Relationship between DNA adduct formation and sister chromatid exchange induction by [3H]8-methoxypsoralen in Chinese hamster ovary cells

Copyright @ 1980 Sy Academic Pieas. ix. ,411 rigkls of reproduction in any form reserved 0014-4827i80/0i0015-08s02,00io

Experimental

ELATIONSHIP

Cell Research 128 (1988) 15-22

BETWEEN

DNA A TER CHROMATID EXC XYPSORALEN IN CHINESE DOUGLAS M. CASSEL’ and SAMUEL

A. LATT

Division oj’Genetics and Mental Retardation Ceher, Children’s Hospital Medical Center, and the Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA

SUMMARY The combination of [3H]8-methoxypsoralen ([3H]8-MeOP) plus near .uitraviolet (UV) light was used to study sister chromatid exchange induction and [3H]8-MeOP-DNA adduct formation in Chinese hamster ovary (CHO) cells. Approx. 2.2 adducts/cellierg/mm2 light were Formed under conditions leading to the induction of 0.011 sister chromatid exchanges/erg/mm2 light during hbe two cvcles following DNA damage. Based on these data, the adduct to sister chromatid exchange ratio is nearly 2OO.Both DNA monoadducts and crosslinks were produced by the illumination employed; the relative effectiveness of these S-MeOP-DNA reaction products in sister chromatid exchange induction remains to be determined. The yield of 8-MeOP-DNA adducts did not show appreciable cell cycle dependence, and the efficiency of adduct formation, unlike sister chromatid exchange induction, was the same for DNA which replicated early or late in the DNA synthesis phase. However, substitution of 5-bromodeoxyuridine (BUdR) for thymidine in the DNA led to a nearly 2-fold increase in adduct formation. It is concluded that, while sister chromatid exchanges are typically a much more sensitive index of DNA damage than are chromosome aberrations, these exchanges may still correspond to only a small fraction of the total damage produced.

Sister chromatid exchanges (SCEs) appear to be highly sensitive and reliable indices of the effect of mutagen-carcinogens on chromosomes [l-5]. These exchanges are at least 200 times as frequent as chromosome structural aberrations [I, 2, 61 under a wide variety of conditions, such as exposure of ceils to alkylating agents [l , 2, 6]~ However, details about the actual molecular events leading to SCE formation remain to be elucidated [7-91. Of particular interest is the quantitative relationship between NA damage created by chemical agents the increment in sister chromatid exchanges formed in responses to this damage. e combination of 8-methoxypsoralen 2-801803

(8-MeQP) plus near UV light c~~st~t~tes a well defined system for a~a~ysi~~the stoichiometric relationship between damage and SCE ~~duct~o~, SC by this combtne served in many organisms and cell types [l&13], with the exte t of SCE fQrmatiQ~ dependent upon both the 8tration and the illuminatio eQP, as well as many related compounds, rms chemically stable mono-adducts a.nd crosslinks wit

i Present address: Department of Radiology, Sianford University Medical School, Stanford, CA 94305. USA.

16

Case1 and Latt

these compounds, including the study of chromatin [ 19-241 and the treatment of psoriasis [25]. Preferential reactivity with double helical nucleic acids presumably reflects the ability of psoralens to bind by intercalation [28], in an orientation compatible with adduct formation across the 5,6-pyrimidine double bond. Finally, radioactive psoralen derivatives can be prepared [17, 291, and the extent of psoralen-DNA adduct formation quantitated by radioactivity measurements. In the present experiments, tritiated Smethoxypsoralen. ([3H]S-MeOP) has been prepared and photo-reacted with Chinese hamster ovary [30] (CHO) cells, forming both mono-adducts and crosslinks with DNA. The total amount of 8-MeOP reacted with CHO cell DNA under different conditions was quantitated and compared with SCE formation during the subsequent two replication cycles. The results indicate that SCEs, in spite of their relative sensitivity compared with other cytological tests, may still reflect only a small fraction of genetic damage to a cell. MATERIALS

AND METHODS

[3H]8-methoxypsoralen [3H]8-MeOP 8-methoxypsoralen, a generous gift of the Paul B. Elder Co., Bryan, Ohio, was tritiated by catalytic exchange by New England Nuclear Co. Exposure of 100 mg of 8-MeOP to 25 Ci of tritium (under non-reducing conditions) yielded 425 mCi of crude product. This material was purified by thin layer chromatography (Mallinckrodt Chrom AR plates) using CHCI, as solvent. Material moving as a fluorescent band with R,-0.5 was eluted with methanol and filtered. The final yield was approx. 0.2 mM 8-MeOP at a specific activity of 0.32 mCi/pmole. 8-MeOP concentrations were calculated from absorptivity measurements and molar extinction coefficient of 1.25~ lo* M-l cm-’ at 298 nm in methanol, based on dry weight. Radioactivity was measured in a Beckman model LS-330 scintillation counter, using an energy window set to collect virtually all the tritium radiation. Aqueous samples were counted in Aquasol and non-aqueous samples in Liquifluor. Counting efficiencies, determined with standard tritiated water or toluene, in Exp Cell Res 128 i/%0)

Aquasol or Liquifluor, were determined to be 42 and 48 %, respectively. CHO cells, the generous gift of Dr Arthur Pardee, were cultured at 37°C in modified Ham’s F-10 medium as described previously [ 12, 3 11.Cells, grown as monolayers in rectangular based T-25 flasks, were exposed to 8-MeOP (typically a 100x stock solution in methanol) and, 1 h later, irradiated from below with 4 W, UV21 lamps (LJV Products, San Gabriel, Calif.) through a 5 mm Corning 7-51 (360 nm wide band pass) filter. DNA solutions were reacted under the same conditions. Lamp intensity, measured as before [12] with an International Light (Newburyport, Mass.) IL-500 photometer, refer to flux received by the cells in the interval between 320-370 nm. The same lamp intensity measurement convention was used for both DNA-adduct and SCE induction experiments. For measurement of adduct formation, cells were harvested by trypsinization, washed three times in Hanks’ buffered salt solution, and vortexed in 0.01 M KCl, 0.0015 M MgCl,, 0.01 M Tris, pH 8.5, 0.5% NP40, to obtain a crude nuclear preparation. This material was digested with 1 mg/ml nuclease-free pronase (Biorad) (preheated to 80°C) in 0.1 M NaCl, 0.01 M EDTA, 0.03 M Tris pi 8.0, 2% sarcosyl at 37”~ for at least 18 h, followed by 100 yg/ml pancreatic RNAse (previously boiled) for a few hours at 37°C [32]. This material was spotted onto Gallard-Schlessinger paper filters either directly or after CsCl centrifugation (Beckman L265B, SW 50.1 rotor, 30 000 rpm). Before treatment and analysis, samples tested for crosslink formation were also subjected to phenol extraction, followed by CHCI, extraction and dialysis versus pH 7 buffer. All manipulations were performed in room lighting excluding wavelengths <550 nm; otherwise samples were protected from light. Filter paper discs containing DNA were dried at 37”C, washed three times in ice-cold 5% trichloroacetic acid, in ethanol, and then dried, before radioactivity measurement in Liquifluor. Virtually identical results were obtained when these discs were counted in Aquasol. The effect of attachment of [3H]8-MeOP to DNA on counting efficiency, compared with that of free 8-MeOP, was determined by measuring the effect of DNAse II (Worthington) hydrolysis on counting efficiency (in Aquasol). Complete digestion (assessed by reduced acid precipitability) caused a 2.2fold increase in counting efficiency, while binding of intact DNA to filters had a negligible effect on radioactivity detection. When calculating numbers of r3H]8-MeOP molecules bound to DNA, radioactivity measurements on DNA plus filter-bound [3H]8-MeOP were thus multiplied by this factor of 2.2. At a specific activity of 0.32 mCi/pmole and an overall counting efficiency of 22%, one molecule of DNA-bound [3H]8-MeOP yielded 0.26~ 1O-9cpm. Preparative centrifugation in CsCl (Varlacoid) gradients (0.1 M Tris, 0.01 M EDTA, pH 8), employing different internal density standards, was used to assess the accuracy of the filter paper assay and to separate [3H]8-MeOP bound to DNA substituted to different extents with 5-bromodeoxyuridine (BUdR). Similar gradients, also containing 0.167 pg/ml propidium diiodide [33] (Calbiochem), were used to resolve [3H]8-MeOP bound to native and denatured

DNA adduct.formation

avid sister c~~rornat~d exchange ~~d~~ct~(~~

DNA. Analytical CsCl centrifugation utilized a Suinco Model E ultracentrifuge with a photoelectric scanner (kindlv made available bv Dr Ezio Merler). Densities were determined [34] relative to M. lysodekkus DNA (1.731 gm/cm3) and amounts of DNA calculated from areas under densitometer tracings (absorptivity at 268 am), weighted for radius-dependent cell sector area. BUdR substitution was calculated assuming that it was proportional to density increment, with 100% substitution of BUdR for dT (58% A-T composition) giving a density shift of 0.108 g/cm3 [35]. DNA measurements utilized a modified diaminobenzoic acid procedure [36j. The relative amount of [9I]&MeOP forming monoadducts and crosslinks following treatment, was determined by shearing phenol-extracted DNA to approx. 3X IQ” D (determined in 5-20 % sucrose sedimentation vefociiy gradients with internal standards), immersing the DNA in boiling water for 10 min. quick cooling in ice, and centrifugation at 34000 rpm in CsCl (average density 1.54 gicmY) containing 0.167 pg/mi propidium diiodide. Positions of (denser) denatured and native DNA in such gradients were previously determined in centrifugations with known native or denatured “H or 14CCHO DNA BUdR (Sigma) was added to cells at 2.5~10~~ M (for SCE measurement) or 5~ IO-” M (for inducing buoyant density shifts). Cells incorporating BUdR were protected from light, except when deliberately irradiated to produce 8-MeOP adducts. For SCE analysis, 8-MeOP plus light treatment preceded BUdR addition, and cell culture, harvest, slide preparation, photography, and SCE scoring followed previously described procedures [37, 381. 33258 Hoechst was the generous gift of Dr H. Loewe. Hoechst AC. Frankfurt am Main.

1’7

Light dose (eigs/mm2x lO”i

Fig. 1. DNA adducts in CHQ ceils (X 90-j) or DNA (X1o-6) exposed to [3H]8-MeOP (7.4X10d6 M) plus near UY light. Monolayers of CHO cells or solutions of 6~ !QF M calf thvmus DNA were treated with doses of 320-370 nm illumination. Cells were then harvested? and nuclei isolated and subjected to pronase-RNAse digestion. Each data point represents the average of two separate experiments, each performed at ieast in dup!icate. The number of [“H]8-MeOP-DNA adducts/cell was calcuiated from the amount of radioactivity nrecipitabie on naner fiiters by trichloroacetic acid, as de&bed in Methods. The sl;pe of the lower ) is 2.1810.06 (adducts/ceil)/(ergs/mn?). line ( The scale of the upper line (3--O), reflecting adducts/ 7 pg DNA (Cl-cell equivalent) has been reduced by l/10, i.e. DNA reaction exceeded that of ceils by 20-3@-fold.

NA adducts were based sacetuz acid-precipitable radhof chromatin digested activity onto fil NAse or of DNA from with gronase an CsCl gradients. The dependence of ad uct formation on Covalent binding of [3H]8-MeOP to CL-IO NA was routinely quantitated by a fil- light exposure was determined with mater paper binding assay. Approx. 90% of terial exposed to 7.4X 1OV ~:~~]8-~~e~~ igher concentrations of [“H]8ioactivity measured this way from a eOP (up ts at least 7.4X10-” M) caused Ase digest of nuclei crude pronase, following CsCl centri- roughly ~~o~o~t~onate~y greater adduct could be recover gation, as a single band with buoyant formation (data not showfi), although this nsity appropriate for CHO cell DNA (fig. was not investigated in depth. Adduct for1). Washed whole cells, when spotted on mation with free DNA was perha filter papers, yielded 2-3-fold as much greater than that with DNA in in radioactivity, and were not utilized. Filter probably resecting both reduced DSA acpaper and CsCI analysis yielded com- cessibility in chromatin an parable values for [W]8-MeOP bound to tion of intact cells by 8-M NA; these S-MeOP-DNA of adduct formation on 1 calf thymus adducts were resistant to repeated CHCI, a~~~~xi~at~~~ linear, an extraction. All subsequent measurements straight lines are drawn assuming such a

18

Cassel and Latt Table 1. Ratio of DNA adducts to SCE in CHO cells following treatment with r3H]8-MeOP plus light” DNA adductslcell SCE/Z cell? AdductsiSCE o

e Light

2’

’

4’

dose (ergs/mm’x

’

6’

J

8

103)

Fig. 2. SCE induction in CHO cells by S-MeOP or r3H18-MeOP nlus near UV light (320-370 nm). CHO monolayers were exposed to kcreasing doses’of light 1 h after additions q , 7.4~10-~ M 8-MeOP: n , 7.4~ 10e6 M [3H]8-MeOP; or 0, nothing. The cells were then placed in fresh medium containing 2.5X10W5M BUdR and allowed to replicate twice (-28 h) prior to harvest at metaphase. Slides were then subjected to a 33258 Hoechst plus Giemsa procedure and SCE per cell counted. Each data point represents the average of four separate cultures, from which approx. 25 cells were scored. The [3H]8-MeOP was at least 85% as effective as the unlabelled 8-MeOP in inducing SCEs.

proportionality (fig. 1). Analysis of adduct formation in cells as a second order function of light dose indicated that the quadratic term accounted for less than 20% of the total at the highest light exposure shown (and less than 3 % of the total at 2x lo4 ergs/mm2, the highest exposure used for SCE induction). The ability of r3H]8-MeOP to induce SCEs in CHO cells was comparable to that of non-radioactive 8-MeOP, while light alone had no significant effect (fig. 2). Tritiation, which had no detectable effect on the absorption spectrum or chromatographic mobility of 8-MeOP, also had little if any effect on its ability to act as a potent inducer of SCE formation. The combination of 8-MeOP plus near UV light produced many more DNA adducts than SCEs in CHO cells (figs 1, 2). Assuming a linear proportionality between either adduct or SCE induction and light exposure, an adduct/SCE ratio of 195 can be computed (table 1). Optimal conditions conflicted in these experiments. SCE exEq, Cd Rrs 128 (IYW)

2.18+0.06/(ergs/mm2) 0.011i0.001/(ergs/mm2) 19.5+2Y

B 7.4X1O-6 M [3H]8-MeOP, near UV light (320-370 nm); table entries derived from slopes of lines in figs 1 and 2. * Each cell treated before BUdR addition (and scored directly for adduct formation) yields two second division metaphases scored for SCE induction. ’ Experimental uncertainty in determination of the absolute detection efficiency of the scintillation counter, as well as internal quenching due to binding to DNA would add perhaps another +20% uncertainty to this ratio.

periments utilized low light doses (at lo4 erg/mm2, viability was approx. 50%, but it decreased rapidly at higher doses). However, biochemical experiments required higher doses of UV to obtain adequate amounts of bound radioactivity. Therefore, the light exposure range used in the biochemical experiments exceeded (though overlapped at its lower bound) that used for the SCE induction. 8-MeOP-DNA adduct formation did not show significant S-phase dependence. These measurements were performed for two reasons. First, SCE analyses were timed to select for cells just about to enter S phase prior to treatment, while adducts were measured on a more heterogeneous cell population. Second, SCE inducibility varies during S [12, 401, though not appreciably during Gl [41]. In contrast to the SCE data, no appreciable difference in adduct formation (based on total DNA) was seen for synchronized [ 121cells at the start, near the middle, or at the end of S (table 2). Similarly, unsynchronized cells and cells blocked at Gl/S reacted identically (data not shown).

19

BM.4 ndduct formation and sister c~lr~rn~t~d exchange ~~d~c~~~~ Reactivity oj’ CHO ceil DNA with eOP plus near UV light at various times within S phase” Time

GP-S 4 h into S 8 h into S

Table 3. I&l. replicating C plus near e/V light A, BUdR O-4.5 h; dT 4.5-9 h, 8-~e~~~~~~bt starting 9 h after release; B, BUdR @-4.5 h; followed by gMeOP+light; C, dT O-4.5 h, %Ud iight starting 9 h after reiease -

[3H]8-MeOPb 7 pg DNA 3.07x 106 2.68X 10s 2.86X 106

--

o CM0 cells were subjected to isoleucine-hydroxyurea synchronization [3 I], released, and treated with 2.2x lo-” M [W]&MeOP followed 1 h later (at times within S indicated) by 3 x IO5ergs/mm’ near UV light. * Molecules of [3H]8-MeOP bound to DNA, based on measurement of radioactivity: DNA analysis as described in Methods. 7 pg=DNA content/CHO cell in 61 [39].

Prorocoi Replication within S of BUdR-substituted DNA (BT) DNA” (TT) DNA Average

Early

Early

Late

0.73

I 08

0.27

1.75

1.49

1.46

reaclivify

[H]8MeOP/(%T) [3H]8-MeOP/(TT) [8H]8-MeOP/early [3H]8-h4eOP/late

DNA” DNA DNA DNA

1.75 I.69 0.56 Corroborative data indicating comparaeOP adduct formation with early 0 Relative amount of BT and TT DNA, based in after CsCl centrifugation; average of two experireplicating DNA were obtained by OD,,, ments. introducing BUdR at the start or end of S b Relative amount of [“H&MeOP adducts formed with (unifilarily substituted) and TT (~~~s~bsf~tute~) and analyzing [3J3]8-MeOP bound to unsub- BT DNA, based on radioactivity; average of two experistituted or unifilarly substituted DNA, re- ments and normalized for rel. DNA content. pycnic CsCl centrifugation rences observed could, by examining NA lling sequence, be attribteiy one or two entire cycles uted to an increase in 8-MeOP reactivity associated with BUdR substitution, rather o the timing of DNA replication. enhancement of 8-MeOP reactivity Swars also observed in separate experiments

Table 4. Effect elf BUdR substitution on CHO DNA reactivity with [ uv iight” DNA contentC BUdR” (hours)

0

19 44

Rel. reactivity 4.4x loj 7.7x 105 10.3x loj

1.00 1.20 1.61

1.00 0.42 0.06

0.00 0.47 0.49

0.00 0.11 0.45

n SXlOW M BUdR, present during preharvest interval indicated; preharvest exposure to 7.5X10-” M [W]S-MeOP, followed 1 h later by 3.6~ lo5 ergs/mm* near UV light. b Molecufes of [W]g-MeOP bound to DNA, based on measurement of radioactivity. c BUdR substitution approx. 80% (each polynucleotide chain involved), based on buoyant density analysis [34, 35] and utilizing 14C @e DNA as a ~=I.740 density marker. TT, %T, and BB refer to DNA substituted with BUdR in 0, 1 or 2 polynucleotide chains, respectively.

20

Case1 and Latt

form of crosslink was estimated, based on the associated rapid renaturability conferred on DNA fragments. With 1x lo5 and 2.5~ lo5 ergs/mm2 light exposure, the proportion of [3H]8-MeOP in rapidly renaturing (presumed to be predominantly crosslinked) DNA increased from 40 to 60% (fig. 3). In contrast, r3H]dT-substituted, phenol-extracted DNA from CHO cells not exposed to 8-MeOP plus light showed no peak, resolvable above background, identifiable as native DNA following a heat and quick cool cycle.

200

t

.I Aa, ,i :;;.A;0 20

E E z :: 5

30

The present experiments show that, while SCE induction in CHO cells by S-MeOP plus light greatly exceeds induction of chromosome breaks (an increment of less than 0.05 per cell was seen even at the highest treatment used), the SCEs in turn reflect less than 1% of the total 8-MeOP-DNA adduct formation. Assuming both adducts and SCE to be linearly proportional to light dose (over their respective exposure range), an adduct/SCE ratio of 195 (with an overall uncertainty of perhaps 30%) is determined. Non-linearity in light response would not alter appreciably the general magnitude of this estimation of the adduct/ SCE ratio. Selective scoring of viable cells for SCE analysis would bias these data towards cells with lower damage, but cell viability was high in the O-l x lo4 erg/mm2 dosage interval. Similarly, while SCEs formed after the second cycle were not scored in the present experiments, and SCE induction by a single dose of 8-MeOP plus light can occur for at least three cycles [12], the third cycle contribution was ~20 % of that in the previous two cycles [12]. Thus, an adduct/SCE ratio much greater than 1 for this treatment in Err, CrilRrs

128(1980)

50

200 150 100 .:

d

5o B 00IA**

TT

I IO

20 Fraction

DISCUSSION

40

30

40

50

no

3. Rel. amounts of [3H]8-MeOP reacted with CHO cell DNA in the form of mono-adducts and crosslinks. DNA from CHO cells exposed to 7.4~10~” M [3H]8-MeOP and (A) 1.0~ lo5 ergs/mm’, or (B) 2.5X lo5 ergs/mm’ near UV light was isolated, sheared, heated and quick cooled as described in Materials and Methods. The DNA was centrifuged at pH 8 in CsCl containing 0.167 pg/ml propidium diiodide. r3H]8-MeOP bound to native, renatured DNA (II) is presumably in the form of crosslinks, while that bound to DNA which did not renature (I) is presumably in the form of monoadducts. Material in peak (II), i.e. crosslinks, accounted for an average (2 expts) of approx. 40 and 60% of the total [3H]8-MeOP in (A) and (B), respectively.

Fig.

CHO cells seems established. A similar excess of mitomycin C-DNA adducts versus SCEs in human fetal lung fibroblasts can be estimated from data (at one [3H]mitomycin C treatment level) of Schneider & Monticone [42], although the primary thrust of these latter experiments was different than that of those presented here. It will be interesting to determine whether the ratio of DNA damage to SCE induction is greatly in excess of unity for other types of clastogenic agents. Data indicating the correspondence between SCE induction and mutagenesis have now been obtained [43], although the exact proportionalities depend on the agent used. Even greater variation is observed when chromo-

DNA adduct formation

and sister chromntid

some breakage and SCE induction in reNA damage are compared in different cell types for different agents [9, 44, 451. One interpretation of these results is that SCEs and breakage reflect very low probability outcomes of DNA damage. SCEs and chromosome aberrations may or may not correspond to the same DNA damage, with SCEs perhaps reflecting a small but biologically significant component of the original damage. A lV-105-fold ratio of DNA damage to chromosome breaks provides a wide interval within which the ratio NA damage to SCE formation can fall, the actual relationship between SCEs chromosome breaks might vary signifient on details of the DNA

cibility throughout S [ 121,indicates that altered inducibility reflects cellular response to damage and not the extent of damage. The increased adduct formation of DNA substituted with may reflect either enhanced binding en or enhanced reactivity pyrimidine double bond. A ement of psoralen reactivity was recently reported for RNA containing uoro-uracil [27]. The mechanism of this halogen atom effect on psoralen reactivity as not yet been determined. The present data indicate that, at high “) illumination levels, much damage by S-MeOP is in ct form, even though this agent ally crosslink DNA. Other studies of psoraien-DNA reactions, at high hght exposures, similarly indicate an adixture of DNA mono-adducts and crosslinks [14-Z!]. The nature of the monoadducts, which might ultimately be controlled by altering the excitation wavelength

exchmge

inducrion

21

[IS], has not yet been deter based on recent data indicati nificant ~~~~~rti~~ of psoralen ducts are in the form of cros low levels of 365 nm ~~~~rni~~t~o~ [46], it is premature to speculate about the relative ucts present in cells subes of Bight (G IO4 ergs/ oyed for SCE induction.

of this

question

will

respire

further

st~idy.

of most agents capable of inducing %X:9, and there exists 2 need for DNA re following damage for SCE mduction :o occur [B2, 411, some caution is probably advisable in neglecting t SCE ~~d~~t~o~of damage to celluiar somponents other than DNA [e.g. 4.71~ The expert technical assistance of Mr Michael Risenhard and MS Lois Juergens is greatly appreciated. This research was supported by funds from the NIH (GM21 121) and the ACS (CD36D). D, M. C. was the recipient of a National Foundation March of Dimes Summer Feilowship, and S. A. L. is the recipient of a Research Career Deveiopment Award (C&%00122) from the National institute of General 52edicai Cciences

1. Eatt, S A, Rot natl acad sci US 71 (~1974)3162. Perrv. P & Evans. H 9. Nature 2% Cl9751121. Wolff: S, Ann rev’genet 1: (1977) 183. Perry, P, Chemical mutagens (ed A Mollaender & F DeSerres) vol. 6. Plenum Press, New York (1979). 5. Lat:, S A, Schreck, R R, Loveday. K S & Shuier, (3 F, Cytogenetic testing of environmental mutagens (ed T C Msu). In press. 4. iatt, S A, Science 185 (1974) 74. 7. Latt, S A, Allen. J W. Shuler, C. Loveday. K S $i Monroe, S H, Molecular human cytogenetics (ed R S Sparkes, D E Comings & C F Fax). VII ICI%UCLA symposium on moiec&r & cellu!ar biology, p. 315. Plenum Press, New York (1977). 8. Galloway, S, DNA repair processes (ed W W Nichois B D G ~~~~y) p. 191. Symposia specialists, Miami, Fla (1977). 2. 3. 4.

22

Case1 and Latt

9. Wolff, S, DNA repair mechanisms (ed P C Hanavalt, E C Friedberg & C F Fox). IX UCLAICN symposium on mo&lar & celiular biology, D. 751. Academic Press, New York (1978). 10. Carter, D M, Wolff, K & Schnedl; W,‘J invest dermatol67 (1976) 548. 11. Waksvik, H, Brogger, A & Stene, J, Human genet 38 (1977) 195. 12. Latt, S A & Loveday, K S, Cytogenet cell genet 21 (1978) 184. 13. Mourelatos, D, Faed, M J W & Johnson, B E, Experientia 33 (1977) 1091. 14. Cole, R, Biochim biophys acta 254 (1971) 30. 15. Musajo, L, Bordin, F, Caporale, G, Marciani, S & Riaatti. G, Photochem ohotobiol6 (1967) 711. 16. Johnston, N H, Johnson, M A, Moore, C B & Hearst, J E, Science 197 (1977) 906. 17. Ou, C N, Tsa, C H, Tapley, K J, Jr & Song, P S, Biochemistry 17 (1978) 1047. 18. Chattejee, P K & Cantor, C R, Nucleic acids res 5 (1978) 3619. 19. Hanson. C V. Shen. C K J & Hearst. J. Science 193 (1976) 62.’ 20. Cech. T R & Pardue. M L. Proc natl acad sci US 73 (1976) 2644. ’ 21. - Cell 11 (1977) 631. 22. Isaacs, S T, Shen, C K J, Hearst, J E & Rapoport, H, Biochemistry 16 (1977) 1058. 23. Hyde, J E & Hearst, J E, Biochemistry 17 (1978) 1251. 24. Hallick. L M. Yokota. H A. Bartholomew. J C & Hearst,‘J E, J virol27’(1978) 127. 25. Parrish. J A. Fitznatrick. T B. Tannenbaum. L & Pathak; M A, New eng jmed 291 (1974) 1207. 26. Hearst, J E & Thiry, L, Nucleic acids res 4 (1977) 1339. 27. Ott, C N & Song, P S, Biochemistry 17 (1978) 1054. 28. Dall’Acqua, F, Rodighiero, G & Musajo, L, Rend sci fis mat nat 40 (1966) 411.

E,rp Cell Res 128 (1980)

29. Cassuto, E, Gross, N, Bardwell, E & HowardFlanders, P, Biochim biophys acta 475 (1977) 589. 30. Tjio, S M & Puck, T T, J exp med 108 (1958) 259. 31. Hamlin, J L & Pardee, A B, Exp cell res 100 (1976) 265. 32. Davidson, R L & Bick, M C, Proc natl acad sci US 70 (1973) 138. 33. Grossman, L I, Watson, R & Vinograd, J, J mol bio186 (1975) 271. 34. Thomas, C A & Berns, K I, J mol biol3 (1961) 277. 35. Luk, D C & Bick, M D, Anal biochem 77 (1977) 346. 36. Einstein, L P, Schneeberger, E E & Colten, H R, J exp med 143 (1976) 114. 37. Perry, P & Wolff, S, Nature 251 (1974) 256. 38. Latt, S A, Allen, J W, Rogers, WE & Juergens, L A, Handbook of mutagenicity test procedures (ed B Kilbey, M Legator, W Nichols & C Ramel) p. 275. Elsevier, Amsterdam (1977~). 39. McBride, 0 W & Peterson, E A, J cell biol 47 (1970) 132. 40. Kato, H, Nature 252 (1974b) 739. 41. Wolff, S, Bodvcote, J & Painter. RB. Mutat res 25 (1974) 73. _ 42. Schneider, E L & Monticone, R E, Exp cell res 115 (1978) 269. 43. Carrano,‘AV, Thompson, L J, Lindl, P A & Minkler, J L, Nature 271 (1978) 551. 44. Ikushima, T, Nature 268 (1977) 235. 4.5. Sasaki, M S, Nature 269 (1977) 623. 46. Dall’Acqua, F, Research in photobiology (ed A Castellani) p. 24.5. Plenum Press, New York and London (1977). 47. Ewig, R A G & Kohn, K W, Cancer res 38 (1978) 3197. Received June 27, 1979 Revised version received January 24, 1980 Accepted January 30, 1980

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