating that both gap-6lling and elongation and joining up
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FIG. 4. Sedimentation pro&s from cells pulse-labeled and chased. with 160 ergs/mma ((d), (e) and (f)) were Cells unirradiatad ((a), (b) and (c)) or u.v.-irradiated incubated for 46 min, then pulse-lrrbeled for 30 min ((a) and (d)), chased for 4 hr in the absence ((b) and (e)) or presence ((c) and (f)) of 2 x 10m3 aa-hydroxyures. Total cts/min: (a) 1296, (b) 9800, (c) 13,600, (d) 2106, (e) 2303, (f) 2060.
were prevented by this inhibitor. (The small amount of increase in molecular weight observed in the presence of hydroxynrea was not always seen and may represent synthesis occurring before the hydroxyurea has been taken up into the cells.) Figure 5 shows the kinetics of the chase. Most of the gap-tilling (type III --t type II) was completed within the first hour and the subsequent elongation and rejoining took a further 4 hours. (d) Recombination versus de novo synthesis The gap-filling process could take place either (a) by a recombinational exchange mechanism, whereby the gap is filled in by the corresponding piece of parental DNA from the sister duplex (Howard-Flanders et al., 1968). Presumably the resulting gap in the parental strand is subsequently filled-in by de rwvo synthesis ; or (b) by direct synthesis of new material in the daughter-strand gap. In E. coli, Rupp et al. (1971) have recently produced evidence favoring the former mechanism. The two mechanisms may be distinguished by following the fate of the parental DNA strands and of the DNA made after U.V. irradiation. On the recombinational
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2 hr Chase
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Pm. 5. Sedimentation profiles from irradiated cells pulse-labeled and ohased for various times. Cells irradiated with 110 ergs/mma, incubated for 45 min then pulse-labeled for 30 min (a), followed by a cold abase of 1 hr (b), 2 hr (a), or 4 hr (d). Total cts/min: (8) 2910, (b) 4433, (c) 3831, (d) 3300.
model daughter-strand gaps are filled in with pre-existing parental mate&l and subsequently the parental DNA strands will contain stretches of material synthesized after U.V. irradiation. On the other hand, direct synthesis will result in the daughter strands containing material synthesized subsequently, the parental strands remaining intact throughout. These predictions were tested in L5178Y cells by two types of experiment, both taking advantage of the selective photolysis of bromouracil-containing sections of DNA by U.V. light. It has been shown (Lion, 1970; Hutchinson & Hales, 1970; Hewitt & Marburger, 1971, Biophys. Sot. Ann. Meeting, Abstr. 11, p. 27a) that U.V. light introduces single-strand breaks (or alkali-labile bonds) into BrU-substituted DNA. This effect has been studied in detail in this laboratory by Setlow, Regan & Ley, who have found that the extent of damage to BrU-substituted DNA verse unsubstituted DNA is greater at s, wavelength of 313 nm than at 254 nm. This technique has been used for measuring the extent of repair replication in mamm&an cells (Regan et al., 1971) and in E. cdi (Ley & Setlow, 1972). (i) Fate of the daughter-strand gaps Cells were U.V. irradiated (254 run), pulse-labeled, and then incubated in the presence of BrdUrd for 4 to 5 hours, followed by exposure to 313~run light. The expected effects on the DNA molecules are shown in Figure 6. With u&radiated cells (Fig. 6(a)), the initial [3H]thymidine pulse labels the growing strands (A), giving a type II profile. On incubation the growing strands are elongated with BrU-containing material and joined up to adjacent replicons (B), giving a type I profile. Light at 313 nm breaks down the BrU-containing sections (C), returning to a type II protile (only the strands containing a section marked with a wavy line in Fig. 6 are identified).
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A -B --
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N8-mmmam
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--
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A /Y--+G!l
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U.V. irradiated Profile type m
h-----V Chase+Brd
Urd
I I
B -
B -
,!
C II
l
*:: w
I
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-mu-
r
C
313nm III
m -M--Mm Recombination
de now
Synthesis
FIG. 6. Expected effect of 313-nm light on unirradiated (a) and u.v.-irradiated (b) cells. Celle pulse-labeled (A) and ohaaed with BrdUrd (B) followed by treatment with 313-nm light (C). (b) Left, gaps filled in by reaombination; r&ght, gaps flllad in by de W.WOayuthesis. (-) DNA synthesized before pulse label; (-) DNA synthesized during pulse label; m DNA containing BrU; (X) pyrimidine dimers (only relevant dimers shown); (Y) long BrU-containing stretoh of DNA joining up adjeoent replicons. Note: For clarity, the replicating process taking plaoe on only one strand of the DNA duplex is shown.
With irradiated cells (Fig. 6(b)), the initial profile should be of type III, which on incubation gives a type I profile after gap-filling and elongation and joining up. There are now two possibilities. (1) According to the recombinational mechanism postulated by Rupp et al. (1971) for E. coli, the gaps are filled in by parental material, and the new material (BrU-containing) fills in the resulting gap in the unlabeled parental strand. Hence, treatment with 313-nm light would not be expected to re-form the discontinuities in the (labeled) daughter strands, as shown in Figure 6(b), left. Thus the effect of 313-nm light would be identical to that in the initially m&radiated case (type I+ type II). (2) Alternatively, the gaps would be filled in by de 120~0 synthesis. In this case the gaps would be filled in by BrU-containing material. The 313-nm light should introduce breaks into these sections (Fig. 6(b), right), and the small pieces observed after the pulse of [“Hlthymidine should be re-formed, resulting in a type III protIle. Figure 7 shows sedimentation profiles from experiments with 313-nm light-induced degradation. The left-hand diagrams in Figure 7 show the profiles after a chase with BrdUrd following U.V. exposures of 0,110, and 220 ergs/mm2. Although the elongation and rejoining process was not quite complete after the chase following the initial exposure of 220 ergs/mm2 (Fig. 7(c)), there was no trace of the initial low molecular weight material seen after the pulse (compare Fig. 2(b)), demonstrating that the gaps had been filled in. The right-hand Figures are profiles after a subsequent 313-nm exposure of 3.4 X lo6 ergs/mm 2. With the initially m&radiated cells there was a return to a type II profile as expected (compare Fig. l(b)) ; with the 110 ergs/mm2
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12
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FIG. 7. Sedimentation prof?les from u.v.-irradieted cells after 313-nm exposure. Cells were irradiated at 264 nm, inaubatad for 46 min. puke-labeled, chased with BrdUrd for 4 to 6 hr (left) and then exposed to 3.4 x lo5 ergs/mm~ et 313 nm (right). (13)and (b) No initial irradiation, 30-min pulse, 4*6&r chase; (0) and (d) 110 ergs/mma, SO-min pulse, 46hr chase. (e) and (f) 220 ergs/mm’, 30-min pulse, B-hr chase. Total cts/min: (a) 9800, (b) 4886, (a) 3006, (d) 3663, (6) 2087, (f) 1609.
sample the peak at fractions 17 and 18 began to reappear (compare Fig. 2(a)) ; and with the 220 erge/mma sample there was the clear reappearance of the low molecular weight peak at the same position as that obtained immediately after the pulse (type III, compare Fig. 2(b)). Thus in each case there was a return to a profile type similar to that seen after the pulse label with no further treatment. Thus the gaps in the DNA synthesized after the initial U.V. exposure were reformed on exposure to 313-nm light after the chase with BrdUrd, indicating that they had been filled in with BrUsubstituted DNA (i.e. by de nova synthesis (Fig. 6(b), right)). In a control experiment, in which the chase was carried out with thymidine instead of BrdUrd, no breakdown was observed on exposure at 313 ,nm, a type I profile remaining even at very high exposures.
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The results of all the experiments with 313-nm light-induced degradation are shown in Figure 8, where they are plotted in two different ways: as the amount of radioactivity in the region of 5 to 55 x lo6 d&as (i.e. the low-molecular weight region) divided by the total radioactivity of the DNA with molecular weight above 5 x lo6 (Fig. 8(a)); and as the measured weight-average molecular weight (Fig. 8(b)). (Since the DNA molecules are not uniformly labeled this does not give a true weightaverage molecular weight.) The data have been plotted somewhat arbitrarily in this manner in order to demonstrate the following points : (1) in all cases at low exposures of 313~nm light (0 to 0.2 x lo5 ergs/mm2) there was a
. .
0 (b) P
IO-> Exposure to313nm light (ergs/mm’) FIG. 8. Re-formation of low molecular weight pieces by 313-nm light. (8) Fraction of the radioaotivity of molecular weight in excess of 5 X 10s lying between 5 and 55 x 10s daltons after different doses of 313-nm light. (b) Measured weight-average molecular weight (2 counts x moleoular weight/ 2 counts). Cells were initially irradiated et 254 nm with 0 for 30 min. (-O-O-), 110 (-•-•--) or 220 (-m--m--) ergs/ mma and pulse-labeled (-n-n-) Unirradiated cells pulse-labeled for 16 min. For both (a) and (b) the points at the right are those obtained immediately after pulse-labeling, with no subsequent treatment with BrdUrd or 313-nm radiation (i.e. these represent DNA shown in the upper diagrams (A) of Fig. 6(a) and (b)).
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rapid degradation to a certain level. This was the result of breakdown of the long BrU-containing piece of DNA that joined up adjacent replicons, marked Y in Figure 6, producing a conversion of type I to type II profiles. (2) With initially unirradiated cells after higher exposures at 313 nm there appeared to be a further small amount of degradation. (3) With initially irradiated samples there was a reproducibly greater amount of breakdown in the range O-2 to 3 x lo5 ergs/mm2. This resulted from the re-formation of the filled-in gaps. Above 3 x lo5 ergs/mm2 there was a further small amount of degradation similar to that seen in initially m&radiated cells. (4) With initially irradiated samples, after high doses of 313-nm light, the fraction of low molecular weight material and the measured weight-average molecular weights were similar to those obtained after the pulse-label without further treatment (shown at the right of the Figure). Because of the degradation seen in initially u&radiated cells, the differences between the profiles from cells initially unirradiated and those u.v.-irradiated with 110 ergs/mm2 were not large. Since the rate of synthesis after an exposure of 110 ergs/mm2 was about one-half that in u&radiated cells, it was essential to compare the profiles from the irradiated cells with those from unirradiated cells that were both pulselabeled for 05 hour and chased for 4 hours (pulse and chase times the same as for u.v.-irradiated cells) and pulsed for 15 minutes and chased for 2 hours. In the latter case the lengths of label were the same as in the u.v.-irradiated cells, so that labeling artifacts were avoided (Lehmann & Ormerod, 1969). There was more low molecular weight material produced by 313-nm light with the 15-minute pulse than with the 30-minute pulse, but this was also seen immediately after the initial pulse (Fig. 8, open squares). With the 110 ergs/mm2 sample, the 313-nm light-induced breakdown was still reproducibly greater, and with the 220 ergs/mm2 sample, the breakdown was clearly much greater. If the cells were pulse-labeled before the initial U.V. irradiation, the amount of degradation by 313-nm light was the same in irradiated and u&radiated cells (see Table 2), showing that the degradation is specific for DNA synthesized after the U.V. irradiation and does not merely reflect a greater sensitivity of u.v.-irradiated DNA to 313-nm light. These results show that in all cases there was a return to the profiles obtained immediately after the thymidine pulse, along with a small amount of background TABLE
2
Effect of 313~nm Eight on cells ultraviolet-irradiated Initial u.v. exposures (ergs/mm”) 0 110 0 110
10-s x exposure to 313-nm light (ergs/mm”) 1.9 1.9 3.8 3.8
Percentage low molecular weight material 30 31 34 35
after pulse labeling Measured weightaverage mol. wt (x 106) 89 86 82 80
Cells pulse-labeled for 30 min were u.v.“irradiated, incubated for 4 hr in the presence of BrdUrd and then irradiated with 313-nm light. Measured weight-average mol. wt is defined in the legend to Fig. S(b).
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LEHMANN
degradation. This shows that the gaps were filled-in (at least partly), with BrUcontaining DNA, i.e. by de nova synthesis (Rig. 6(b), right). (ii) Cdibration
of 313-nrn light-indeed
depdation
From the exposure of 313~nm light at which the low molecular weight peak appeared it is possible to calculate the size of the BrU-containing piece of DNA inserted into the gaps. In order to do this it is necessary to measure the amount of breakdown of BrU-containing DNA by 313~nm light. In these experiments, cells (unirradiated) were incubated with [3H]BrdUrd under identical conditions to those used in the chase experiments and subsequently exposed to 313-nm light. Under these conditions a linear effect of dose was obtained as shown by a plot of l/number-average molecular weight against dose at 313 run (Fig. 9). The dose-effect line gives a value of 1 break/ 2.4 x lo5 daltons/105 ergs/mma, which is similar to the values obtained by Ley &
10m3 x Exposure (ergs/mm2)
FIU. 9. The effeot of irradietion at 313 nm on the moleoular weight of BrU-substituted DNA. Cells were labeled for 4 hr with 20 pCi [3H]BrdUrd/ml. et 8 total conoentration of 6 pg/ml. 8nd irredi8ted with different exposures of 313 nm. Numberaverage mol. wt (ikf,,) of sediment8tion proGlee o&utsted 88 described in the legend to Teble 18nd in Lehm8nn & Ormerod (1970).
Setlow (1972) for irradiation of fully BrU-substituted DNA in E. di and Chinese hamster cells. In Figure 8 most of the release of low molecular weight fragments (type II --t type III) occurs over the exposure range of 313~mu light of about 0.5 to 2 x lo6 ergs/mma. As calculated in the Appendix, this release pattern corresponds to the pattern that would be expected if the size of the filled-in gap were about 800 nucleotides (though this value could well be in error by a factor of 2). (iii) Fate of parental DNA The above results indicate that the daughter-strand gaps are filled in with a piece of new DNA comparable in length to the size of the gap in E. coli (Iyer BERupp, 1971). They support the hypothesis that the gap is filled-in completely by de novo synthesis, and that the stretches of BrU-substituted DNA do not just represent small amounts of repair synthesis. An alternative explanation, not entirely ruled out, is that duriug the chase with BrdUrd recombinational exchanges of large pieces of daughter and parental DNA took place so that all strands of DNA contained pieces of labeled thymidine-containing DNA attached to stretches of BrU-substituted DNA. In order to test for this, experiments analogous to those described above were performed on parental DNA. Cells were labeled with [14C]thymidine for 12 hours and then treated with or without U.V. irradiation. This was followed by incubation in BrdUrd for a further 12 hours.
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uv, no BrdUrd 313 nm
1 (b)
uvBrdUrd
no313nm
(d 1 uv. BrdUrd 313 nm c
IO Bottom
Fraction
no
.20 Top
FIQ. 10. Breakdown of parental DNA by light at 313 nm. Cells were lebeled for 12 hr with 0.5 @i [W]thymidine/ml., followed by (8) u.v., 12 hr in thymidine, exposed 8t 313 nm; (b) u.v., 12 hr in BrdUrd, no exposure 8t 313 nm; (c) no u.v.. 12 hr in BrdUrd, exposed 8t 313 nm; (d) u.v., 12 hr in BrdUrd, exposed 8t 313 nm. U.V. exposure 110 ergs/mma; 313 m-n-exposure 4.0 x lo6 ergs/mma; BrdUrd or thymidine 8t 2 x 10m5 M. In (d) the aounts in fractions 1 to 9 8re superimposable on the oounts in fractions 1 to 9 of (e), if these 8re divided by 1.7. The deahed lines in fractions 10 to 20 are obtained by dividing the counts of the corresponding f&&ions in (8) by 1.7. The dotted lines are obtained by subtracting these values from the total counts in the oorresponding frsctions in (d).
Finally the cells were exposed at 313 nm. If u.v.-induced recombination had taken place, the labeled parental strands should contain stretches of BrdUrd and degradation of the labeled DNA should be observed in the initially u.v.-irradiated cells but not in the unirradiated cells. Figure 10 shows the results of this experiment. Controls omitting the 313~nm irradiation, or incubating the cells in thymidine instead of BrdUrd showed little degradation (Fig. 10(a) and (b)). With initially u&radiated cells, incubated in BrdUrd and irradiated at 313 nm, appreoiable degradation of the parental DNA was observed (Fig. 10(o)). This suggests that a DNA molecule substituted in one strand with BrU is fragmented by 313 nm light to a small extent even in the opposite unsubstituted strand (about 1% of the breaks in the BrU strand). With initially u.v.-irradiated oells (Fig. 10(d)), a bimodal distribution was obtained. Because of the u.v.-induced lengthening of the generation time some cells will not yet have replicated all their DNA during the 12 hours in BrdUrd. Unreplicated DNA in these cells did not contain BrU and was therefore not broken down. DNA in the rest of the cells contained BrU and was broken down. Figure 10(d) can therefore be regarded as a composite of two separate profiles, as shown by the dotted lines. It can be seen that the extent of breakdown of the BrU-substituted population is the same
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A. R. LEHMANN
as, but not greater than, that of the initially unirradiated cells. The absence of any specific u.v.-induced breakdown of parental strands again suggests that there are no recombinational exchanges involving the parental strands, although the resolution in this experiment was somewhat lower than hoped for, owing to the breakdown in unirradiated cells. 4. Discussion The experiments described above show that in L5178Y cells, as in E. coli, the newly synthesized DNA formed after U.V. irradiation of the cells contain gaps, probably opposite the pyrimidine dimers, and these gaps are filled in on further incubation. This is in agreement with the findings for Chinese hamster cells (Cleaver & Thomas, 1969) and with preliminary reports of Rupp et al. (1970) and Zipser & Rupp (1970, Bbphys. Hoc. Ann. Meeting, Abstr. 10, p. 261a), also using L6178Y cells. This process may therefore represent the principal method whereby some mammalian cells (with the exception of primate-derived lines, which can also excise dimers) are able to tolerate the production of a large number of dimers in their DNA. The filling-in of gaps is prevented by hydroxyurea (Fig. 4), which inhibits the formation of deoxyribonucleoside triphosphates (Pfeiffer & Tolmach, 1967). This is of interest since the process of repair replication in human cells, as well as the small amount in Chinese hamster cells, is insensitive to hydroxyurea (Cleaver, 1969, 1970; Regan et al., 1971). Superficially both these processes are similar, in that each is a filling-in of gaps in DNA strands. Figures 2(a) and 3 show that not all the labeled DNA synthesized after 110 ergs/mma is of low molecular weight. Table 2 showed that the average interdimer distance after 110 ergs/mm2 was lo7 daltons, a length of DNA that is synthesized in about 15 minutes in these cells (Lehmann & Ormerod, 1970). In fact in some cases, since there is a distribution of interdimer distances, it will sometimes take appreciably longer to synthesize the DNA between two dimers. It is possible then that in these cases a gap is filled in behind the replication fork before it has reached the next dimer. Some of the labeled material would then be of high molecular weight. Further support is given to this hypothesis by the data of Figure 5, which show that most of the gaps are filled in within 1 hour of their formation. The experiments described in section (d) of Results utilized light of wavelength 313 nm, which specifically degrades and thereby identifies sections of DNA containing BrU. The results obtained suggest that the gaps in the daughter strands are filled in de novo and that recombinational exchanges are not involved. When the daughter strands were radioactively labeled and the gaps filled in, in the presence of BrdUrd, 313 nm light reformed the low molecular weight pieces observed immediately after the pulse-label. The possibility that these results could be explained by the action of an endonuclease which degrades sections of DNA from the ends of strands during exposure at 313 nm is rendered unlikely, since (1) the size of the pieces produced is dependent on the initial U.V. exposure and is the same as the size of the interdimer pieces formed immediately after the pulse-label, (2) when the length of labeled DNA in unirradiated cells is the same as that in the u.v.-irradiated cells, more low molecular weight material is still released from the u.v.-irradiated cells, and this is not merely a consequence of any instability imparted to the DNA by the presence of pyrimidine dimers (Table 2), and (3) leaving the samples in ice for a further few minutes after 313~nm exposure does not produce any more breakdown.
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These results demonstrate that the daughter strand gaps are f&d in with BrUcontaining DNA. The size of the BrU-containing stretch of DNA in the filled-in gap (about 809 nucleotides) is much larger than would be expected for a small amount of repair synthesis at the ends of the gaps. Furthermore with radioactively labeled parental strands (section d(iii)) and post-u.v. synthesis in BrdUrd, no BrU could be detected in the parental strands, suggesting that they were not involved in recombinational exchanges. We conclude therefore that recombinational exchanges are not necessary for repair of daughter-strand gaps in L5178Y cells, in contrast to the results obtained for E. co.%(Rupp et al., 1971). DNA appears to be synthesized de nova in the gap and somehow the dimer is bypassed. It is still possible that a very small part of the gap could be filled in with parental material, the rest being filled in with newly synthesized DNA, but gross recombinational exchanges, as detected in E. coli by Rupp et al. (1971) seem unlikely in L6178Y cells. The approximate value (866 nucleotides) for the size of the filled-in gaps, deduced in the Appendix, is quite similar to the size of the gap in E. coli (1096 to 1866 nucleotides) estimated by Iyer & Rupp (1971) using chromatographio techniques. (The values obtained by both methods are susceptible to quite large experimental errors.) This is of the order of the size of Okazaki fragments of DNA replication (Okazaki, Okazaki, Sakabe, Sugimoto t Sugino, 1968), as pointed out by Iyer t Rupp (1971), who also speculated on the possible significance of this observation. The model of postreplication repair in L5178Y cells that emerges from this work is that gaps of about 800 nucleotides are formed opposite pyrimidine dimers. These gaps are filled in quite soon afterwards by a process that involves de TWVOsynthesis and is inhibited by hydroxyurea. Some of the important questions which remain to be answered are : (1) What is the nature of the nucleotides inserted directly opposite the pyrimidine dimer Z Are they random nucleotides, or does the repair machinery have some mode of recognition of the distorted pyrimidines ? (2) Is postreplication repair in E. coli different from that in mammalian cells 1 Rupp et al. (1970,1971) have produced evidence to show that newly synthesized DNA is associated end-to-end with parental DNA strands after U.V. irradiation of E. coli, suggesting that recombinational exchanges are in fact involved in E. coli. My results show that it is probably not involved in mouse cells. (3) If the gap is filled in by de nova synthesis, why is a gap left in the first place ‘1 Presumably the enzyme system which fills in the gap is different Corn the replication machinery. (4) What is the nature of the site beyond the dimer, at whichsynthesisisreinitiated?
APPENDIX
Estimate of the size of filled-in gaps Table 3 shows that the number of pieces per growing strand formed during the pulse time is quite small (one or two), so that in general, one 313~nm light-induoed break in a f&d-up gap reforms the low molecular weight pieces. 22
A.R.LEHMANN
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TABLET Cal42.d4dionof number of end1 pieces synthesized during pulse label --U.Y-7. Dose (erl3 s/mm”)
Replication rate (106 daltonq min/strand)
Amount of DNA made in 30 mm by one replioation fork ( x 108)
0 110 220
oq 0*4$ 0*2$
24 12 6
Approx. size of interdimer spacing§ (106 daltons)
10 6
Average no. of short pieoea made in 30 mm
-1 -1
t Based on Lehmann & Ormerod (1970). $ Based on unpublished incorporation studies, assuming that the u.v.-induced decrease in the rate at eaoh replication rate of uptake of [3H]thymidme is caused by a reduation in replication fork rather than a decrease in the number of replicating forks. $ From Table 1.
Ifthe probability of forming a break between two nucleotides in the B&J-containing stretch is p and the number of nucleotides in this stretch is N, the probability of forming one or more breaks in this stretch is equal to 1 -eepN. This is approximately equal to the fraction of small pieces that are re-formed, F. Figure 11 shows a plot of F against Np. When Np = 1, F w 0.6.
Probability of forming o break x size of pop
FIG. 11. Theoretical
release of low molecular
weight pieces by irradiation
at 313 nm.
The BrU-containing material marked Y in Figure 6(b) is broken down at comparatively low exposures of 313~nm light (see Fig. 8). The further release of low molecular weight material from initially irradiated cells shown in Figure 8(a) corresponds to the re-formation of small pieces and is analogous to Figure 11.60% of this further release is obtained at a dose of about lo5 ergs/mm2. At this dose p = 1 break/2*4~ lo5 daltons, so that N (the size of the gap) w 2.4 x lo5 daltons or about 800 nucleotides. This value must be regarded as an approximation because of the background degradation observed in initially unirradiated cells, because in some cases two breaks are required to re-form the low molecular weight pieces and because of the arbitrary nature of the ordinate chosen for Figure 8(a). I am most out, and Dr J. K. Setlow, W. D. Rupp U.S. Atomic
grateful to both Dr R. B. Setlow, in whose laboratory this work was carried R. D. Ley for helpful discussions and encouragement, to Drs R. B. Setlow, and M. L. Randolph for critical comments about the manuscript, and to Dr for discussion of his unpublished results. This research was supported by the Energy Commission under contract with the Union Carbide Corporation.
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REFERENCES Care, L. (1965). l’&oZogy, 25, 226. Carrier, W. L. & Setlow, R. B. (1971a). AlutZ@. B&o&em. 43, 427. Carrier, W. L. & Setlow, R. B. (19716). In Methods in EnzymcZogy, ed. by L. Grossman & K. Moldave, vol. 21, p. 230. New York: Academic Press. Cleaver, J. E. (1969). Radiation Rea. 37, 334. Cleaver, J. E. (1970). Photo&em. Photobiol. 12, 17. Cleaver, J. E. & Thomas, G. H. (1969). Biochem. Biophys. Res. &mm. 36, 203. Cleaver, J. E. & Trosko, J. E. (1969). I&. J. Radiution Biol. 16,411. Cook, J. S. (1970). In PhotophysioZogy, ed. by A. C. G&se, vol. 5, p. 191. New York: Academic Press. Elkind, M. M. (1971). B&o&/e. J. 11, 602. Elkind, M. M. & Kamper, C. (1970). Riophys. J. 10, 237. Fischer, G. A. & Sartorelli, A. C. (1964). Methook Med. Res. 10,247. Hanawalt, P. C., Pettijohn, D. E., Pauling, E. C., Brunk, C. F., Smith, D. W., Kanner, L. C. L Couch, J. L. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 187. Howard-Flanders, P., Rupp, W. D., Wilkins, B. M. t Cole, R. S. (1968). Cold SF. Hurb. Symp. Quunt. Biol. 83, 196. Huberman, J. A. BERiggs, A. D. (1968). J. Mol. Bid. 32, 327. Hutchinson, F. L Hales, H. B. (1970). J. Mol. BioZ. SO, 69. Iyer, V. N. & Rupp, W. D. (1971). B&him. biophys. Acta, 228, 117. Klimek, M. (1966). Photo&m. Photdiol. 5, 603. Lehmann, A. R. (1970). Ph.D thesis, London University. Lehmann, A. R. & Ormerod, M. G. (1969). Nature, 221, 1053. Lehmann, A. R. BEOrmerod, M. G. (1970). B&&m. biophye. Acta, 204, 128. L&t, J. T., Klucis, E. S. L Sun, C. (1970). Biophye. J. 10,277. Lion, M. B. (1970). Biochim. biophys. Acta, 209, 24. McBurney, M. W., Graham, F. L. & Whitmore, G. F. (1971). B&him. Biophye. Res. comm. 44, 171. McGrath, R. A. BGWilliams, R. W. (1966). Nature, 212, 534. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. & Sugino, A. (1968). Proc. Nat. Acad. A%., Wash. 59, 548. Onnerod, M. G. & Lehmann, A. R. (1971). Biochim. biophye. Acta, 240, 369. Painter, R. B. & Cleaver, J. E. (1969). Radiu.t&m Rec. 37, 461. Pfeiffer, S. E. & Tohnach, L. J. (1967). Cancer Res. 27, 124. Rauth, A. M. (1970). In Current Topics in Radiation Research, ed. by M. Ebert & A. Howard, vol. 6, p. 195. Amsterdam: North-Holland Publishing Co. Regan, J. D., Setlow, R. B. & Ley, R. D. (1971). Proc. Nat. Acud. Sci., Wwh. 68, 708. Regan, J. D., Trosko, J. E. & Carrier, W. L. (1968). Biophys. J. 8,319. Rupp, W. D. L Howard-Flanders, P. (1968). J. Mol. Biol. 31, 291. Rupp, W. D., Wilde, C. E., Reno, D. L. & Howard-Flanders, P. (1971). J. Mol. Bid. 61, 25. Rupp, W. D., Zipser, E., von Essen, C., Reno, D., Prosnitz, L. & Howard-Flanders, P. (1970). In Time and Dose Re.k%mehip in Radicltion Biology aa Applied to Radiothevupy, p. 1. Brookhaven Natl. Lab. Publication BNL 50203 (C-57). Setlow, J. K. (1967). In Compehetive Biochemietry, ed. by M. Florkin & E. H. Stotz, vol. 27, p. 167. Amsterdam: Elsevier. Setlow, R. B. (1968). Progr. Nucleic Acid Res. & Mol. Biol. 8, 257. Setlow, R. B. & Carrier, W. L. (1966). J. Mol. Biol. 17, 237. Smith, K. C. (1971). In Photophysiology, ed. by A. C. Giese, vol. 6, in press. New York: Academic Press. Steward, D. L. & Humphrey, R. M. (1966). Nature, 212, 298. Trosko, J. E., Chu, E. H. Y. & Carrier, W. L. (1965). Radiation. Res. 24, 667.
Postreplication
Repair of DNA in Ultraviolet-irradiated Mammalian Cells A. R. LEHMMN~
Biology Division, Oak Ridge National Laboratory Oak Ridge, Tenn. 37830, U.X.A. (Received 21 April 1971, and in revised form 9 December 1971) DNA synthesized in ultraviolet-irradiated L5178Y mouse lymphoma cells is smaller than that made in u&radiated cells, ae shown by pulse-labeling with tritiated thymidine, followed by sedimentation of the DNA in alkaline sucrose gradients. Hence, DNA synthesized on ultraviolet-irradiated DNA templates contains discontinuities. The estimated distance between these discontinuities is comparable to the average spacing between pyrimidine dimers in the parental strands, suggesting that the replication machinery leaves gaps opposite the dimers. On subsequent incubation these discontinuities are Clled in by a process which is inhibited by hydroxyurea. In order to determine whether the gaps are filled in by a recombinational mechanism or by de 7aoe)osynthesis, irradiated cells were pulse-labeled with radioactive thymidine, followed by incubation in bromodeoxyuridine and exposure to light of wavelength 313 nm. Bromuracil-containing sections of DNA are selectively disrupted by light of this wavelength. Thus if the gaps in the radioactively labeled daughter strands are filled in by exchange with thymidine-containing DNA from the parental strands (recombinational exchange), they will not contain bromuracil, and the molecular weight distributions of the radioactive DNA from initially irradiated and unirradiated cells will be similar after exposure to 313-nm light. On the other hand, if the gaps are filled in by bromuracil-substituted DNA (arising from de nOvo synthesis), the small pieces of radioactive DNA synthesized in the initially irradiated cells during the pulse-labeling will be reformed on exposure to 313-nm light, and the molecular weight distribution from those cells will have a lower average molecular weight than that from similarly treated unirradiated cells. The results are in accordance with the second alternative, indicating that the gaps in the daughter strands opposite the pyrimidine dimers are filled in with newly synthesized DNA. Furthermore parental DNA does not seem to be involved in the gap-filling process. This excludes many of the recombinational models which have been put forward to account for similar gap-Clling phenomena observed in bacteria. The results give an approximate value of ,800 nucleotides for the size of the filled-in gaps.
1. Introduction A surprising aspect of the effect of ultraviolet light on mammalian cells is that, despite the fact that mammalian DNA offers a target about 1000 times as big M the DNA in bacteria, the U.P. sensitivities of mammalian cells and bacteria are very similar. This is in contrast to the situation with X-rays, to which mammalian cells are in general 10 to 100 times more sensitive. A typical U.V. survival curve for mammalian cells has a shoulder and a D, value both of about 50 to 100 ergs/mm2 (see review by t Present address: Biology England. 21
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Rauth, 1970). In this dose range there are about 10e pyrimidine dimers per cell (assuming a total mass of DNA per cell of lo-l1 g and a percentage of pyrimidines as dimers of 0.02 to 0.04). This clearly implies that mammalian cells are capable of tolerating the production in their DNA of a large number of such alterations without being killed. In bacteria there are three known types of repair processes for removing or circumventing pyrimidine dimers: (1) photoreactivation (see review by J. K. Setlow, 1967). This process has been detected in marsupials but in no other mammals (see review by Cook, 1970). (2) Excision-repair of dimers, whereby a section of the DNA containing the dimers is excised by the cell’s repair machinery (see review by R. B. Setlow, 1968). The resulting gap is then f&d in by a process termed repair-replication (see review by Hanawalt et al., 1968). Human and other primate cell lines excise ditners efficiently from their DNA (Regan, Trosko & Carrier, 1968) but very little dimer excision (Trosko, Chu & Carrier, 1966; Steward & Humphrey, 1966; Klimek, 1966; Cleaver & Trosko, 1969) and only very low levels of repair-replication (Painter & Cleaver, 1969 ; Cleaver, 1970) have been detected in rodent cells. (3) Postreplication repair (“recoli which was unable to excise combinational repair”) ; using a strain of Edwichia thymine dimers yet could still survive a U.V. dose producing about 50 dimers per cell, Rupp & Howard-Flanders (1968) showed that the DNA synthesized in u.v.-irradiated cells had a lower molecular weight (as measured in alkaline sucrose gradients) than that from unirradiated cells, suggesting that this newly synthesized DNA contained gaps. Subsequently the gaps were filled in, as demonstrated by an increase in size of the newly synthesized material during further incubation. In later work Rupp and co-workers presented data which suggested that the gaps in the new strands were opposite pyrimidine dimers in the parental strands (Howard-Flanders, Rupp, Wilkins & Cole, 1968) and estimated that the size of the gaps was about 1000 to 1600 nucleotides in length (Iyer & Rupp, 1971). They have recently presented evidence (Rupp et al., 1970; Rupp, Wilde, Reno & Howard-Flanders, 1971) for end-to-end association of daughter and parental DNA after U.V. irradiation, supporting their hypothesis that recombinational exchanges are involved in the filling-in of the gaps in E. coli. The process of postreplication repair has been detected in all strains of E. coli tested, exoept for those carrying the recA mutation (see review by Smith, 1971). The existence of similar processes in Chinese hamster cells and mouse lymphoma L5178Y cells has been briefly reported by Cleaver t Thomas (1969) and Rupp et al. (1970). This paper describes the process in detail in mouse lymphoma cells (L5178Y) and presents evidence that in these cells postreplication repair does not involve recombinational exchanges.
2. Materials and Methods The cell line used in all experiments was the mouse lymphoma line L6178Y (generously provided by Dr G. A. Fischer), which was grown in suspension culture at 37°C in Fischer’s medium (Fischer & Sartorelli, 1904) supplemented with 10% foetal calf serum. The doubling time was 11 to 12 hr.
(a) Ultraviolet
irradiation
Cells were spun down, washed in PO, saline (phosphate buffered saline: 8.0 g NaCl, 0.2 g KCl, 1.15 g Na,HPO,, 0.2 g KH2P0,, 0.1 g CaCl,, 0.1 g MgCl, . 6H,O/l.) and resuspended in 5 to 10 ml. PO4 saline at a cell concentration of about 3 x 105/ml. The cell suspension was stirred in a Falcon Petri dish and irradiated with U.V. light (from a mercury germicidal lamp) at 264 nm with an incident U.V. exposure of 110 or 220 ergs/mm2.
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(b) Radioache lxzb&ng After irradiation the cells were resuspended in Fischer’s medium, and Biter 8 certain interval of time, ,25 or 50 &i [naet~~Z-3H]thymidin+nl. (lSCi/m-mole, Schwarz BioResearch, Inc.) were added and the cells incubated usually for O-5 hr. (c) Ckz8e eampe9immt8 In ch8sse experiments, the cells were spun down after radioactive labeling, washed, and resuspended in medium containing 25 M deoxycytidine/ml. 8nd either 2-5 pg thymidine/ml. or 5 pg BrdUrd/ml., and incubsted for various periods of time. (d) Degmdatkn wing 313~nm light In these experiments, after incubation with BrdUrd the cells were spun down, washed in PO4 saline, and resuspended in this buffer at 8 concentrstion of 2 to 3 x lo6 cells/ml. The suspension ~8s then exposed in 8n ice-cooled l-cm-path quertz microcuvette to light of wavelength 313 nm from 8 18rge, qu8rtzprism Hilger monochrom8tor illuminated with 8 Philips 1000 W, high-pressure, water-cooled mercury arc. A thin Myl8r film W&Binserted between the monochromator and the sample to eliminate 8ny scattered shortw8velength light. The exposure rate 8s measured by a calibrated thermopile ~8s about 1.3 x lo5 ergs/mmz/min, end the incident exposure was between 0 and 6 x IO5 ergs/mma. Cells were incubated for 4 to 5 hr with 20 PCi [3H]BrdUrd/ml. at total concentration of 5 pg/ml. end 2.5 CcQdeoxycytidine/ml. and then treated exactly as in the shove section, but irrsdiated at 313 mn in the exposure range of 0 to IO4 ergs/mma. The exposure r&e w&8 5 to 10 x lo3 ergs/mma/min. A 3.9~ml. gradient of 5 to 20% sucrose, O-1 M-N&~, O-1 M-N~OH wss formed in a 4ml. polyallomer tube. One-tenth ml. of 2% sodium dodeoyl sulfate, 0.02 as-EDTA wae layered over this. In all experiments, immediately before lysis and centrifugation, the cells, resuspended in PO4 saline, were irmdi8ted with 2 kred of X-rsdietion (see Results, se&ion (8)) delivered from a 250 kVpf30 mA, G. E. k&xitron X-ray msohine (+-mm Al filter), and a volume containing 5 to 15 x lo3 cells (i.e. about 0.1 ccgof DNA) WBBthen 18yered into the sodium dodecyl sulf8te on top of the gradients. These were centrifuged immediately et 20°C in the SW56 rotor in 8 Beckman model L ultracentrifuge, usually at 40,000 rev./min for 75 min. After centrifug8tion the bottoms of the tubes were pierced with 8 Buchler tube piercer, and 26 to 29 &drop fractions were collected on whatman no. 17 peper strips (1 cm width), 8 modification of the filter-paper disk technique 8s described by Carrier & Setlow (19718). The strips were washed in 5% trichloroacetic &d, twice in ethanol, and fhmlly in 8cetone. When dry, they were cut up into scintillation vi&~ and the radioactivity ~8s measured in 8 Packard liquid-scintillation spectrometer, with toluene containing 4 g 2,5-bis-2-(S-tert.-butylb enzoxazolyl)-thiophene/l. 8s ecintilletor. Calculations of moleoulsr weights and percentage counts for each fraction were performed by a computer program which used the empiric&y determined relationship between molecular weight, M, and the mtio of the distanoe sedimented to the length of the liquid column, D: M = 5-02 x lo3 (loll D/wat)a’6, where w is the rotor speed (rev./mm) and t is the centrifug&ion time (mm). In experiments in which accurate molecular weights were required, marker DNA from h phsge ~8s used, aesuming a single-strend molecular weight of 16 x lo6 d&ons (Caro, 1965).
3. Results (B) Problems and interpretations of sedimentation pro$les of DNA from mammalian cell8 Presently available data suggest that 8 replicating DNA molecule in 8 mammalian cell hes the structure shown in Figure 1(e) (Huberman & Riggs, 1968 ; Lehmann, 1970 ; Lehmann t Ormerod, manuscript in prep8ration). Replicating units (replicons) are
A. R. LEHMANN
322
(d)
(e)
2 0
Bottom
20
IO Fraciion
Top
no.
FIU. 1. Sedimentation profiles of DNA from unirmdiated celk labeled for various periods of time and the DNA struotures they represent. The DNA of the cells was labeled with radioactive thymidine, after which the cells were X-irradiated 8nd lysed in detergent on top of mlkmline sucrose gradients. These were then centrifuged for 75 min 8t 40,000 rev./min. (8) Cells labeled with 0.5 pi [‘*C]thymidine/ml. for IS hr; (b) and (c) cells lsbeled with 25 PCi [3H]thymidine/mJ. for 30 min and 16 min, respectively. Total cts/min: (8) 4225, (b) 9661, (c) 3779. (d) Replicated DNA, (e) replicating DNA mt different stages of the S-period. The discontinuous verticel lines delineate separate replicons. Sediment&ion in all Figs is from right to left.
joined end-to-end and are approximately lo* daltons in length (Huberman & Riggs, 1968; Lehmann & Ormerod, manuscript in preparation). In a pulse-labeling experiment onIy the growing DNA strands (the inside strands in Fig. l(e)) become radioactively labeled. However, the much longer parental strands, though unlabeled, are unfortunately present, and in alkaline sucrose they entangle with the growing strands, rendering any interpretation of the profiles meaningless (Lehman, 1970; LehnmM & Ormerod, manuscript in preparation). This can be circumvented by degrading the DNA to a small extent, so that the long parental strands are somewhat broken down but the shorter, growing strands are hardly affected. Although this can be done chemically, with strong alkali (Regan, Setlow & Ley, 1971; Elkind & Kamper, 1970; Lett, Klucis t Sun, 1970), a low dose (1 to 3 krads) of X-rays (Lehmann BEOrmerod, manuscript in preparation; Lehmann, 1970; Elkind & Kamper, 1970) has been found to be much more reproducible, and can be more accurately controlled. It also takes less time, and is therefore more convenient. Hence at the end of all treatments cells were X-irradiated for 5 seconds (2 brads)---a dose that produces approximately one single-strand break per 200x lo6 daltons (Lehmann & Ormerod, 1970). The growing
POSTREPLICATION
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strands, being mostly shorter than this, were hardly affected. It should be emphasized that the sole purpose of this X-irradiation was to fragment the parental DNA strands and thus prevent entanglement. The cells were always lysed immediately (i.e. within seconds) after this irradiation. All the experiments described below utilized mouse lymphoma L5178Y cells, which do not excise pyrimidine dimers from their DNA after U.V. irradiation (results to be published elsewhere). Figure 1 shows sedimentation profiles of DNA from unirradiated cells under different circumstances together with the structures of the DNA they are presumed to represent. The first (Fig. l(a)), which will be referred to as type I, represents “joined-up DNA” (as shown in Fig. l(d)) and is typical of a profile from DNA labeled for several hours. Its main characteristics are a peak with a sharp front at &f = 150 to 200 x lo6 daltons and a low molecular weight tail. As discussed in some detail in an earlier paper (Ormerod & Lehman, 1971) and as reported by McBurney, Graham & Whitmore (1971) and Elkind (1971), this sharp front is caused by a sedimentation anomaly and is seen in all profiles of DNA with molecular weights greater than 2 x 10s daltons, whether from bacteria or mammalian cells, if the DNA is centrifuged at high rotor speeds (20,000 rev./min or more) (e.g. McGrath & Williams, 1966; Rupp & Howard-Flanders, 1968; Lett et al., 1970; Elkind & Kamper, 1970; Ormerod & Lehmann, 1971; Regan et al., 1971). The material at this peak represents an accumulation of DNA of molecular weight between 15 and 10 x 10s daltons. The second and third types (type IIA in Fig. l(b) and IIB in Fig. 1(c)) represent “growing DNA” (Fig. l(e)). They are characterized by broad, flat-topped profiles stretching across the middle of the gradient. The distribution arises because in an asynchronous population there are growing strands of all sizes between zero and the size of the replicating unit (about 1O8) such as those shown in Fig. l(e). After a l&minute pulse (Fig. I(c), type IIB), a small peak is seen at fractions 15 to 17 (M w 4 x 107) ; this arises from a population of relatively small replicating units and is less marked in the 30-minute pulse (Fig. l(b)), type IIA), when much of the DNA in the small units has elongated and joined up to the DNA in adjacent units. These profiles have also been discussed in detail elsewhere (Lehmann, 1970; Lehmann BE Ormerod, manuscript in preparation). Prolonged incubation after a short pulse label would be expected to convert type II prollles to type I, corresponding to elongation of the growing strands and joining up of adjacent replicons after synthesis. (b) Formation of gaps after ultraviolet irradiation Pulse-labeling cells after U.V. irradiation produces completely different profiles as shown in Figure 2. These proflles are referred to as type III. There is a pronounced peak near the top of the gradient at a molecular weight of 15 to 25 x lo6 daltons after 110 ergs/mm2 and 5 to 15 x lo6 daltons after 220 ergs/mm2, showing that the DNA synthesized after U.V. irradiation is indeed shorter than that synthesized without prior U.V. exposure. This confirms the results of Cleaver & Thomas (1969) and a preliminary report of Zipser & Rupp (1970, Biophys. Sot. Ann. Meeting, Abstr. 10, p. 261a). In order to test whether the gaps in the DNA synthesized after U.V. exposure were opposite pyrimidine dimers, cells were labeled for 16 hours with O-5pCi [14C]thymidine/ ml. (50mCi/m-mole) and U.V. irradiated. Half of the irradiated cells were examined for dimer content as described by Carrier & Setlow (1971b). The rest were incubated and pulse-labeled with [3H]thymidine, and a sedimentation experiment was carried
A. R. LEHMANN
324 12 (a)
110 ergs /mm2 M=25x106
cn
Uottom
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Fraction no.
FIG. 2. Sedimentation profiles from u.v.-irrediated oells. cells were incubated (b) 220 ergs/mm a. After irradiation, 30 min with (a) 25 pCi [sH]thymidine/ml. or (b) 60 pCi carried out et 40,000 rev./mm for (a) 76 min or (b) 86 min.
Exposures were (a) 110 ergs/mms and for 46 min and then pulse-labeled for [3H]thymidine/ml. Centrifugation was Total ots/min: (a) 4259, (b) 2398.
out as described in Materials and Methods. In Table 1 the number-average molecular weights for the low molecular weight pieces of the newly synthesized 3H-labeled DNA (calculated from the sedimentation experiment) are compared with the interdimer spacings in the parental strands (calculated from the fraction of l*C radioactivity in pyrimidine dimers). Good agreement was obtained, suggesting that the gaps might indeed be opposite the dimers. The replication mechanism envisaged by Rupp & Howard-Flanders (1968) for u.v.-irradiated E. coli was as follows: in an irradiated cell the replication machinery continues to elongate the growing DNA strand until it reaches a dimer. It then leaves a gap and synthesis recommences at some point beyond the dimer. In my experiments, as shown above, the average interdimer distance is about lo7 daltons after 110 ergs/mnP. As shown in earlier work (Lehmann t Ormerod, 1970), the replication rate in these cells is about O-8x lo6 daltons/minute. Hence it should take on the average 1245 minutes for the replication machinery to proceed from one dimer to the
TABLE
1
Convpari8on of dimer spacing and size of piece8 synthesized in irradiuted cell8 U.V. exposure @f3d=*)
Percentage of W radioactivity as dimers
Calculated interdimer spacing? (108 daltons)
110
0.019
9.5
220
0.032
54
Measured ikf,, of3 low molecular weight pieces ( x 10s) 12.0 6.6
t Based on assumptions: (1) ratio of dimers in E. COGDNA, T-T/T-C/Cc = 6:4: 1 (Setlow & Cerrier, 1966); (2) thymine content of mouse DNA = 30% henae; (3) ratio of dimers in mouse DNA, T-T/T-C/C-C= 6*2:3.3:05. t. Calculated from the slope of a plot of log (number of molecules in eaoh fraction) against moleoular weight (Lehmann & Ormerod, 1970).
POSTREPLICATION
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325
next. Thus it might be expected that during the first few minutes immediately after irradiation, in some replicons the replicating machinery would not yet have reached a dimer, so that the labeled DNA would not be in short pieces. This prediction is borne out by the experiment shown in Figure 3, in which cells were pulse-labeled for 16 minutes at 0, 10, and 20 minutes after irradiation. With increasing times before labeling, the amount of low molecular weight material increased. However, after 20 minutes there was still a high molecular weight tail, and this remained even if the cells were labeled 30 to 60 minutes after irradiation. Possible explanations for this are given in the Discussion. In subsequent experiments, in order to obtain maximum amounts of low molecular weight DNA, the cells were always incubated for 30 to 60 minutes between U.V. irradiation and pulse-labeling.
Pulse after
aottom Fraction
no.
Top
FIG. 3. Sedimentation profiles from cella pulse-labeled at various times efter irradiation. Cells were exposed to U.V. light of 110 er&nma, incubated for 0, 10, or 20 min and then pulselabeled for 16 min with 26 $i [3H]thymidine/ml. Total ctsjmin: 3080 (-), 4898 (---), 2571 (. . .).
(c) Filling-in
of gaps
Figure 4 shows results from pulse-chase experiments. With unirradiakl cells, a 0*5-hour pulse (Pig. 4(a)) gave, as expected, a typical type II pro6le (seePig. l(b) and discussion thereof). A subsequent 4 hour incubation in unlabeled thymidine converted this to a type I pro& (Pig. 4(b)), indicating elongation of the growing strands and joining of the strands in adjacent replicons. This process was prevented by 10e3 Yof DNA synthesis (Pfeiifer & Tolmach, hydroxyurea (Pig. 4(c)), an inhibitor 1967). With u.v.-irradiated cells the straight pulse gave a type III profile (pig. 4(d)). Pilling-in of the gaps should be manifested by a conversion of a type III to a type II profile and subsequent elongation and joining up by conversion of type II to type I. Both processes occurred during a 4-hour incubation in unlabeled thymidine, and a type I profile was obtained (Pig. 4(e)). In the presence of hydroxyurea (Pig. 4(f)) a type III profile remained, demonstrating that both gap-6lling and elongation and joining up
326
A. R. LEHMANN
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(c ) Chase t hydroxyurea
0 Bottom Fraction
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Tap
FIG. 4. Sedimentation pro&s from cells pulse-labeled and chased. with 160 ergs/mma ((d), (e) and (f)) were Cells unirradiatad ((a), (b) and (c)) or u.v.-irradiated incubated for 46 min, then pulse-lrrbeled for 30 min ((a) and (d)), chased for 4 hr in the absence ((b) and (e)) or presence ((c) and (f)) of 2 x 10m3 aa-hydroxyures. Total cts/min: (a) 1296, (b) 9800, (c) 13,600, (d) 2106, (e) 2303, (f) 2060.
were prevented by this inhibitor. (The small amount of increase in molecular weight observed in the presence of hydroxynrea was not always seen and may represent synthesis occurring before the hydroxyurea has been taken up into the cells.) Figure 5 shows the kinetics of the chase. Most of the gap-tilling (type III --t type II) was completed within the first hour and the subsequent elongation and rejoining took a further 4 hours. (d) Recombination versus de novo synthesis The gap-filling process could take place either (a) by a recombinational exchange mechanism, whereby the gap is filled in by the corresponding piece of parental DNA from the sister duplex (Howard-Flanders et al., 1968). Presumably the resulting gap in the parental strand is subsequently filled-in by de rwvo synthesis ; or (b) by direct synthesis of new material in the daughter-strand gap. In E. coli, Rupp et al. (1971) have recently produced evidence favoring the former mechanism. The two mechanisms may be distinguished by following the fate of the parental DNA strands and of the DNA made after U.V. irradiation. On the recombinational
POSTREPLICATION
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2 hr Chase
Bottom
IO
20
IO
0 Fraction
20
)P
no.
Pm. 5. Sedimentation profiles from irradiated cells pulse-labeled and ohased for various times. Cells irradiated with 110 ergs/mma, incubated for 45 min then pulse-labeled for 30 min (a), followed by a cold abase of 1 hr (b), 2 hr (a), or 4 hr (d). Total cts/min: (8) 2910, (b) 4433, (c) 3831, (d) 3300.
model daughter-strand gaps are filled in with pre-existing parental mate&l and subsequently the parental DNA strands will contain stretches of material synthesized after U.V. irradiation. On the other hand, direct synthesis will result in the daughter strands containing material synthesized subsequently, the parental strands remaining intact throughout. These predictions were tested in L5178Y cells by two types of experiment, both taking advantage of the selective photolysis of bromouracil-containing sections of DNA by U.V. light. It has been shown (Lion, 1970; Hutchinson & Hales, 1970; Hewitt & Marburger, 1971, Biophys. Sot. Ann. Meeting, Abstr. 11, p. 27a) that U.V. light introduces single-strand breaks (or alkali-labile bonds) into BrU-substituted DNA. This effect has been studied in detail in this laboratory by Setlow, Regan & Ley, who have found that the extent of damage to BrU-substituted DNA verse unsubstituted DNA is greater at s, wavelength of 313 nm than at 254 nm. This technique has been used for measuring the extent of repair replication in mamm&an cells (Regan et al., 1971) and in E. cdi (Ley & Setlow, 1972). (i) Fate of the daughter-strand gaps Cells were U.V. irradiated (254 run), pulse-labeled, and then incubated in the presence of BrdUrd for 4 to 5 hours, followed by exposure to 313~run light. The expected effects on the DNA molecules are shown in Figure 6. With u&radiated cells (Fig. 6(a)), the initial [3H]thymidine pulse labels the growing strands (A), giving a type II profile. On incubation the growing strands are elongated with BrU-containing material and joined up to adjacent replicons (B), giving a type I profile. Light at 313 nm breaks down the BrU-containing sections (C), returning to a type II protile (only the strands containing a section marked with a wavy line in Fig. 6 are identified).
328
A. R. LEHMANN (0)
Unirmdiated
A -B --
//
“5\ I
--
Chase+BrdUrd --
II Y
C --
I
Y
313 nm I
N8-mmmam
Profile type It
--
-.
II
(b) Profile III
A /Y--+G!l
type
U.V. irradiated Profile type m
h-----V Chase+Brd
Urd
I I
B -
B -
,!
C II
l
*:: w
I
-
313Ynm
-mu-
r
C
313nm III
m -M--Mm Recombination
de now
Synthesis
FIG. 6. Expected effect of 313-nm light on unirradiated (a) and u.v.-irradiated (b) cells. Celle pulse-labeled (A) and ohaaed with BrdUrd (B) followed by treatment with 313-nm light (C). (b) Left, gaps filled in by reaombination; r&ght, gaps flllad in by de W.WOayuthesis. (-) DNA synthesized before pulse label; (-) DNA synthesized during pulse label; m DNA containing BrU; (X) pyrimidine dimers (only relevant dimers shown); (Y) long BrU-containing stretoh of DNA joining up adjeoent replicons. Note: For clarity, the replicating process taking plaoe on only one strand of the DNA duplex is shown.
With irradiated cells (Fig. 6(b)), the initial profile should be of type III, which on incubation gives a type I profile after gap-filling and elongation and joining up. There are now two possibilities. (1) According to the recombinational mechanism postulated by Rupp et al. (1971) for E. coli, the gaps are filled in by parental material, and the new material (BrU-containing) fills in the resulting gap in the unlabeled parental strand. Hence, treatment with 313-nm light would not be expected to re-form the discontinuities in the (labeled) daughter strands, as shown in Figure 6(b), left. Thus the effect of 313-nm light would be identical to that in the initially m&radiated case (type I+ type II). (2) Alternatively, the gaps would be filled in by de 120~0 synthesis. In this case the gaps would be filled in by BrU-containing material. The 313-nm light should introduce breaks into these sections (Fig. 6(b), right), and the small pieces observed after the pulse of [“Hlthymidine should be re-formed, resulting in a type III protIle. Figure 7 shows sedimentation profiles from experiments with 313-nm light-induced degradation. The left-hand diagrams in Figure 7 show the profiles after a chase with BrdUrd following U.V. exposures of 0,110, and 220 ergs/mm2. Although the elongation and rejoining process was not quite complete after the chase following the initial exposure of 220 ergs/mm2 (Fig. 7(c)), there was no trace of the initial low molecular weight material seen after the pulse (compare Fig. 2(b)), demonstrating that the gaps had been filled in. The right-hand Figures are profiles after a subsequent 313-nm exposure of 3.4 X lo6 ergs/mm 2. With the initially m&radiated cells there was a return to a type II profile as expected (compare Fig. l(b)) ; with the 110 ergs/mm2
POSTREPLICATION
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329
12
1-J
0
n
I
110 ergs/mm2
110eras/mm2
(0
(e) 220 ergs/mm2
4
0~ Bottom
I
(d)
(c)
t 8i-
I
IO
20
t 220 ergs/mm*
i.:-r’”
0 Fraction No
IO
20
‘P
FIG. 7. Sedimentation prof?les from u.v.-irradieted cells after 313-nm exposure. Cells were irradiated at 264 nm, inaubatad for 46 min. puke-labeled, chased with BrdUrd for 4 to 6 hr (left) and then exposed to 3.4 x lo5 ergs/mm~ et 313 nm (right). (13)and (b) No initial irradiation, 30-min pulse, 4*6&r chase; (0) and (d) 110 ergs/mma, SO-min pulse, 46hr chase. (e) and (f) 220 ergs/mm’, 30-min pulse, B-hr chase. Total cts/min: (a) 9800, (b) 4886, (a) 3006, (d) 3663, (6) 2087, (f) 1609.
sample the peak at fractions 17 and 18 began to reappear (compare Fig. 2(a)) ; and with the 220 erge/mma sample there was the clear reappearance of the low molecular weight peak at the same position as that obtained immediately after the pulse (type III, compare Fig. 2(b)). Thus in each case there was a return to a profile type similar to that seen after the pulse label with no further treatment. Thus the gaps in the DNA synthesized after the initial U.V. exposure were reformed on exposure to 313-nm light after the chase with BrdUrd, indicating that they had been filled in with BrUsubstituted DNA (i.e. by de nova synthesis (Fig. 6(b), right)). In a control experiment, in which the chase was carried out with thymidine instead of BrdUrd, no breakdown was observed on exposure at 313 ,nm, a type I profile remaining even at very high exposures.
A.
330
R. LEHMANN
The results of all the experiments with 313-nm light-induced degradation are shown in Figure 8, where they are plotted in two different ways: as the amount of radioactivity in the region of 5 to 55 x lo6 d&as (i.e. the low-molecular weight region) divided by the total radioactivity of the DNA with molecular weight above 5 x lo6 (Fig. 8(a)); and as the measured weight-average molecular weight (Fig. 8(b)). (Since the DNA molecules are not uniformly labeled this does not give a true weightaverage molecular weight.) The data have been plotted somewhat arbitrarily in this manner in order to demonstrate the following points : (1) in all cases at low exposures of 313~nm light (0 to 0.2 x lo5 ergs/mm2) there was a
. .
0 (b) P
IO-> Exposure to313nm light (ergs/mm’) FIG. 8. Re-formation of low molecular weight pieces by 313-nm light. (8) Fraction of the radioaotivity of molecular weight in excess of 5 X 10s lying between 5 and 55 x 10s daltons after different doses of 313-nm light. (b) Measured weight-average molecular weight (2 counts x moleoular weight/ 2 counts). Cells were initially irradiated et 254 nm with 0 for 30 min. (-O-O-), 110 (-•-•--) or 220 (-m--m--) ergs/ mma and pulse-labeled (-n-n-) Unirradiated cells pulse-labeled for 16 min. For both (a) and (b) the points at the right are those obtained immediately after pulse-labeling, with no subsequent treatment with BrdUrd or 313-nm radiation (i.e. these represent DNA shown in the upper diagrams (A) of Fig. 6(a) and (b)).
POSTREPLICATION
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rapid degradation to a certain level. This was the result of breakdown of the long BrU-containing piece of DNA that joined up adjacent replicons, marked Y in Figure 6, producing a conversion of type I to type II profiles. (2) With initially unirradiated cells after higher exposures at 313 nm there appeared to be a further small amount of degradation. (3) With initially irradiated samples there was a reproducibly greater amount of breakdown in the range O-2 to 3 x lo5 ergs/mm2. This resulted from the re-formation of the filled-in gaps. Above 3 x lo5 ergs/mm2 there was a further small amount of degradation similar to that seen in initially m&radiated cells. (4) With initially irradiated samples, after high doses of 313-nm light, the fraction of low molecular weight material and the measured weight-average molecular weights were similar to those obtained after the pulse-label without further treatment (shown at the right of the Figure). Because of the degradation seen in initially u&radiated cells, the differences between the profiles from cells initially unirradiated and those u.v.-irradiated with 110 ergs/mm2 were not large. Since the rate of synthesis after an exposure of 110 ergs/mm2 was about one-half that in u&radiated cells, it was essential to compare the profiles from the irradiated cells with those from unirradiated cells that were both pulselabeled for 05 hour and chased for 4 hours (pulse and chase times the same as for u.v.-irradiated cells) and pulsed for 15 minutes and chased for 2 hours. In the latter case the lengths of label were the same as in the u.v.-irradiated cells, so that labeling artifacts were avoided (Lehmann & Ormerod, 1969). There was more low molecular weight material produced by 313-nm light with the 15-minute pulse than with the 30-minute pulse, but this was also seen immediately after the initial pulse (Fig. 8, open squares). With the 110 ergs/mm2 sample, the 313-nm light-induced breakdown was still reproducibly greater, and with the 220 ergs/mm2 sample, the breakdown was clearly much greater. If the cells were pulse-labeled before the initial U.V. irradiation, the amount of degradation by 313-nm light was the same in irradiated and u&radiated cells (see Table 2), showing that the degradation is specific for DNA synthesized after the U.V. irradiation and does not merely reflect a greater sensitivity of u.v.-irradiated DNA to 313-nm light. These results show that in all cases there was a return to the profiles obtained immediately after the thymidine pulse, along with a small amount of background TABLE
2
Effect of 313~nm Eight on cells ultraviolet-irradiated Initial u.v. exposures (ergs/mm”) 0 110 0 110
10-s x exposure to 313-nm light (ergs/mm”) 1.9 1.9 3.8 3.8
Percentage low molecular weight material 30 31 34 35
after pulse labeling Measured weightaverage mol. wt (x 106) 89 86 82 80
Cells pulse-labeled for 30 min were u.v.“irradiated, incubated for 4 hr in the presence of BrdUrd and then irradiated with 313-nm light. Measured weight-average mol. wt is defined in the legend to Fig. S(b).
A. R.
332
LEHMANN
degradation. This shows that the gaps were filled-in (at least partly), with BrUcontaining DNA, i.e. by de nova synthesis (Rig. 6(b), right). (ii) Cdibration
of 313-nrn light-indeed
depdation
From the exposure of 313~nm light at which the low molecular weight peak appeared it is possible to calculate the size of the BrU-containing piece of DNA inserted into the gaps. In order to do this it is necessary to measure the amount of breakdown of BrU-containing DNA by 313~nm light. In these experiments, cells (unirradiated) were incubated with [3H]BrdUrd under identical conditions to those used in the chase experiments and subsequently exposed to 313-nm light. Under these conditions a linear effect of dose was obtained as shown by a plot of l/number-average molecular weight against dose at 313 run (Fig. 9). The dose-effect line gives a value of 1 break/ 2.4 x lo5 daltons/105 ergs/mma, which is similar to the values obtained by Ley &
10m3 x Exposure (ergs/mm2)
FIU. 9. The effeot of irradietion at 313 nm on the moleoular weight of BrU-substituted DNA. Cells were labeled for 4 hr with 20 pCi [3H]BrdUrd/ml. et 8 total conoentration of 6 pg/ml. 8nd irredi8ted with different exposures of 313 nm. Numberaverage mol. wt (ikf,,) of sediment8tion proGlee o&utsted 88 described in the legend to Teble 18nd in Lehm8nn & Ormerod (1970).
Setlow (1972) for irradiation of fully BrU-substituted DNA in E. di and Chinese hamster cells. In Figure 8 most of the release of low molecular weight fragments (type II --t type III) occurs over the exposure range of 313~mu light of about 0.5 to 2 x lo6 ergs/mma. As calculated in the Appendix, this release pattern corresponds to the pattern that would be expected if the size of the filled-in gap were about 800 nucleotides (though this value could well be in error by a factor of 2). (iii) Fate of parental DNA The above results indicate that the daughter-strand gaps are filled in with a piece of new DNA comparable in length to the size of the gap in E. coli (Iyer BERupp, 1971). They support the hypothesis that the gap is filled-in completely by de novo synthesis, and that the stretches of BrU-substituted DNA do not just represent small amounts of repair synthesis. An alternative explanation, not entirely ruled out, is that duriug the chase with BrdUrd recombinational exchanges of large pieces of daughter and parental DNA took place so that all strands of DNA contained pieces of labeled thymidine-containing DNA attached to stretches of BrU-substituted DNA. In order to test for this, experiments analogous to those described above were performed on parental DNA. Cells were labeled with [14C]thymidine for 12 hours and then treated with or without U.V. irradiation. This was followed by incubation in BrdUrd for a further 12 hours.
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uv, no BrdUrd 313 nm
1 (b)
uvBrdUrd
no313nm
(d 1 uv. BrdUrd 313 nm c
IO Bottom
Fraction
no
.20 Top
FIQ. 10. Breakdown of parental DNA by light at 313 nm. Cells were lebeled for 12 hr with 0.5 @i [W]thymidine/ml., followed by (8) u.v., 12 hr in thymidine, exposed 8t 313 nm; (b) u.v., 12 hr in BrdUrd, no exposure 8t 313 nm; (c) no u.v.. 12 hr in BrdUrd, exposed 8t 313 nm; (d) u.v., 12 hr in BrdUrd, exposed 8t 313 nm. U.V. exposure 110 ergs/mma; 313 m-n-exposure 4.0 x lo6 ergs/mma; BrdUrd or thymidine 8t 2 x 10m5 M. In (d) the aounts in fractions 1 to 9 8re superimposable on the oounts in fractions 1 to 9 of (e), if these 8re divided by 1.7. The deahed lines in fractions 10 to 20 are obtained by dividing the counts of the corresponding f&&ions in (8) by 1.7. The dotted lines are obtained by subtracting these values from the total counts in the oorresponding frsctions in (d).
Finally the cells were exposed at 313 nm. If u.v.-induced recombination had taken place, the labeled parental strands should contain stretches of BrdUrd and degradation of the labeled DNA should be observed in the initially u.v.-irradiated cells but not in the unirradiated cells. Figure 10 shows the results of this experiment. Controls omitting the 313~nm irradiation, or incubating the cells in thymidine instead of BrdUrd showed little degradation (Fig. 10(a) and (b)). With initially u&radiated cells, incubated in BrdUrd and irradiated at 313 nm, appreoiable degradation of the parental DNA was observed (Fig. 10(o)). This suggests that a DNA molecule substituted in one strand with BrU is fragmented by 313 nm light to a small extent even in the opposite unsubstituted strand (about 1% of the breaks in the BrU strand). With initially u.v.-irradiated oells (Fig. 10(d)), a bimodal distribution was obtained. Because of the u.v.-induced lengthening of the generation time some cells will not yet have replicated all their DNA during the 12 hours in BrdUrd. Unreplicated DNA in these cells did not contain BrU and was therefore not broken down. DNA in the rest of the cells contained BrU and was broken down. Figure 10(d) can therefore be regarded as a composite of two separate profiles, as shown by the dotted lines. It can be seen that the extent of breakdown of the BrU-substituted population is the same
334
A. R. LEHMANN
as, but not greater than, that of the initially unirradiated cells. The absence of any specific u.v.-induced breakdown of parental strands again suggests that there are no recombinational exchanges involving the parental strands, although the resolution in this experiment was somewhat lower than hoped for, owing to the breakdown in unirradiated cells. 4. Discussion The experiments described above show that in L5178Y cells, as in E. coli, the newly synthesized DNA formed after U.V. irradiation of the cells contain gaps, probably opposite the pyrimidine dimers, and these gaps are filled in on further incubation. This is in agreement with the findings for Chinese hamster cells (Cleaver & Thomas, 1969) and with preliminary reports of Rupp et al. (1970) and Zipser & Rupp (1970, Bbphys. Hoc. Ann. Meeting, Abstr. 10, p. 261a), also using L6178Y cells. This process may therefore represent the principal method whereby some mammalian cells (with the exception of primate-derived lines, which can also excise dimers) are able to tolerate the production of a large number of dimers in their DNA. The filling-in of gaps is prevented by hydroxyurea (Fig. 4), which inhibits the formation of deoxyribonucleoside triphosphates (Pfeiffer & Tolmach, 1967). This is of interest since the process of repair replication in human cells, as well as the small amount in Chinese hamster cells, is insensitive to hydroxyurea (Cleaver, 1969, 1970; Regan et al., 1971). Superficially both these processes are similar, in that each is a filling-in of gaps in DNA strands. Figures 2(a) and 3 show that not all the labeled DNA synthesized after 110 ergs/mma is of low molecular weight. Table 2 showed that the average interdimer distance after 110 ergs/mm2 was lo7 daltons, a length of DNA that is synthesized in about 15 minutes in these cells (Lehmann & Ormerod, 1970). In fact in some cases, since there is a distribution of interdimer distances, it will sometimes take appreciably longer to synthesize the DNA between two dimers. It is possible then that in these cases a gap is filled in behind the replication fork before it has reached the next dimer. Some of the labeled material would then be of high molecular weight. Further support is given to this hypothesis by the data of Figure 5, which show that most of the gaps are filled in within 1 hour of their formation. The experiments described in section (d) of Results utilized light of wavelength 313 nm, which specifically degrades and thereby identifies sections of DNA containing BrU. The results obtained suggest that the gaps in the daughter strands are filled in de novo and that recombinational exchanges are not involved. When the daughter strands were radioactively labeled and the gaps filled in, in the presence of BrdUrd, 313 nm light reformed the low molecular weight pieces observed immediately after the pulse-label. The possibility that these results could be explained by the action of an endonuclease which degrades sections of DNA from the ends of strands during exposure at 313 nm is rendered unlikely, since (1) the size of the pieces produced is dependent on the initial U.V. exposure and is the same as the size of the interdimer pieces formed immediately after the pulse-label, (2) when the length of labeled DNA in unirradiated cells is the same as that in the u.v.-irradiated cells, more low molecular weight material is still released from the u.v.-irradiated cells, and this is not merely a consequence of any instability imparted to the DNA by the presence of pyrimidine dimers (Table 2), and (3) leaving the samples in ice for a further few minutes after 313~nm exposure does not produce any more breakdown.
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These results demonstrate that the daughter strand gaps are f&d in with BrUcontaining DNA. The size of the BrU-containing stretch of DNA in the filled-in gap (about 809 nucleotides) is much larger than would be expected for a small amount of repair synthesis at the ends of the gaps. Furthermore with radioactively labeled parental strands (section d(iii)) and post-u.v. synthesis in BrdUrd, no BrU could be detected in the parental strands, suggesting that they were not involved in recombinational exchanges. We conclude therefore that recombinational exchanges are not necessary for repair of daughter-strand gaps in L5178Y cells, in contrast to the results obtained for E. co.%(Rupp et al., 1971). DNA appears to be synthesized de nova in the gap and somehow the dimer is bypassed. It is still possible that a very small part of the gap could be filled in with parental material, the rest being filled in with newly synthesized DNA, but gross recombinational exchanges, as detected in E. coli by Rupp et al. (1971) seem unlikely in L6178Y cells. The approximate value (866 nucleotides) for the size of the filled-in gaps, deduced in the Appendix, is quite similar to the size of the gap in E. coli (1096 to 1866 nucleotides) estimated by Iyer & Rupp (1971) using chromatographio techniques. (The values obtained by both methods are susceptible to quite large experimental errors.) This is of the order of the size of Okazaki fragments of DNA replication (Okazaki, Okazaki, Sakabe, Sugimoto t Sugino, 1968), as pointed out by Iyer t Rupp (1971), who also speculated on the possible significance of this observation. The model of postreplication repair in L5178Y cells that emerges from this work is that gaps of about 800 nucleotides are formed opposite pyrimidine dimers. These gaps are filled in quite soon afterwards by a process that involves de TWVOsynthesis and is inhibited by hydroxyurea. Some of the important questions which remain to be answered are : (1) What is the nature of the nucleotides inserted directly opposite the pyrimidine dimer Z Are they random nucleotides, or does the repair machinery have some mode of recognition of the distorted pyrimidines ? (2) Is postreplication repair in E. coli different from that in mammalian cells 1 Rupp et al. (1970,1971) have produced evidence to show that newly synthesized DNA is associated end-to-end with parental DNA strands after U.V. irradiation of E. coli, suggesting that recombinational exchanges are in fact involved in E. coli. My results show that it is probably not involved in mouse cells. (3) If the gap is filled in by de nova synthesis, why is a gap left in the first place ‘1 Presumably the enzyme system which fills in the gap is different Corn the replication machinery. (4) What is the nature of the site beyond the dimer, at whichsynthesisisreinitiated?
APPENDIX
Estimate of the size of filled-in gaps Table 3 shows that the number of pieces per growing strand formed during the pulse time is quite small (one or two), so that in general, one 313~nm light-induoed break in a f&d-up gap reforms the low molecular weight pieces. 22
A.R.LEHMANN
336
TABLET Cal42.d4dionof number of end1 pieces synthesized during pulse label --U.Y-7. Dose (erl3 s/mm”)
Replication rate (106 daltonq min/strand)
Amount of DNA made in 30 mm by one replioation fork ( x 108)
0 110 220
oq 0*4$ 0*2$
24 12 6
Approx. size of interdimer spacing§ (106 daltons)
10 6
Average no. of short pieoea made in 30 mm
-1 -1
t Based on Lehmann & Ormerod (1970). $ Based on unpublished incorporation studies, assuming that the u.v.-induced decrease in the rate at eaoh replication rate of uptake of [3H]thymidme is caused by a reduation in replication fork rather than a decrease in the number of replicating forks. $ From Table 1.
Ifthe probability of forming a break between two nucleotides in the B&J-containing stretch is p and the number of nucleotides in this stretch is N, the probability of forming one or more breaks in this stretch is equal to 1 -eepN. This is approximately equal to the fraction of small pieces that are re-formed, F. Figure 11 shows a plot of F against Np. When Np = 1, F w 0.6.
Probability of forming o break x size of pop
FIG. 11. Theoretical
release of low molecular
weight pieces by irradiation
at 313 nm.
The BrU-containing material marked Y in Figure 6(b) is broken down at comparatively low exposures of 313~nm light (see Fig. 8). The further release of low molecular weight material from initially irradiated cells shown in Figure 8(a) corresponds to the re-formation of small pieces and is analogous to Figure 11.60% of this further release is obtained at a dose of about lo5 ergs/mm2. At this dose p = 1 break/2*4~ lo5 daltons, so that N (the size of the gap) w 2.4 x lo5 daltons or about 800 nucleotides. This value must be regarded as an approximation because of the background degradation observed in initially unirradiated cells, because in some cases two breaks are required to re-form the low molecular weight pieces and because of the arbitrary nature of the ordinate chosen for Figure 8(a). I am most out, and Dr J. K. Setlow, W. D. Rupp U.S. Atomic
grateful to both Dr R. B. Setlow, in whose laboratory this work was carried R. D. Ley for helpful discussions and encouragement, to Drs R. B. Setlow, and M. L. Randolph for critical comments about the manuscript, and to Dr for discussion of his unpublished results. This research was supported by the Energy Commission under contract with the Union Carbide Corporation.
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