dna cross-linking in chinese hamster cells exposed to near ultraviolet light in the presence of...

Biochimica et Biophysica Acta, 331 (1973) 181-193 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97853

DNA CROSS-LINKING IN CHINESE HAMSTER CELLS EXPOSED TO

NEAR ULTRAVIOLET LIGHT IN THE PRESENCE OF 4,5 ' ,8-TRIMETHYL-

PSORALEN*

E. BEN-HUR** and M. M. ELKIND***

Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.) and Experimental Radiopathology Unit, Medical Research Council, Hammersmith Hospital, London W12 (Great Britain)

(Received June 20th, 1973)

SUMMARY

Exposure of Chinese hamster cells to near ultraviolet light in the presence of 4,5',8-trimethylpsoralen (psoralen) produces cross-linking of duplex DNA. The production of cross-links in complementary strands is shown using isopycnic sedimentation in CsC1 density gradients, and velocity sedimentation in alkaline sucrose gradients. The latter method is more sensitive since DNA of higher molecular weight can be analyzed and therefore, fewer cross-links per cell detected. Using [3H]- psoralen, it is shown that approx. 11 ~ of the psoralen photoaddition products in complementary strands. This percentage is consistent with a two quantum photochemical induction of cross-links with the overall reaction rate being limited by the rate of conversion of monoadducts to cross-links. During incubation after exposure to psoralen plus near ultraviolet, up to approx. 90 ~ of the covalently bound psoralen disappears from the DNA.

INTRODUCTION

Exposure of DNA viruses 1'2, bacterial cells 3'4 and cultured mammalian cells 5 to near ultraviolet light (300-400 nm) in the presence of furocoumarins (psoralens) produces lethal and mutagenic effects. When applied topically, psoralen (Fig. 1)and its derivatives sensitize mammalian skin to near ultraviolet 6'7, as manifested by the appearance of erytheme and epidermal cell death, dyskeratosis, epidermal cell disorganization, edema and desquamation. Nether psoralens alone nor light alone can produce these effects.

* Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.

** Present address: Department of Cellular Biochemistry, The Hebrew University, Hadassah Medical School, Jerusalem (Israel).

*** Present address: Division of Biological and Medical Research, Argonne National Laboratory, Argonne, I11. 60439 (U.S.A.).

182 E. BEN-HUR, M. M. ELKIND

Fig. 1. Molecular structure of psoralen.

The sensitizing action of psoralen plus near ultraviolet light has been ascribed to photochemical addition of a psoralen molecule to the 5,6 double bond of a pyrimidine base in cellular DNA (refs 8 and 9). Such adducts containing cyclobutane rings have been isolated and identified t ° - ~2 but they have not as yet been shown to take part in cellular inactivation and mutagenesis. Recently the production of cross- links in DNA was shown to occur when bacteriophage 2, Escherichia coli, and mouse leukemia cells L5178Y were exposed to 360 nm light plus 4,5',8-trimethylpsoralen 13, and the yield of cross-links was correlated with the rate of inactivation of repair- deficient mutants of E. coli ~ 4.

In this work we describe the production of psoralen plus near ultraviolet light induced cross-links in the DNA of cultured Chinese hamster cells, measured by the alkaline sucrose gradient ~s, as modified for use with mammalian cells by Elkind and Kamper 16 and Elkind ~7. When cells were exposed to [3H]psoralen plus near ultraviolet light, the disappearance of bound psoralen from cellular DNA was observed during post-irradiation incubation.

MATERIALS AND METHODS

Cell line Chinese hamster fibroblasts, subline V79-753B-3M, were grown as monolayers

in glass petri dishes in a modified Eagle's medium is called EM-15, and which contained 15 % fetal calf serum. They doubled in number in 8-9 h.

Psoralen plus near ultraviolet light treatment Prior to irradiation, the medium in dishes was sucked out and Dulbecco's

buffer containing 1 . 1 0 - 6 M 4,5',8-trimethylpsoralen (Paul B. Elder Co., Bryan, Ohio) was added back for 30 min at approx. 3 °C. Cells were exposed at approx. 3 °C to near ultraviolet light irradiation from two tubular fluorescent sunlamps (FS40 Westinghouse) held in a reflector at a distance of 37 cm from the Cells. The absorbance of the psoralen containing buffer at 300-400 nm is negligible. The fluence of the incident light was 10 ergs • m m - 2 . s-x determined with a thermopile (Eppley Laboratory, Inc.) which was calibrated by a U.S. Bureau of Standards standard lamp.

Light-induced bindin# of psoralen to DNA Near ultraviolet light-induced binding of tritiated 4,5',8-trimethylpsoralen to

cellular DNA was estimated by measuring the specific activity of the DNA. Cells in log phase were exposed to near ultraviolet light in the presence of 1 • 1 0 - 6 M 4,5',8-trimethyl[3H]psoralen, 4.94 Ci/mM, (custrom tritiated by New England Nu- clear, Boston, Mass.), the cells were washed with cold saline, trypsinized and sedi-

D N A C R O S S - L I N K I N G IN CHINESE H A M S T E R CELLS 183

mented in the cold, and their RNAs were digested with 0.1 M NaOH at 37 °C over- night. DNA and proteins were then precipitated with cold 0.3 M HC104 and the DNA extracted from the pellet by heating with 0.5 M HCIO4 at 70 °C for 30 min. DNA concentration was estimated from the absorbance at 260 nm and the amount of bound psoralen from the number of dpm, measured in a liquid scintillation coun- ter. The dpm were calculated, after the counting efficiency was determined, using 3 H 2 0 as an internal standard.

Alkaline sucrose gradient sedimentation To label DNA, cells were grown for approx. 20 h at 37 °C in the presence of 0.5

#Ci/ml [3H]thymidine, 6.0Ci/mM (Schwarz/Mann, Orangeburg, N.Y.). Follow- ing psoralen plus near ultraviolet light treatment, an aliquot of a cell suspension (approx. 5000 ceils) obtained by trypsinization at approx. 3 °C, was pipetted onto a 5-20 % alkaline sucrose gradient. The contents of the lysed cells was centrifuged in a Spinco L2-50HV ultracentrifuge using an SW-50.1 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). 10-drop fractions were collected on glass filters, dried and the radioactivity counted as previously described 16. Extraction of the filters with cold acid improved the counting efficiency but did not change the patterns observed and hence, extraction usually was not performed. Recovery of radioactivity from each gradient was determined by dividing the total counts obtained from it, by the counts contained in the cells layered on the gradient. The latter value was determined from an equal number of whole cells which were lysed and counted under conditions equi- valent to those for the 10-drop samples from the gradient. The recovery was usually larger than 80 %. Since the gradients were fractionated from the top, the direction of sedimentation in the profiles shown is from left to right.

Isopycnic sedimentation in CsCl Cells (approx. 1 • 106) labeled with [3H]thymidine and treated with psoralen

plus near ultraviolet light were X-irradiated (250 kVp, 650 R/min) at ice temperature with 10 or 20 kR, suspended in medium, centrifuged in the cold (to remove serum proteins) and resuspended in 1.0 ml of saline at approx. 3 °C. The cells were lysed and their DNA denatured by the addition of 1.0 ml of 0.2 M NaOH followed by incubation for 150 rain at 37 °C. The pH of the cell lysate was then brought to 11.0 by addition of 0.9 ml of 0.1 M Na2EDTAplus 12.0 ml of a 0.05 M sodium phosphate buffer, pH 11.0 (obtained by titration of Na2HPO4 with NaOH). Samples containing 3.8 ml of this solution were added to 4.9 g CsC1, optical grade (Harshaw Chemical Co., Cleveland, Ohio) in polyallomer tubes. Tube contents were centrifuged in the SW-50.1 rotor at 30 000 rev./min for approx. 64 h at20 °C. 12-drop fractions were collected on glass fiber filters and the radioactivity counted as above. The densities of the CsC1 solutions were measured pycnometrically in 100/~l pipettes.

Calculation of cross-linking rate A. Sedimentation rates in an alkaline sucrose gradient. The number average

molecular weight (M.) was not calculated directly point-by-point, from the distri- tion of radioactivity in the gradients, since a small amount of radioactivity in the top fractions has a very large effect on M n. Instead, the weight average molecular weight (Mw) was first calculated from the equation

M w = ~'fiiMi (1)


where f~ is the fraction of radioactivity in the ith fraction and Mi is the molecular weight of that fraction 34. M, was taken as 0.5 Mw. Since this relation holds only for random DNA distributions, it was applied only for sedimentation profiles showing this pro- perty (estimated as described by Lehmann and Ormerod32). The number of cross- links (N) produced per DNA molecule was determined from the equation

U----- M,* 1 (2) MR

where M n is the number average molecular weight of the DNA from cells not treated with psoralen plus near ultraviolet light and Mn* is the number average molecular weight following treatment. This calculation is based on the assumption that two strands of DNA, held together by a cross-link, sediment at the same rate as a single DNA strand with the same total number of nucleotides 2°.

B. Isopyenic sedimentation in CsCl. This method separates native from denatured DNA and therefore, the fraction of nonrenaturing DNA (molecules not containing complementary strand cross-links) can be estimated. With each isopycnic sedimentation, using a sample of the same cells a determination of M, was made from the sedimentation rate in an alkaline sucrose gradient in order to calculate the rate of cross-linking per dalton.

RESULTS

Sedimentation of cross-linked DNA in alkaline sucrose 9radients In untreated cells, 4 h of lysis at approx. 25 °C in alkali prior to sedimentation

in an alkaline sucrose gradient (36 000 rev./min for 60 min at approx. 12 °C or for 90 min at approx. 0 °C) is generally required to release single-stranded DNA, M, = 2 • 108, from a DNA-containing material which has been referred to as the "complex" (refs 16 and 17). When cells are exposed to near ultraviolet light in the presence of psoralen, and even when cells are lysed for as long as 6 h at approx. 25 °C, a progressive loss of radioactivity recovered from the gradient is observed as a function of irradiation time, if slow speed sedimentation is used (I1 000 rev./min for 17.1 h at 3 °C). Thus, after 4 min exposure approx. 40 ~'o of the DNA is recovered. This is the result of incomplete release of single-stranded DNA from the complex. Slow speed sedimentation for a period long enough to position undamaged single-strands near the middle of the gradient a7 results in the complex spinning to the bottom of the tube.

To demonstrate more directly the effect of cross-linking, the release of single- stranded DNA from the complex was studied in psoralen plus near ultraviolet light treated cells and compared to untreated cells. For the conditions used in Fig. 2, after sedimentation the complex appears near the bottom of the tube while the 2 • 108 dalton material remains near the top 17. Figure 2a shows that 1 h lysis at approx, 25 °C already compromises the integrity of the complex in untreated cells and after 4.5 h lysis almost all the DNA sediments as single-strands.

In cells exposed to psoralen plus 2 rain near ultraviolet light, 8 h are required for release of "cross-linked, single-stranded DNA" (Fig. 2b). (The justification for this designation will be given later.) The mode of the DNA peak after psoralen plus near ultraviolet light is at Fraction 6, compared to Fraction 4 in the case of untreated cells, as a result of cross-linking. The difference in the rate of complex resolution is

DNA CROSS-LINKING IN CHINESE HAMSTER CELLS 185

i i i i i i

(a) 15 2 x 108 | o CONTROL

UNTREATED CELLS /o~ ~ PSORALEN+2min NUV

15 - o i.Oh LYSIS AT 25oc ! / k . PSORALEN+4min NU V J \' 6 ' " 4"5 h LYSIS AT 25°0 IO '- / \

oF I @~ 0 58°/° _ _ _ ' b ol ~ ~¢~ ~ ~ " '""~{b)"~

i fk o CONTROL 0 (b) j~ ~ 15 ~- E-9B

f~ 15 ~- I / 2 min NUV ~

~J ! /1! I I o 2 h ~ 5 ° C ~ ? ~ . 20 rain PSORALEN + NUV

,o o o 5 io 15 2o 25 30 FRACTION NUMBER FRACTION NUMBER

Fig. 2. Resolution of a DNA complex in alkaline sucrose gradients as a function of lysis time. (a) Cells (approx. 5000) were lysed in a 0.25 ml top layer (0.45 M NaOH, 0.5 M NaC1, 0.01 M Na2EDTA, pH 13.2) on top of a 5-20 70 alkaline sucrose gradient (0.3 M NaOH, 0.7 M NaCI, 0.003 M Na2EDTA) at approx. 25 °C for: 1 h, (O); 4.5 h, (0 ) . (b)Cells treated with 1 • 10 -° M 4,5",8-trimethylpsoralen were exposed for 2 min to near ultraviolet (NUV) light and lysed at approx. 25 °C for: 2 h, (O); 8 h, (0 ) . Sedimentation was at 5000 rev./min for 16.25 h at 0 °C. Direction of sedimentation was from left to right.

Fig. 3. Sedimentation in alkaline sucrose gradient of DNA from Chinese hamster cells grown for approx. 20 h with 0.5 #Ci/ml [3H]thymidine. (a) Cells (approx. 5000) were lysed for 2.5 h at 37 °C on top of an alkaline sucrose gradient and centrifuged at 9000 rev./min, approx. 0 °C for 16.25 h. Control, untreated cells, (O); ceils exposed for 2 rain to near ultraviolet (NUV) light in the presence of 1 • 10 -6 M 4,5",8-trimethylpsoralen, (A); cells exposed to psoralen plus near ultraviolet light for 4 rain, (0 ) . (b) Cells were X-irradiated with 20 kR and then lysed as above for 5 h at approx. 25 °C. Sedimentation was at 11 000 rev./min, approx. 3 °C for 17.1 h. Cells were exposed to psoralen plus light for: 0 rain, (O); 5 min (A); 20 min, (O). The arrows indicate the position of DNA of molecular weight 2 • 108.

more pronounced following shorter lysis periods. After a 1 h lysis at 25 °C, a consi- derable amount of the D N A already sediments as single-stranded material when untreated cells are used (Fig. 2a), while in psoralen plus near ultraviolet light-treated cells, essentially all the D N A sediments in the complex even after 2 h lysis at 25 °C.

In order to obtain recovery of essentially all the DNA, cells were either X- irradiated (20 kR) prior to lysis at approx. 25 °C, or the lysis temperature while cells were on the gradients was increased to 37 °C. In both cases essentially all the D N A was recovered. The X-ray dose used reduced the M, of single-stranded D N A from 2 • 10 s to approx. 2.5 • 107 in control cells while a 2.5 h lysis at 37 °C resulted in D N A of approx. 8 • 107. Because of the greater molecular weight of the D N A from untreated cells when lysis at 37 °C was used, this method was more sensitive for the detection of cross-links. Figure 3a shows the sedimentation pattern of D N A from

186 E. BEN-FIUR, M. M. ELKIND

psoralen plus near ultraviolet light-treated cells that were lysed at approx. 37 °C. It is evident that light exposure in the presence of 4,5' ,8-trimethylpsoralen increases the sedimentation velocity o f the D N A peak. Figure 3b shows that when cells were X-irradiated (20 kR) and then lysed at approx. 25 °C, this behavior is also evident but after longer near ultraviolet light exposures. This is interpreted to be the result of the product ion of D N A cross-links between two complementary strands 2°

Fig. 4 shows the dependence of the number of D N A cross-links (closed circles) produced per 1 • 108 daltons as a function of irradiation time with near ultraviolet light (calculated as described under Materials and Methods). (The open circles in Fig. 6 are discussed presently.) Lysis was at approx. 25 °C after 20 kR. Similar results were obtained for lysis at 37 °C without irradiation. In the exposure range of up to 20 min, a linear relationship exists between the incident light and the number of psoralen cross-links produced. After 30 rain there is some indication of saturation. This may be due to the phenomenon observed with T2 D N A , where multiple cross- links per duplex caused the sedimentation velocity to increase up to a saturation value. F r o m the slope of the curve the rate of cross-link product ion for duplex D N A was calculated to be 4.5 • 10-9/min.

4 0 - 4 0 o

.~ 30 ~3o %

l" % ~

z 2o ~ zo

IO I0 ~ g

0 b . f " I ___L l ~ _ . I _ I ~ 0 0 5 I0 15 20 25 30

IRRADIATION TIME ( r a i n )

Fig. 4. Number of DNA. cross-links produced and psoralen molecules bound per 1 • 108 DNA. as a function of exposure to psoralen plus near ultraviolet lights (NUV). The filled circles are cross-links (average of three experiments as shown in Fig. 5b) and their number was calculated as described in Materials and Methods. Vertical bars denote standard errors of the mean. The open circles are psoralen molecules bound to DNA. Estimation of light induced binding of psoralen is described in Materials and Methods.

Isopycnic sedimentation of cross-linked DNA in CsCI gradients Experiments using isopycnic sedimentation in CsC1 density gradients were

performed in order to demonstrate that the psoralen plus near ultraviolet light in-


duced D N A cross-links are indeed formed between the complementary D N A strands (interstrand cross-links).

Interstrand cross-linking can be demonstrated by denaturation of the D N A with alkali or heat followed by quick renaturation. In either case, only cross-linked D N A will renature to form native DNA, which can then be separated from the denatured material because it has a lower density. We used alkali denaturation at pH 12.5 followed by sedimentation in an alkaline CsC1 density gradient at pH 11.0 (renaturation occurs at this pH). Under these conditions a better separation of denatured from native D N A is obtained than when sedimentation is at neutral pH 21. Fig. 5 shows

I T i I

"!° /.1 o CONTROL • PSORALEN~ 4rain NUV

~ 20~- ~ i i ; , ~PSORALEN*IZmin NUV

Io i /

0 ~ . . . . . . . '~" "" ~ t 0 5 l O 15 20 25 3 0

FRACTION NUMBER

Fig. 5. Isopycnic sedimentation in CsCl gradients. Labeled DNA was sedimented in CsCI (see Materials and Methods) at 30 000 rev./min, approx. 20 °C for 64 h. Cells were exposed to near ultraviolet light in the presence of I - 10 -6 M 4,5',8-trimethylpsoralen for: 0 min, (O); 4 rain, (O); 12 rain, (A). p.y. = 75 ~ i 5 ~. Direction of sedimentation was from left to right.

the results of such an experiment. It can be seen that while from control cells only denatured D N A is obtained (p ~-- 1.74 g/cc), light exposure in the presence of psoralen leads to the appearance of native D N A (p ---- 1.70 g/cc) and a progressive loss of denatured DNA. It should be noted that the density of the renatured D N A de- creases with increasing light exposure until it reaches the value 1.70, the density of Chinese hamster cells native DNA. This behavior was observed repeatedly and is probably the result of the random induction of X-irradiation breaks which are ex- pressed after exposure to alkali and which result in bihelical fragments with single- stranded tails 22. The latter structure would have a larger buoyant density than per- fectly renatured D N A and hence, it would result in a net density between 1.70 and 1.74 g/cc. The influence of single-stranded regions would be reduced as the extent of cross-linking increases. This probably also reflects the fact that cross-links will occur first in the longest molecules and the longest molecules are the most likely to have single-strand breaks produced by X-irradiation and the lysis procedure. Thus as in Fig. 5, after small near ultraviolet doses, some single-strandedness is evident but not after large doses. While the extent of cross-linking tends, therefore to be under- estimated by this method because renatured D N A tends to be denser than it should be, this effect becomes negligible with increasing number of cross-links. For example, in Fig. 5 after a 4 min exposure the proportion of D N A which is cross-linked seems already well separated from the denatured DNA.


When the log percentage of nonrenaturing D N A is plotted as a function of near ultraviolet light irradiation time, an apparently exponential curve is obtained (linear on semilog coordinates, Fig. 6). That such a relationship is consistent with a two-step photochemical reaction for the production of cross-links can be seen from the following.

,oot%---~- ~ - , -~ - .7 l

L.. "~_-,\ \ k/k I 50]- ' % ' ~ ' 2 I

z io

' \ ,,-I ! ' \ I

2 | , I L • J I 0 I0 20 30

IRRADIATION TIME (ra in)

Fig. 6. Percent of nonrenaturable DNA from cells treated with 1 • 1 0 - 6 M 4,5',8-trimethylpsoralen and light, as a function of near ultraviolet light dose, determined from isopycnic sedimentation in CsCI. The dashed lines show the curves resulting from equation 6 with the ratios of the rate constants, k"/k', equal to the values shown, and 1/k" = 8.5 min.

The overall reaction is considered to be23:

P s + D N A P s - D N A hv P s -DNA h~ P s - D N A

(pso ra l en+DNA) ~ (complex) ~ " (monoadduct) k-;;~ (cross-link) (3)

Since psoralen is added to cells for 30 min prior to near ultraviolet light exposure, which is long enough to yield a maximum effect for a given near ultraviolet light exposure, it is likely that the production of monoadducts is not limited by the amount of complex. This is borne out by the psora len-DNA binding results in Fig. 4 for exposures up to at least 12 min. The production of cross-links leads to the loss of nonrenaturable D N A (nr-DNA) as derived below, providing there are no light-induced back reactions and that the probability of forming a cross-link with one quantum is very small compared to the other probabilities. Denoting the starting concentration of the complex by (Ps-DNA)o,

(Ps -DNA) = e_k, D (4) (Ps-DNA)o


(Ps-DNA) _ (1--e -k'°) e -k''° (5) (Ps-DNA)o

and, therefore, adding Eqns 3 and 4,

(nr -DNA) _ e_k,O_~e_k,Oe_(k,+k,,)O (6) (Ps-DNA)o

Two exteme relationships between k' and k" are worth considering. If k' ~ k",

(nr-DNA) ~ e_k,, o (7a) (Ps-DNA)o -

and when k' << k"

(nr-DNA) ~ e_k,o. (7b) (Ps_DNA) ° -

I f k ' = k " = k,

(nr-DNA) _ 2 e - kD e_2kO (8) (Ps-DNA)o

In addition to the apparently exponential dose-effect relation traced by the observed points in Fig. 6, three dashed curves are also shown. When k " / k ' = 1, Eqn 8 predicts a curve with a shoulder. However, as the ratio of rate constants increases, the shoulder becomes narrower as shown for k"/k ' ratios of 1, 2 and 3 in Eqn 6. For the solid curve which appears to fit the data, the exposure time to reduce the nonrenaturing DNA fraction by 1/e ( = 0.37) is 8.5 min. The theoretical curves show that a ratio of rate constants of 3 is already large enough to give a good fit of Eqn 5 to the experimental results. For k"/k ' ~-- 4 the theoretical curve is essentially exponential.

The lack of a significant shoulder on the experimental curve in Fig. 6 is consistent with the absence of a zero initial slope for the cross-linking curve in Fig. 4. Neither set of data indicates whether k' or k" is rate limiting. From the following analysis, however, it is concluded that the cross-linking step is rate limiting.

In Fig. 4, in addition to the induction of cross-links (closed circles), there is also plotted the induction of psoralen adducts. The slopes of these curves indicate that 7.8 adducts are formed per cross-link. These adducts, however, must include psoralen molecules bound to DNA which are not cross-linkable. In addition to binding not involving intercalation, if cross-linking is between pyrimidines s'9 in opposite strands and on either side of an intercalated adduct 2a, then there will be regions where the number of cross-linkable sites is fewer than the number of pyrimidines in a single- strand. Pyrimidine sequences of three or more would only have two cross-linkable sites regardless of the number of pyrimidines.

For these reasons, the ratio of 7.8 adducts to cross-links from Fig. 4 is probably an overestimate concerning cross-linkable monoadducts. However, since the theoretical curves in Fig. 6 show that a ratio of rate constants equal to 3 already gives a good agreement between theory and observation, a ratio < 7.8 would be consistent with cross-linking (k") as the rate-limiting step.


In addition, to the foregoing degree of internal consistency, it is worth noting that the cross-linking data in Figs 4 and 6 are in fairly close agreement. As determined from measurements of velocity sedimentation in alkaline gradients, the single-stranded D N A used in Figs 5 and 6 had an M,, = 1.6 • 1 0 7. This means that the cross-linking rate, k", of duplex D N A is 1 / (8 .5×2×1 .6×107) ~ 3.7. 10-9/min; a value only 20 ~ lower than that obtained using velocity sedimentation measurements (Fig. 4). Thus, the data are consistent with a two-step production of cross-links with the cross-linking step being rate limiting.

Disappearance of psoralen bound to DNA Cultured human cells are able to remove ultraviolet induced pyrimidine dimers

f rom their D N A (refs 24 and 25) in a process which may be analogous to the dark repair mechanism in ultraviolet-irradiated bacteria 26'z7. However, excision of pyrimidine dimers was not observed in cultured Chinese hamster cells 28. Fig. 7 shows that the 3H bound to cellular D N A via [3H]psoralen disappears during incubation in growth medium at 37 °C following 1 min exposure to near ultraviolet light in the presence of [3H]psoralen. The loss of label alone does not guarantee that psoralen, its monoadducts, or cross-link products are removed although such removal seems likely. The rate at which psoralen disappears is linear with time and about 90 % of the bound psoralen is removed within 10-11 h post-irradiation incubation at 37 °C. Because this was estimated by measurement of the specific activity of the DNA, it

I 0 0 - ~ T- -

8 0

6

- y ~ 60 ~

° 7

20 -4: a_ i

i 1 o 4 L 6 T I M E A T 3 7 " C ( h r s )

Fig. 7. Removal of bound psoralen from cellular DNA. Chinese hamster cells were exposed to 1 min near ultraviolet light in the presence of 1 • 10 -4 M 4,5',8-trimethyl[aH]psoralen. The psoralen bound to DNA was determined as described in Materials and Methods after incubation of the cells at 37 :C in growth medium for various times. The different symbols represent separate experiments.


has to be corrected for the residual DNA synthesis following psoralen plus 1 min near ultraviolet light. In 10 h, this amounts to only approx. 20 ~o of the DNA con- tent of the cells (Ben-Hur, E., Nine, B. and Elkind, M. M. unpublished). The corrected estimate for removal, 88 ~ , is therefore not significantly different.

DISCUSSION

Our results show that DNA cross-linking by psoralen plus near ultraviolet light in mammalian cells can be analyzed by means of velocity sedimentation in alkaline sucrose gradients. When used as described, this latter method is more sensitive than isopycnic sedimentation in CsCI gradients since usually DNA of higher molecular weight can be analyzed. Although with the velocity sedimentation technique the rate of DNA cross-linking may be overestimated 2°, for qualitative purposes this also would increase the sensitivity of the method.

DNA cross-linking was found to make more difficult the resolution of the complex into single-stranded DNA, as observed with alkaline sucrose gradients (Fig. 2). Introduction of single-strand breaks by X-rays facilitates the process, as it does in untreated cells 16' 17,29,30. The tighter binding of the complex is probably not due to psoralen cross-links between DNA and lipid 29'3°, since no lipid was found to sediment in the DNA peaks from psoralen plus near ultraviolet light-treated cells in CsC1 density gradients when the lipids were labeled with [3H]choline chloride (data not shown). Theinhibition of the resolution of the complex is therefore more likely due to DNA interstrand cross-linking; a point which in turn emphasizes the likelihood that the complex is duplex DNA. (This is in accord with observations with other cross- linking agents to be reported separately.) Resolution of the complex under alkaline conditions is a process, therefore, which results in denatured DNA but possibly also in changes in tertiary structure as well 17'3°'33. Breaks induced by radiation 16'17'3°, 5-bromouracil substitution of thymine in DNA followed by X-irradiation 31 or fluorescent light 19, actinomycin D 33, and elevated temperature all facilitate the resolution of the complex.

The rate of duplex DNA cross-linking by near ultraviolet light in the presence of 1 • 10 -6 M 4,5',8-trimethylpsoralen was found to be approx. 4.1 • 10-9/min (average of the rates observed with alkaline sucrose gradient and isopynic sedimentation). The D 6 exposure (the exposure required to reduce survival by a factor of 1/e along the exponential part of the curve) of the survival curve under these conditions is 4.8 s 5. If the average aggregate amount of DNA per cell in the logarithmic phase of growth is taken as 6- 1012 dalton, it can be calculated that there are approx. 2000 cross-links/cell per lethal hit. It is conceivable that the presence of even one cross- link could result in cell death if it is not repaired or bypassed, since DNA strand separation is probably required for DNA replication. (This assumes that viability requires the replication in a near normal way of the entire genome which might not be the case.) In repair deficient mutants of Escherichia coli this indeed seems to be the case 14, and, therefore, it is reasonable to assume that Chinese hamster cells can tolerate such a large number of cross-links only if they possess a repair mechanism(s) which removes the cross-links from the DNA. Our observation that the cells are probably able to remove psoralen photoadducts from their DNA (Fig. 7) is consistent with the existence of such a process. Also, since about 90 ~o of the bound psoralen can be


removed, and since approx. 11% of this psora len is in the fo rm of cross-l inks (as- suming 37 and 4.1 • 10-9/ ra in for psora len b inding and cross- l inking, respect ively) it is possible tha t at least some o f the cross-l inks have also been removed. Indeed, using velocity sed imenta t ion techniques, some evidence for the removal o f cross- links has been observed, but this was not easy to detect because o f a concomi t an t b r eakdown of D N A (Ben-Hur and Elkind, unpubl i shed results). I f excision repa i r is invoked in the removal of b o u n d psoralen, it p robab ly involves only a few nucleotides per psora len p h o t o a d d u c t (or wha t is excised is reused) because no significant degrada t ion of cel lular D N A to an acid-soluble form was observed dur ing the t ime required to remove abou t 90 % o f the b o u n d psora len (10-11 h). Final ly , it should be made clear that the quant i ta t ive results app ly specifically to 4 ,5 ' ,8- t r imethylpsora len a l though the general features are l ikely to be appl icable to o ther psoralens.

ACKNOWLEDGEMENT

We are indebted to Dr S. Lacks for his comments dur ing the p repa ra t ion of the manuscr ip t and to Miss B. Nine for an excellent technical assistance. This work was suppor t ed in par t by a U .S .Na t iona l Ins t i tu te of Hea l th Fe l lowship No. 1-F03- C A 52437-01, f rom the Na t iona l Cancer Insti tute.

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