J. Mol. Biol. (1977) 114. 141-151

Localization

of Inhibition of Replicon Initiation Regions of DNA

to Damaged

Irradiation of synchronized S pl~a,so c’hitlese Ilanlster o\.ary cells with 313 nm wavelength light inhibited t)llr initiation of rrpliculs in DNA substituted with bromodeoxyuridine but did not affect tile rc,plication of unsubstitutpd DNA occurring simultaneously in the same ccslls. Tlri. Y result suggests that initiation is inhibited only in the region of the chromoso~~~c~ that sl&ins damage. Calculations of the frequency of damaged sites sugg& tlrat tllis inllibition, and probably a similar efFect induced by X-rays, coldd be mediated by conformational changes in regions of individual D-Ud rnol1~11~~s “l> to se\,eral hundred pm it1 lengt,h.

1. Introduction Several agents that damage DNA, including Xi-ran-s (Makino & Okada, 1975: 1976). methylmethanesulfonate Walters & Hildebrand, 1975 ; Painter & Young. (Painter, 1977), and bromodeoxyuridine plus 313 nm wavelength light (Povirk & Painter, 1976), have been shown to inhibit the initiation of replicons in the DNA of cultured mammalian cells. Because the 313 nm light-induced inhibition requires the specific sensitization of DNA by BrdUrdt (Hutchinson, 1973), it is likely that all these agents inhibit replicon initiation by damaging .DKA molecules. This damage could lead to inhibition in at least two nays : it could induce a conformational change that prevents initiation of the damaged DXA molecule. or it could trigger a metabolic regulatory mechanism to shut down the initiation of new replicona in t’he entire cell. To distinguish between these two possibilities, cells having half their DNA substituted with BrdUrd were obtained and the replication of unsubstituted DNA was examined at a time when replication of t,he substituted DX.4 was inhibited by treatment with 313 nm light.

2. Materials and Methods (a) Cells and labeling

mnteritrls

Chinese hamster ovary cells from a stock maintaint~tl in tllis laboratory were grown in a 5% CO2 atmosphere in Eagle’s mirlimum essential supplemented with 15% (v/v) fetal calf serlun (GIBCO). L”H]Thymidine [14C]tjhymidine (50 Ci/mol) were obtained from New EngIant Nuclear. t Abbreviation

used: BrdUrd,

brornocleoxyuridint,. l-11

for several years medium (GIBCO) (11 Ci/mmol) and

bottles (Belco Class) hav-inp 750 an2 sklrfhw iwm altti kvcw grobvrr for 2 to 3 days in 40 ml of medium at 0.3 rev~s/mitr. Nest, to rcrnovc 100x aud dying rc~lls. tire bottles were and tlw medinm was removed imd rcplaccd wittr 20 ml rotated for 5 min at 250 ro\s/mitr. of fresh medium. At 1 11 intervals for up t,o 1,.) h thrreaft’er, the bottles were rot,atcd at 200 revs/min for 5 min (Klevecz & Kapp, 1973) and the medium wittl mitotic cells was removed and replaced wit,11 fresh medium. The mitotic cells from the first 2 selectious were discarded and those from subseyurnt selections were divided into 1 to 4 cultures and plated into Petri dishes (Falcon Plastics). Ttlc mitot,ic index was 65 to 750,. Cells selected by mitotic shakeoff remained synchronized for at least 2 gcnorations (Fig. 1). Fxporiments to measure DNA syntllesis xvere pcrformod beginning at tbr: peak of the first S please (7 II, 95% labeled nuclei) or ttrr: second S phase (20 11. K6qi, labeled rrrrclri).

-1L.-4

8

I-

12

t ~~~_ 1_

16

20

24

28

32

Time offer shokeoff (h) FIG. 1. Synchrony of Chinese hamster ovary cells as measured by [3H]thymidine uptake as a function of time after mitotic shakooff. Cells growing in a single roller bottle were shaken off every hour for 12 h and plated into Petri dishes. At various times after shakeoff, cells were incubated in the presence of 1 @i [3H]thymidine/ml for 20 min. Cells were then scraped off dishes into ice-cold SSC. One portion (approx. IO5 cells) was immediately sonicated, the DXA was precipitated, and its radioactivity counted just as for gradient fractions. Cells from the other portion were treated with trypsin (0.12% (w/v), 12 min at 37°C) and counted in a Coulter counter. Each point represents cells from a single Petri dish.

(c) Bromodeoquridine

substitution

To obtain cells with BrdUrd in about half their DNA, the protocol shown in Fig. 2 was used. Assuming that the chromosomes segregated randomly (Prescott & Bender, 1963; Heddle et al., 1967) and that most of the cells progressed through 1 cell cycle within 20 h after mitotic selection, most of the cells should have contained, at the t’ime of irradiation, both BrdUrd-substituted [3H]DNA and unsubstituted [r4C]DNA. (d) Irradiations The 313 nm wavelength was selected from the emissions of a high-pressure mercury arc lamp (Phillips SP-500) (P ovirk & Painter, 1976). Cultures were irradiated at room temperature without any liquid covering. The intensity was 20 to 30 J/m2 per s and the range of doses was 33 to 560 J/m 2. The frequency of single-strand breaks produced by this system in fully BrdUrd-substituted DNA in Chinese hamster ovary cells, as measured by velocity sedimentation in alkaline sucrose gradients, was 2 x IO-lo breaks/dalton per J/m2. This value is comparable to, though somewbat lower than, the value of 4x lo-10

INHIBITION

OF

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143

Shakeoff 4:

>’ I

S (0)

I b)

idi

(Ci

(e!

FIG. %. Experimental protocol for obt,aining tolls with M(vO BrdUrd in half their DNA. Cells were first grown in a roller bottle for several generations in medium containing 0.01 $i [lV]thy200/, BrdUrd (2 x 1O-6 M), [3H]midine/ml; t,hen grown for 1 generation in medium containing thymidine (8 x 10d6 M, 0.15 @i/ml), and fluorodroxyuridinr (FdUrd) (10 -6 M); and then shaken off at mitosis. The cell layer was washed twice with salincl, and thymidine chase medium was added 1 h before the final mitotic shakeoff. At the time of shakeoff, all the DNA should have had one strand labeled with 14C and the other strand labeled with 3H and substituted with BrdUrd (0). The mitotic cells were then plated into Petri dishes and grown for 20 h in thymidine (10e6 M). half of the DNA should have had 1 strand labeled with l*C After 1 generation-time in thymidine, and the other half should have had 1 strand labeled with 3H and substit,uted 20% with BrdUrd (d). During the next S phase, cells were irradiated with 313 nm light, and incubated for 2 h in the presence of 1000’ BrdUrd and fluorodeoxyuridinr. The fractions of unsubstituted [‘W]DNA and 20% BrdUrd-substituted [3H]DNA that replicated during t.hr incubation period were measured separately by the shifts of radioactivity to hybrid density on crsium chloride equilibrium density gradients. -, Normal unlabeled DNA strand; W, [14C]DNA strand; Il, 200,; loo:/, 13rtlUrd.su)-,stit,uted unlabeled DN:\ BrdUrd-substituted [3H]DNA strand; m,

strand.

breaks/dalt,on

per J/m2 obtained by Lehmann suspension at O”C, using a monochromator wit,h ZOo/o BrdUrd in one strand only, t,he frcquencp

or 2 x IO - l1 breaksldalton

(197”), wl~o irradiated L5178Y cells in the same lamp. In DNA substituted with of breaks would br ono-tenth t,his value,

per J/m2.

(0) Cesiutn

chloride

equilibrium

gradients

To determine the proportions of 207; BrdUrd-substituted and unsubstitutod DNA that shifts of radioactivity replicated during the 2-h post irradition incubation in 100 SO BrdUrd, to hybrid density on cesium chloride gradients were measured (Gerner et al., 1974). Cells were scraped off the Petri dishes into 3 ml of SSC (SK! is 0.15 M-sodium chloride, 0.015 Msodium citrate, pH 7.4), lysed with 0.1% (w / v ) so d’mm dodecyl sulfate, and treated wit!h RNase (50 pg/ml) for 1 h at 37°C and then with Pronnse (500 pg/ml) for 2 h at 37°C. The lysate was extracted twice with chloroform/isoamyl alcohol (24: 1). The aqueous layer containing DNA was dialyzed overnight against SSC and sheared in a Virtis homogenizer to reduce the size of the DNA to about lo7 daltons. A total of 4.5 ml of this solution was added to 5.8 g of cesium chloride and centrifuged for 40 11at 40,000 revs/min in a Beckman Ti50 rotor (Gautschi et al., 1972). Then 15-drop fractions were collected from the bottom of the tubes. A total of 1 ml of 200 pg carrier DNA/ml in 0.1 M-NaOH and 0.5 ml of 6% (w/v) Na,P,O, in 1.5 M-HCl were added to each fract,ion and the precipitated DNA was fixed to Whatman GFjC filters. The filters were washed wit,h 5% (w/v) trichloroacetic acid and 70% and 100% (v/v) ethanol and counted in a Packard liquid scintillation spectrometer in toluene containing Omnifluor (New England Nuclear).

I-14

I,.

1,‘. I’OL’ I IC 1;

(t’) .-I /rfol,rrtlio!lrtllJ/,!I var. detornlillat io11 of t 1111pv”“““1;~p 01 Inlwlml Illlolt~i (i.(,. tllo fr;rc+ic,ll of cells in S ptlase) 7 01’ 20 11 aftf?r Sllillil’Otf’. Irlitcltic, cc~ll~ \\‘s, tllc lnctlium \vi\s rc~placc~l \vitjtr one cvntaining 20 &i [3H]tllymidinc/ ml and the cells wcrc i~~c~lbatc~tl for I5 rllill. Tlrv wlls w~rr: thou waslwtl and fixed wit11 ctlranol/acetic acid (3: I) ant1 tlrivtl. To cxaminc chromosome sr~greg~dtiori iitrd labcling, s~rlchmnizctl tolls were blocked in t,lreir second mitosis by irlc>lIbatioll it1 tllc pressrice of 10V6 ar-(lolcemid (Ciba) for 8 11 beginning 20 11 a,ft,cr mitotic sl~akv off. Tl~cb cells \vcr(x removed from the dishes by vigoroIls pipetting. pelletttltl 1)~ (~(:rltrifrlgatioll, swollen in 0.075 X-KC1 (4 min, 22”(J), apaiu pclletted, and iixc,d wit11 c~ll~~ol ‘acetic acitl (3 : I ) (\Volff et al., 1976). This suspension was dropped orrt)o slitlcs arltl allowc~l to tlr~,. Slides for botll expcrilmvts \\-(‘I’(’ dippcvl it1 Kodak NTIS-2 emulsion, oxposed for 10 t,o 12 days, developed, and stainvtl wit’11 (: iwllsa.

(g) Alkaliile

sw~mse gradieds

To examine the inhibition of illitiat,ion, DNA was pulse-labeled with [3H]tllymidine 30 min after irradiation and analyzed on alkaline sucrose gradients. A total of 0.5 ml of a lysis solution (0.2 &I-NaOH, 0.02 1\1-EDTA, 0.17; (w/v) Nonidet NP-40 (Shell Oil)) was 0.9 M-NaCl, layered on top of a 36-m& 5:/A t)o iLO”/, a lkaline sucrose gradient (0.1 ix-NaOH, 0.02 M-EDTA). Tllen lo5 cells in 0.5 1n1 of SSC were added and allowed to lyse at room temperature for 3 11. Gradient,s IV~VX:cent,rifuged at 27,000 revs/min for 2.5 h in a Beckman SW27 rotor at 20°C. Calibratioll of t.llis gradient system has been described elsewhere (Clarkson 8z Painter, 1974). &a&ions wcrc filtered and counted as for cesium chloride acitl was used instead of trichloroacetic gradients, except tllat ice-cold 5Sh (1.1~) pcrcllloric acid.

3. Results (a) Replication

of hronlodeor?Ju,i(li,~l,~-,~ub~~t,~tuted

and unsubstituted

DNA

When DNA from cells harwstc~d immcdiatcly after mitotic shakeoff (Fig. 2) was analyzed on cesium chloride ccpilibrium density gradients, the 3H radioactivity, from DNA labeled before from DNA containing HrdCrd, and the IT! radioactivity. 32 28 24 20 16 12 8 4 0

12 Fraction (0)

(b)

16 20

24

number (c)

FIG. 3. Cesium chloride equilibrium density gradients showing replication of HrdUrd-substituted and unsubstituted DXA, Gng the protocol described in the legend to Fig. ?. Density increases from right to loft. (a) Cells harvcastctl at mitotic shakeoff; (b) rmirradiated and (c) irradiated (560 J/m2 of 313 nm light) cells harvestf~tl after the final incubation in BrdUrd. ---i/:---I’_.-- , [‘T]DNA; --O-o-, ZO% BrdUrd-substituted [3H]DSA. Percentagesindicate fractions of radioactivity at hybrid density.

INHIBITION

OF

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146

INITIATION

BrdUrd was added, banded at the same position (Fig. 3(a)), indicating that nearly all the DNA (about 93%) had replicated once in the medium containing 20% BrdUrd. When the same cells were allowed to grow in a thymidine chase for 20 hours after shakeoff and then grown in lOOo/o BrdUrd for two hours, the main peaks of 3H and 14C radioactivit’y became distinct (Fig. 3(b)). The 14C peak had nearly the same height and half-width as the 3H peak, indicating that nearly all t’he [14C]DNA had replicat’ed again during the thymidine chase and had returned to normal densit’y. The 3H peak remained displaced slightly to the heavy side of the l*C peak, ab a, position consistent with 20% BrdUrd substitution in one strand. The two-hour incubation in 100 (+$BrdUrd during the second S phase after shakeoff produced two additional, smaller peaks at hybrid density. These peaks provide a measure of the fract,ion of DNA that had replicated during that, incubation (Gerner et al., 1974). This fract’ion was nearly the same for both unsubstitut)ed [14C]DNA and BrdUrdsubst,ituted [3H]DEA, indicating that the 200A, BrdUrd substit’ution did not itself In cells irradiated with 313 nm light just interfere wit’h the replication process. before the two-hour incubation with lOOo/, BrdUrd, replication of 20% BrdUrdsubst,ituted DNA was inhibited by 25%, whereas replication of unsubstituted DNA rrmained at control levels (Fig. 3(c)). The dose response for this effect (Fig. 4) was

b 2

00 -

E : E e a

70-

E

60-

1

0

200

I

400 Dose of 313 nm light ( J/m21

600

Pro. 4. Fraction of DliA replicated as a, function of dose of 313 nm light, plotted as percentage of the fract,ion replicated in unirradiated controls (0.170 tc, 0.181). The protocol is described in the legend to Fig. 2. Open and closed symbols denote 2 wptwatt~ experiments. [14C]DKA (!*i, A); 20% BrdUrd-subst,ituted [3H]DFAA (0, 0).

biphasic. This pattern was verified in other experiments (data not, shown) and is t’ypical of inhibition of DNA synthesis by both X-rays (Lajtha et al., 1958, Painter AZYoung. 1975; Walters & Hildebrand, 1975) and RrdCrd plus 313 nm light (Povirk 6 Painter, 1976, and unpublished data). Because there is still some controversy about whether low doses of X-rays inhibit DNA synthesis in cells already in S phase, I repea,ted the above experiments using X-rays and cells with unsubstit,uted DNA. In synchronized S phase cells (7 h after mitotic shakeoff. 95% labeled nuclei), 500 and 1000 ra,ds produced decreases of 11 “/b and 18 ‘A, respectively, in the fraction of DNA that acquired hybrid density during a subsequent two-hour incubation in BrdUrd. This result indicates that low X-ray doses do inhibit DNA synthesis in S phase cells. The conflicting results of Gerner et al. (1974) and Gerner (1976) using Chinese hamster ovary cells and nearly the same methods may be due to differences in cell strain or culturing conditions.

1,.

14G

(b) S%mdtan.eity

of replication,

F.

I’O\~llCli

of Ornmod~oxy~~riditt,~-.9.t~lt.stif~f~d mad

urwuhtituted

I)Nd

Autoradiographic experiments were done to verify that, in the equilibrium cesium chloride gradient experiments, substituted DKA and unsubstituted DNA were being replicated simultaneously in the same cells. When cells were grown for one generation in L3H]thymidine and BrdUrd, and collected in mitosis two generations later, autoradiography revealed that, in most cells about’ half t’he chromosome lengt’h was labeled and that all cells cont,ained substant’ial portions of both labeled a,nd unlabeled chromosomes (Fig. 5). Thus? in the previous S phase, when replication was measured, they must have ha’d both substitut’ed and unsubst,ituted parental DNA. This result is consistent’ with random segregat’ion of chromosomes at mitosis (Prescott & Bender, 1963; Heddle et al., 1967) and shows that there was no anomalous tendency for BrdUrd-containing chromosomes to segregate to a single daughter cell.

FIG. 5. Autoradiogram of cells grown for 1 generation in F3H]thymidine and 20% BrdUrd, grown for 2 generations in thymidine, and arrested in mitosis. Substantial port,iona of both labeled and unlabeled chromatin are pretient in each ~11. Many of the chromosomes have only part of their length labeled, indicating that sister chromatid exchanges occurred during either the generation before or the generation after mitotic selection. The very light labeling on some chromosomes was probably caused by small amounts of [3H]DNA precursors contaminat,ing the thymidine chase.

When cells were pulse-labeled with [3H]thymidine for 15 minutes, 20 hours after mitotic shakeoff and collected in the next mitosis, the chromosomes of most cells appeared to be uniformly labeled, within the limits of resolution of the autoradiography (data not shown). This result is what would be expected from Stubblefield’s studies (Stubblefield, 1975) in Chinese hamster DON cells, showing that all chromosomes except the X and Y replicate throughout the S phase (see also Hsu: 1964). Thus, because substantial portions of the pulse-labeled regions in the Chinese hamster ovary cells contained only BrdUrd-substituted DNA and others contained only unsubstituted DNA, most cells were synthesizing both types of DNA at the time of irradiation.

INHIBITION

OF

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INITIATION

147

qf the inhibition of rep&con initiatiw2 in bromodeoxyuridine-substitutedDNA

(c) Verification

Inhibition of replicon initiation can be dist’inguished from inhibition of chain elongation by observing profiles of DNA pulse-labeled with [3H]thymidine 30 minutes after irradiation. Assuming that synthesis of each nascent strand begins at a single origin and continues bidirectionally at the rate of 0.5 to 1.0 micrometre per minute (Edenburg & Huberman, 1975), DNA strands initiating 30 minutes before pulselabeling, i.e. at the time of irradiation, will be 3 x 107 t’o 6 x lo7 daltons in lengbh by the time of pulse-labeling. Nascent strands that initia,te before irradiation will bc somewhat longer than this, and those thab initiate after irradiation will be somewhat shorter. If the irradiation in the experiments reported here caused a substantial number of DNA4 lesions that acted as blocks to the elongaGon machinery, each growing strand would have a certain probability of encountering such a lesion during the 30-minute interval between irradiation and pulse-labeling, in which case it would not incorporate label. This would result in decreased radioactivity throughout the DNA profile, with incorporation into smaller molecules being somewhat less affected, since they would have been growing for a shorter time and thus would have a lower probability of encountering a lesion. This is the result thnt is seen after ultraviolet light irradiation (Lehmann, 1972), which has been shown in autoradiographic experiments to block DNA elongation (Edenberg, 1976). Although some very long replicons require up to 90 minutes to complete replication (Painter & Young, 1976). one would expect to see at least some effect wit’hin 30 minutes if elongat,ion were substantially inhibited. If, on the other hand, irradiation inhibited initiation of new growing strands but did not affect elongation of strands already growing at the time of irradiation, one would expect a normal amount of incorporation into molecules larger than 6 x lOI daltons (those that initiated before irradiation) and a decrease in incorporation into molecules smaller than 3 x lo7 daltons (those that initiat’ed after irradiation). This was the result obtained after irradiation with 313 nm light of cells that, having been grown for one generation in the presence of BrdUrd and then irradiated in the first S phase after mitotic shakeoff, had 20y0 BrdUrd substitution in one st’rand of all their DKA (Fig. 6(a)). Th e c 1ose coincidence of t’he profiles in the high molecular weight. region indicates that nearly all nascent strands that were growing at the time of irradiation were still growing 30 minutes later and had at’tained the same length as those in control cells. Thus, very few strands could have had their elongation blocked by DNA lesions. However, incorporation into small nascent strands was severely suppressed, even at very low doses, indicating that fewer nascent strands u ere initiat,ed after irradiation. Cells with DKA containing no BrdUrd were much less sensitive and showed only a slight inhibition (Fig. 6(c)), possibly caused by a small amount of DNA base damage resulting from 313 nm light alone (P. V. Hariharan, Florida State University, personal communication). Cells irradiated in the second S phase (Fig. 6(b)), which had BrdUrd in half their DNA, exhibited an intermediate level of inhibition, a result that is consistent with the finding from cesium chloride gradients that replication was substantially inhibited in BrdUrd-substituted DNA only. Because 30 minutes

the higher molecular weight nascent strands were still growing normally aRer irradiation in all cases, it can be assumed that nearly all the smaller

12. v.

14x

PO v 1 Ii Ii

Molecular

12 I2

0.5 lllll I 3 5 IO 20 20I

40 40

20

5

lllll

weight

(x IO-‘)

0.5 I 3 5 IO7--r-20

40

20

5

O-51 I 3 5 o-5 --TT~T~~

7--r-

I

IO

20 20 r-T

40 40 ..-

IO 8 6 4 2 0

25

15

IO

25

15

IO

Fraction (01

25 25

20 20

15 I5

IO

5

number (b)

(cl

FIG.

6. Alkaline sucrose gradients of DNA from synchronized S phase cells pulse-labeled with [3H]thymidine beginning 30 min after exposure to various doses of 313 nm light; (0 ( A) ; 33 (0) ; 88 ( n ); 100 (0) J/m2). The cells had all (a), half (b), or none (c) of their DNA substituted with BrdUrd. Cells growing in roller bottles were incubated for 12 h in the presence ((a), (b)) or the absence (c) of 2 x lOmE ix-BrdUrd, 8 x 1Om6 nr-thymidine, and 1O-6 M-fluorodeoxyuridine. Mitotic containing 10e6 Mcells were shaken off and incubated for 7 h ((a), (c)) or 20 h (b) in medium thymidine. Cells were then irradiated, incubated for 30 min in regular medium, incubated for 10 min in medium containing 10 rCi [3H]thymidine/ml, and harvested. In order to correct for possible variations in cell number between gradient,s, cells were labeled with [‘%]thymidine for at least 4 h before irradiation, and the total amount of 14C incorporat,ed was taken as a measure of the relative number of cells in each dish. The 3H radioactivity in each fraction of the gradient 14C radioactivity in the control from irradiated cells was multiplied by the ratio of the total gradient to the total 14C radioactivity in the gradient from irradiated cells, and t)hon divided by the total 3H radioactivity in the control gradient.

replicons (: 10mB mz/J. In solut’ion, the cross-sect’ions both for thr induction of strand breaks in partially substituted DNA and for damage to molecular bromouracil are 60 to 160 times lower at 313 to 315 nm than at 254 to 280 nm. Assuming a similar wavelength dependence for base damage in DNA, the crosssection for base damage in DNA by 313 nm light would be (5 to 9) x 10m5/(60 t,o 160) = (3 to 15) x 1O-7 m2/J. The 20% unifilarly substituted DNA has one BrdUrd nucleotide per lo4 daltons. Thus, 33 J/m2, a dose that reduced initiation to about 37’); of the control level (Fig. 6(a)), would be expected to induce 33 J/m2. (3 to 15) Y 1O-7 m2/J*(1/10* daltons) = one lesion per (2 to 10) x 108 daltons = one lesion per 100 to 500 pm of DNA. Alternatively, it can be calculated (see Materials and Methods) that 33 J/m2 would induce about one single-strand break per 750 pm of DNA. Both of these estimates of the average distance between lesions in DNA at this dose are several t,imes larger than estimates for the average size of replicons in Chimse hamster ovary and other mammalian cell s. which range from 30 to 50 pm (Hubcrman & Riggs, 1968; Gautschi et al., 1972: Edenberg & Huberman, 1975). Thus. these calculations indicat’e that init’iation of a replicon can be prevented by a DNA lesion that, is not in the immediate vicinity of the initiation site and that probably does not need to be in the same replicon. Target, theory calculations for t’he X-my-induced inhibition of replicon initiat’ion lead to similar conclusions. assuming bhat’ DNA is the target (Painter & Rasmussen. 1964: Makino & Okada. 1975). This analysis suggests that initiation of replicons requires a segment of chromatin to have a specific conformation, whose formation and/or maintenance is extremely radiosensitive and can be disrupted by a lesion anywhere within its DNA. These segments appear to be larger than a replicon and may correspond to olust’ers of synchronously operating replicons that have been observed autoradiographically (Huberman & Riggs, 1968; Hand, 1975) and cytologically (Stubblefield, 1975). This phenomenon may serve to reduce mut,agenesis t)y delaying replication of damaged DNA until the damage can be repaired. Further speculation about the exact mechanisms of this effect is hampered by the lack of agents producing single types of lesions. BrdUrd plus 313 nm light induces strand breaks, cross-links, and base damage. Furthermore, repair of any damage is likely to involve induction of strand breaks and excision of bases by repair enzymes (Smets & Cornelis, 1971; Mattern et al., 1973; Verly et al., 1973; Cleaver, 1974). However, two plausible scenarios are (1) that a single-strand break in DNA would relieve superhelical tension, which may be required for initiation of its repliconx, perhaps by changing the affinity of DNA for some other molecule (Davidson. 1972)

L. I‘. POVIHK

150

and (2) that a DNA-protein cross-link may prevent dissociation or movement of a protein, thus inhibiting some altera,tion in nucleoprotein structure necessary for initiation. Two recent st,udies tend to corroborate the first scenario. Gellert et al. (1976) have obtained evidence in Escherichia coli that DNA gyrase, an enzyme which puts superhelical turns into DNA, is required for DI\‘A replication. Furthermore, Cook & Braze11 (1975) have inferred from sedimentation of HeLa cell nuclei that each single-strand break in mammalian D1’;A relaxes a segment of DNA about 500 pm in length (see also Benyajati & Worcel, 1976). The second scenario, however, receives some support from the suggest,ion put forward by Tolmach & Jones (1977) that a model involving cross-links would be more compatible with the time-course of recovery of replicon initiation. The evidence that the inhibition of replicon initiation described in this paper results from the same lesions and by the same mechanisms as that caused by X-rays remains circumstantial. However, the similarities of sedimentation patterns of pulselabeled DNA, of the dose-response curves, of the time-course of inhibition, and of the relative sensitivities of different cell lines (Walters & Hildebrand, 1975 ; Painter & Young, 1976; Povirk & Painter, 1976, and unpublished data), combined with the similarity of the initial DNA lesions produced (Hutchinson, 1973; Ward, 1975), make it seem likely that these agents act by the same biological processes. This conclusion would tend to exclude, for Chinese hamster ovary cells at least, models for the X-ray effect that involve changes in metabolism of small molecules, such as phosphorylation of nucleotides (Ord & Stocken, 1958; Looney et al., 1965; Gautschi et al., 1973). I thank Dr Robert B. Painter, in whose laboratory these experiments were performed, for helpful advice and suggestions. This work was performed under the auspices of the United States Energy Research and Development Administration. REFERENCES Benyajati, C. & Worcel, A. (1976). Cell, 9, 393-407. Clarkson, J. M. & Painter, R. B. (1974). M&at. Res. 23, 107-112. Cleaver, J. E. (1974). Advan. Radiat. Biol. 4, l-75. Cook, P. R. & Brazell, I. A. (1975). ,J. Cell Sci. 19, 261-279. Davidson, N. (1972). J. Mol. BioZ. 66, 307-309. Edenberg, H. J. (1976). Biophys. J. 16, 849-860. Edenberg, H. J. & Huberman, J. A. (1975). Annu. Rev. Genet. 9, 245-284. Gautschi, J. R., Young, B. R. & Painter, R. B. (1972). Biochim. Biophys. Acta, 281, 324-328. Gautschi, J. R., Kern, R. M. & Painter, R. B. (1973). J. Mol. BioZ. 80, 393-403. Gellert, M., O’Dea, M. H., Itoh, T. & Tomizawa, J. (1976), Proc. Nat. Acad. Sci., U.S.A. 73, 44764478.

Gerner, E. W. (1976). Radial. Res. 67 (Abstr.), 531. Gerner, E. W., Meyn, R. E. & Humphrey, R. M. (1974). Radial. Res. 60, 62-74. Hand, R. (1975). J. Cell BioZ. 64, 89-97. Heddle, J. A., Wolff, S., Whissell, D. & Cleaver, J. E. (1967). Science, 158, 929-931. Hsu, T. C. (1964). J. Cell BioZ. 23, 53-62. Huberman, J. A. & Riggs, A. D. (1968), J. MoZ. BioZ. 32, 327-341. Hutchinson, F. (1973). Quart. Rev. Biophys. 6, 201-246. Klevecz, R. R. & Kapp, L. N. (1973). J. Cell BioZ. 58, 564-573. Lajtha, L. G., Oliver, It., Berry, R. & Noyes, W. D. (1958). Nature (London), 1788-1790. Lehmann, A. R. (1972). J. Mol. BioZ. 66, 319-337.

182,

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OF

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Localization of inhibition of replicon initiation to damaged regions of DNA.

J. Mol. Biol. (1977) 114. 141-151 Localization of Inhibition of Replicon Initiation Regions of DNA to Damaged Irradiation of synchronized S pl~a,s...
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