JOURNAL OF BACTERIOLOGY, Mar. 1972, p. 979-986Copyright 0 1972 American Society for Microbiology

Vol. 109, No. 3Printed in U.S.A.

Repair of Pyrimidine Dimer Damage Induced inYeast by Ultraviolet Light

MICHAEL A. RESNICK' AND JANE K. SETLOWBiology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Received for publication 29 October 1971

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivatingenzyme. The amount of competition is taken as a measure of the level of cyclo-butyl pyrimidine dimers in the yeast DNA. A calibration of the competitionusing UV-irradiated calf thymus DNA indicates that an incident UV dose(1,500 ergs/mm2) yielding 1% survivors of wild-type cells produces between 2.5x 104 to 5 x 104 dimers per cell. Wild-type cells irradiated in the exponentialphase of growth remove or alter more than 90% of the dimers within 220 minafter irradiation. Pyrimidine dimers induced in stationary-phase wild-type cellsappear to remain in the DNA; however, with incubation, they become less pho-toreactivable in vivo, although remaining photoreactivable in vitro. In contrast,exponentially growing or stationary-phase UV-sensitive cells (rad2-1 7) showalmost no detectable alteration of dimers. We conclude that the UV-sensitivecells lack an early step in the repair of UV-induced pyrimidine dimers.

The sensitivity of Saccharomyces cerevisiaeto ionizing and ultraviolet (UV) radiations isunder extensive genetic control involving morethan 25 loci (summarized in reference 4). Somemechanisms for the dark repair of damageinduced in yeast deoxyribonucleic acid (DNA)are presumably similar to the excision repairand recombination repair mechanisms de-scribed in bacteria (see references 6, 18, 22 forreviews). However, it has been difficult tocharacterize the yeast repair systems becauseit has not been possible to label the DNA spe-cifically with radioactive deoxyribonucleosides,due in part to the absence of a thymidine ki-nase (5), although progress has been made inexamining repair after large UV doses usingthe general label l4C-uracil (Unrau, Wheat-croft, and Cox, submitted for publication). Re-cently it was reported that some strains ofyeast may be labeled with radioactive thymi-dine monophosphate (7); however, the cellscan incorporate label only during a short pe-riod of exponential growth (Brendel andHaynes, submitted for publication). The abilityor inability to label some strains and not othersis attributable to the presence or absence of asingle gene function.

I Present address: Department of Radiation Biology andBiophvsics, University of Rochester School of Medicine andDentistry, Rochester, N.Y. 14642.

In this study we describe a new method forestimating the number of dimers in yeastDNA. Crude extracts of UV-irradiated, ascompared to unirradiated, cells are shown toinhibit the ability of photoreactivating (PR)enzyme to monomerize UV-induced pyrimi-dine dimers in Haemophilus influenzae trans-forming DNA. The competition, which is con-cluded to result from pyrimidine dimers in thecrude yeast cell extracts, has been standard-ized and has been used to measure dark repairin exponential and stationary cells of a wild-type and a UV-sensitive strain at doses of UVthat result in greater than 1% survival of thewild-type cells.

MATERIALS AND METHODSYeast strains and growth. The two strains of S.

cerevisiae used were S288c, which is wild type, andPR10-ld, which is UV-sensitive due to a mutation inthe uvs9 gene (13). According to recent nomencla-ture (4), this gene is referred to as rad2, and the par-ticular allele previously identified as uvs9-3 (13) isnow rad2-17.The growth medium contained 2% peptone; 2%

dextrose, and 1% yeast extract. Stationary-phasecells were obtained from cultures which had beenincubated with shaking at 30 C for 3 to 5 days. Ex-ponential cells were harvested after growing over-night from a small inoculum to 5 x 106 to 10 x 106cells/ml. The cells were centrifuged and washedtwice with cold M/15 phosphate buffer, pH 7, and

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suspended in the cold buffer at 3 x 107 cells/ml.H. influenzae growth and competence. Growth

of the Rd strain (sensitive to streptomycin) has beendescribed previously (17). Cells were made compe-tent by the method of Steinhart and Herriott (21).UV irradiation of cells. A 25-ml amount of yeast

cells was irradiated in a petri dish (15-cm diameter)surrounded by ice. The exposure rate was 300 ergsper mm2 per min (254 nm).

Photoreactivation of cells. Five General Electric15-W "Blacklight" bulbs, filtered so as to pass onlylight of wavelength greater than 325 nm, were usedto photoreactivate the cells. The exposure rate wasabout 7,000 ergs per mm2 per min. Stirred samples(20 ml) of yeast cells in a petri dish (9-cm diameter)were exposed to the light for 40 min in an incubatorat 30 C. Maximum photoreactivation of freshly irra-diated cells was observed in this period. If cells hadpreviously been incubated in growth medium, theyfirst were harvested, washed twice in warm buffer,and resuspended in the buffer at 3 x 107 cells/ml.

Survival. Afrer appropriate dilution, yeast cellswere plated on petri dishes containing 2% peptone,2% dextrose, 1% yeast extract, and 2% agar. Colonieswere counted after 2 to 3 days of incubation at 30 C.

Postirradiation incubation of cells. To irra-diated yeast cells a 4x concentration of cold growthmedium was added; the final concentration was thesame as that of the normal growth medium. Thesuspension was then heated rapidly (2 to 3 min) to30 C and incubated in the dark with shaking.Crude extracts. Yeast cells which had been incu-

bated in growth medium were harvested by centrifu-gation, washed twice in cold buffer, and suspendedat 2 x 101 to 5 x 108 cells/ml. Cells which were al-ready in buffer were concentrated to this titer. Crudeextracts were made by passing the cell suspensionstwo times through a French pressure cell at 15,000psi. The crude extracts were then centrifuged toremove debris, and the supernatant fluid was ad-justed to an optical density at 260 nm (OD260) of0.90.Transforming DNA. DNA was obtained from

streptomycin-resistant H. influenzae cells in themanner described previously (17). The DNA was di-luted in buffer to 17 gg/ml and exposed to 3,000ergs/mm2 at 254 nm (0.3% survival of the trans-forming activity) as has been reported (17). It wasthen diluted to 0.01 ug/ml.PR enzyme from yeast. The isolation and purifi-

cation of this enzyme was done by the method ofMuhammed (10). Before dilution in the followingcompetition assay, the protein concentration was 40gg/ml.

Competition assay. To 0.5 ml of extract at 0 Cwas added 0.5 ml of irradiated transforming DNAand 0.5 ml of PR enzyme. The solution was thenheated for 30 sec to 37 C, and four samples (0.3 ml)were distributed to four depressions (2-cm diameter)on a spot plate maintained at 37 C. Three sets ofcompetition solutions (representing three differentextracts) were placed on each spot plate (a total of12 spots); the time required to distribute the threemixes was about 3 min. One of the four spots (for a

given competition solution) was not exposed toblacklight, and the other three were exposed for var-ious periods (exposure rate was 5,000 ergs per mm2per min). After photoreactivation, each sample onthe spot plate was added to 0.5 ml of buffer. A 2-mlamount of competent cells was then added with sub-sequent shaking in a 37 C water bath for 30 min toallow uptake of the transforming DNA. The subse-quent procedure for determining transformation hasbeen described previously (21).

Sepharose 4B columns. The columns (10 by 0.6cm) used in this study have been described by Car-rier and Setlow (3). To each column was added 0.25ml of extract. The crude extract did not affect themovement of radioactively labeled Escherichia coliDNA or of mononucleotides.

RESULTSCompetition assay for pyrimidine dimers.

Experiments by Rupert (14) demonstrated thatthe photoreactivability of H. influenzae trans-forming DNA by PR enzyme from yeast is in-hibited by UV-irradiated transforming andheterologous DNA. Setlow et al. (15) showedquantitatively that such competition resultsfrom UV-induced cyclobutyl pyrimidine di-mers in DNA. Thus it is expected that thecompetition between irradiated transformingand heterologous DNA species for photoreacti-vating enzyme could form the basis of an assayfor determining the presence of dimers in theDNA of yeast cells. As shown in Fig. 1, a crudecell extract of irradiated yeast inhibits thephotoreactivability of UV-irradiated trans-forming DNA more than does an extract ofunirradiated yeast. This competition (definedbelow) can be removed by first exposing thewhole cells (Fig. 1) to blacklight or by exposinga corresponding crude extract in the presenceof PR enzyme to photoreactivating light (Fig.2); the competing factor, therefore, is photo-reactivable and is concluded to be pyrimidinedimers in the yeast DNA.

In addition to the competition attributableto dimers, there appears to be another factorpresent in the extracts which generally de-presses the ability to photoreactivate trans-forming DNA (Fig. 1). This factor is heat-stable at 70 C and is not affected by repeatedfreezing and thawing. To test whether this in-hibitor degrades PR enzyme or the trans-forming DNA, an extract was incubated withtransforming DNA and PR enzyme in thedark, and at various times samples were pho-toreactivated. Since there was no significantchange in the transforming capability of thedark-incubated mix or of the photoreactivabil-ity of the transforming DNA, it is concludedthat the inhibitor does not act by degrading the

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4 /I0

2800- ,U.

6001

400-

z

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80 160 240

PHOTOREACTIVATION (sec)FIG. 1. Photoreactivation of UV-irradiated trans-

forming DNA in the presence of extracts from sta-tionary-phase, unirradiated wild-type cells (A), irra-diated cells (a), and irradiated cells which had beenphotoreactivated (0), or in the absence of extracts(0). The UV dose to the cells was 1,500 ergs/mm2.The procedures used for the competition assay aredescribed in the text.

1.01

0.8'

0.6-

XOA-

0.2-

20 40 60 80 00

PHOTOREACTIVATION (sec)

FIG. 2. Competition by an extract of UV-irra-diated (1,500 ergs/mm 2) wild-type cells which hadbeen exposed to photoreactivating light in the pres-ence of photoreactivating (PR) enzyme prior to thecompetition measurements. The PR enzyme (corre-sponding to the concentration used in the competi-tion assay) was added to the extract, and sampleswere exposed to photoreactivating light. The UV-ir-radiated transforming DNA was then added andcompetition was measured.

DNA or the PR enzyme (Table 1). The relativeamounts of PR enzyme, crude cell extract, andtransforming DNA used in the assay werechosen because of the ease in determiningcompetition (Fig. 1), the short times necessary

TABLE 1. Photoreactivability of transforming DNAin the competition assay after incubation at 37 Ca

Time of incubation No. of transformants

before photoreac- Notivation (min) phot Photoreactivation

pooreactivation

0 210 10342 214 12314 212 12146 195

a After preincubation, samples of the competitionassay mixture, which contained extracts of unirra-diated wild-type cells, were exposed to photoreacti-vating light for 2 min. Samples taken at the indi-cated times but not exposed to light were used ascontrols (no photoreactivation).

for exposure of the assay mixture to photoreac-tivating light, the small amount of enzymerequired, and the relatively small degree ofinhibition by extracts of unirradiated cells(with one-fourth the amount of enzyme, thereis approximately four times as much inhibi-tion).To quantitate the relative competition by

extracts of irradiated as compared to unirra-diated yeast cells, we needed a function that isindependent of the initial transforming ability(before photoreactivation) of the competitionmix containing PR enzyme, cell extract, andUV-irradiated transforming DNA. Such afunction is based on the additional time re-quired to photoreactivate the assay mixture tothree times the initial transforming abilitywhen the crude cell extract is from irradiated(B) rather than unirradiated (A) cells. Thecompetition function we have used is definedas C = (B - A)/100. In Fig. 1, the competitionis 0.80, which corresponds to values of 134 and54 min for B and A, respectively.

As seen in Fig. 3, which summarizes thedata of several experiments, competition in-creases with increasing exposures of cells toUV and is independent of whether cells are instationary phase or exponential growth. Al-though the data scatter (within individual ex-periments they are consistent), there appearsto be an approximately linear relationshipbetween the incident dose to cells and compe-tition by the corresponding extract. However,a difference is apparent between the twostrains used in these experiments. Amongother possibilities, it might have resulted fromdifferences between ribonucleic acid (RNA)-to-DNA ratios of the strains, since the concentra-tion of extracts used is dependent on theamount of RNA present.

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To determine the relationship between theactual number of dimers present in an extractand the degree of competition, irradiated calfthymus DNA was added to a normal competi-tion mix containing crude extract of unirra-diated yeast cells, irradiated transformingDNA, and PR enzyme; competition was thenmeasured (Fig. 4). Since the calf thymus DNAhad been irradiated under the same conditionsas the H. influenzae DNA and since their basecompositions are similar [43% guanine + cyto-sine (20), and 38% guanine + cytosine (1), res-pectively], the induction of dimers in the twoDNA species should be approximately equal.The abscissa therefore corresponds to the rela-tive number of dimers in the calf thymus DNA

1.2-a 0 b

1.0-

o 0.80 ~~~00

06 0 100100050 1000 5020.2-0 00~~~~~~~~

0PR~~~~~

0R PR

500 1000 1-00 2000 500 10001500 2000UV DOSE (ergs/mm2)

FIG. 3. Competition by extracts of irradiatedcells. Summary of experiments with exponentiallygrowing (0) and stationary-phase (0) cells, wildtype (a) and UV-sensitive (rad2-17) (b). Competitionby extracts of irradiated cells which had been photo-reactivated is indicated by OPR.

14-

12-

1.0z0

08-w0.!06-

04-

02 -1

8 16 24 32 40 48 56 64 72RATIO OF CALF THYMUS DNA TO TRANSFORMING DNA

FIG. 4. Competition by UV-irradiated (3,000ergs/mm2) calf thymus DNA in a competition assaycontaining an extract of unirradiated wild-type yeastcells. The abscissa corresponds to the ratio of calfthymus DNA to UV-irradiated transforming DNA(3,000 ergs/mm 2, 0.001 gg/ml).

as compared to the transforming DNA. Thecompetition by an extract of yeast cells ex-posed to 1,500 ergs/mm2 is 0.59 in this experi-ment, which in Fig. 4 corresponds to the com-petition by calf thymus DNA containing ap-proximately 30 times the number of dimerspresent in the H. influenzae DNA used in thecompetition mix. We conclude that, since thecalf thymus and yeast DNA species also havesimilar base compositions [36% guanine + cy-tosine in S. cerevisiae (20)] there are, in thecompetition mixture used, approximately 30times as many dimers in the yeast DNA as inthe H. influenzae DNA (which reflects the ex-cess of yeast DNA).

Postirradiation changes in competition:exponentially growing cells. If UV-inducedpyrimidine dimers in yeast are subject to re-pair processes, it is expected that competitionshould decrease in extracts of cells taken atsucceeding times after irradiation. To test this,wild-type cells (S288c) were exposed to 1,500ergs/mm2, corresponding to approximately1.2% survival. A portion of the cells was incu-bated in growth medium on a shaker in thedark at 30 C. At hourly intervals, samples werewashed and resuspended in buffer. One-half ofthe washed sample was exposed to photoreac-tivating light and the remaining portion waskept in the dark. After photoreactivation, boththe photoreactivated and nonphotoreactivatedcells were used for the competition assay.Dark incubation of cells resulted in the loss

of competing ability by cell extracts (Fig. 5).Approximately 90% of the pyrimidine dimerswere altered so as not to compete with dimers

08 *

06-z

04o X D

0.2- X0.~~~~~~~~~~~

2 3 4INCUBATION (hr)

FIG. 5. Competition by extracts of exponentiallygrowing wild-type cells which had been either irra-diated (0) or irradiated and photoreactivated (0).After irradiation of the cells (1,500 ergs/mm 2),growth medium was added and the cells were incu-bated. Samples of the cell suspension were removedat intervals, centrifuged, and resuspended in buffer.Half of each sample was further incubated in thedark and half in the light for 40 min.

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in the transforming DNA (Fig. 3, 5). Theability to photoreactivate these dimers is lessthan in the stationary cells of Fig. 1, presum-ably because exponentially growing cells con-tain much less photoreactivating enzyme thando cells in stationary phase (2). The ability torender the dimer damage noncompeting is as-sociated with the ability to survive low dosesof UV. The UV-sensitive mutant PR10-ld(rad2-17) is 20 to 30 times more sensitive tokilling than the wild-type (S288c) strain (Fig.6), and, correspondingly, there is no change incompeting ability of extracts obtained fromPR10-ld after irradiation (Fig. 7), nor is there

toI

500 1000 1500Uv DOSE (ergs/mm2

FIG. 6. Survival after UV irradiation of wild-type(0) and UV-sensitive (rad2-17) (0) strains.

1.0-

08-

06-

0.4-0

0.2-

a change in ability to photoreactivate the py-rimidine dimers in vivo.Postirradiation changes in competition:

stationary-phase cells. Competition by cellextracts from UV-irradiated stationary-phasecells incubated in growth medium after expo-sure was determined in the manner discussedabove. An extract from freshly irradiated sta-tionary-phase cells exhibits the same competi-tion (Fig. 8) as a corresponding sample fromlog-phase cells (Fig. 5). Unlike the exponen-tially growing cells for which competition de-creases with time of incubation after irradia-tion, there is very little change in competitionby the stationary-phase cell extracts (Fig. 8).However, the ability to photoreactivate thepyrimidine dimers in vivo decreases with time,indicating that the regions of DNA containingthe dimers are altered in some way. A corre-sponding loss of photoreactivability is not ob-served in the UV-sensitive cells (Fig. 9), al-though there appears to be some loss.

Size of DNA that contains dimers. Theresults in the previous section indicated thatin stationary-phase cells the dimers might beexcised as part of oligonucleotides (16) sincethey were still capable of competition in vitro.Such oligonucleotides, which would have to begreater than 18 nucleotides (16), might not bephotoreactivable in vivo because of their loca-tion. To test this hypothesis, extracts of unir-radiated and irradiated cells, as well as cellswhich had been incubated after irradiation,were passed through Sepharose 4B columns todetermine whether comDetition could be de-

i.2 -

1.0-

08-z0

' 06-

0

04 -

* g18

02-

2 3 4INCUBATION (hr)

FIG. 7. Competition bY extracts of UV-sensitive(rad2-17) exponentially growing cells which had beeneither irradiated (0) or irradiated and photoreacti-vated (0). The same conditions were used as in Fig.5.

2INCUBATION (hr)

3 4

FIG. 8. Competition by extracts of stationary-phase wild-type cells which had been either irra-diated (0) or irradiated and photoreactivated (0).The same conditions were used as in Fig. 5.

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RESNICK AND SETLOW

tected in low-molecular-weight material. Asseen in Fig. 10, all of the competition is re-covered in the void volume for extracts of bothincubated and nonincubated irradiated cells,indicating that the dimers are in regionsgreater than 600 nucleotides in length.Photoreactivability of survival. The de-

crease in ability to remove competition by invivo photoreactivation suggested that photo-reactivability of survival in stationary cellsmight be similarly affected. Irradiated wild-type cells (1,500 ergs/mm2) were thereforephotoreactivated at hourly intervals up to 3 hrafter irradiation and plated immediately afterexposure. Dark-held cells were also plated forsurvival at the same times. Photoreactivationimmediately after UV irradiation increased thepercentage of cells surviving from 0.9 to 24%(Fig. 11). The ability to photoreactivate lethaldamage is progressively lost until, at 3 hr, sur-vival can be increased only to 2.4%.

DISCUSSIONThe UV induction of pyrimidine dimers in

the DNA of yeast cells had been indicatedpreviously by the photoreactivability of cellkilling and by the fact that photoreactivat-ionless mutants lacked photoreactivating en-zyme (12). In the present study, the existenceof UV-induced dimers in the DNA of yeastwas inferred by showing that extracts offreshly irradiated cells compete with UV-irra-diated transforming DNA for photoreactivat-ing enzyme and that this competing abilitywas abolished by previous in vivo (Fig. 1) or invitro (Fig. 2) photoreactivation.To calibrate the competition assay, UV-ir-

radiated calf thymus DNA was added to theassay mixture containing unirradiated yeastcrude extract and the competition was deter-

0.6-

zO 0.4-HH

O 0.2-0

0 0

_

0

-I I~-

1 2INCUBATION (hr)

FIG. 9. Competition by extracts of st(sensitive (rad2-17) cells which had beerdiated (0) or irradiated and photorea(The same conditions were used as in Fig

z0

w 0.2-0~

0.1

00

1 2 3 4FRACTION NO.

FIG. 10. Competition by extracts of stationary-phase wild-type cells passed through a Sepharose 4Bcolumn. Extracts of irradiated (a) or unirradiatedcells, as well as irradiated cells which had been incu-bated in growth medium for 220 min after irradia-tion (0), were passed through Sepharose 4B columnsand successive 0.5 ml fractions were collected afterthe first 0.9 ml was discarded. Fraction numbers cor-respond to the first, second, third, and fourth frac-tions collected. The competition is relative to that ofthe corresponding fraction obtained for the unirra-diated extract. In the competition assay, the 0.5-mlfraction replaced the 0.5-ml crude extract.

10-

-J

,U

l-

2INCUBATION (hr)

3 4

FIG. 11. Loss of photoreactivability of stationarywild-type cells with time of incubation after irradia-tion (1,500 ergs/mm2). The photoreactivated (0) andnonphotoreactivated (0) cells were used for the ex-periment of Fig. 8.

mined (Fig. 4). Based on this calibration, the, , irradiated H. influenzae transforming DNA3 4 (0.001 ,ug, 3,000 ergs/mm2) in the competition

assay contains approximately 30-fold fewer

ationary UV pyrimidine dimers than the extract from yeastn either irra- cells (approximately 4 x 106 cells) exposed toctivated (0). 1,500 ergs/mm2. This result is in reasonable.5. agreement with an independent estimate in-

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dicating the presence of approximately 15- to20-fold fewer dimers in the H. influenzae DNA.The latter estimation was made by using theresult of Setlow and Carrier (19) that 2,000ergs/mm2 at 265 nm produced 0.27 x 10- 2dimer per nucleotide in H. influenzae DNA.By correcting to 1,500 ergs/mm2 at 254 nm andassuming that the base compositions of H. in-fluenzae and S. cerevisiae are the same, andassuming further that the incident dose tothe yeast cells is twice that to the yeast DNA,the estimate of 15 to 20 times fewer dimers isobtained.

Since the UV dose to the transforming DNAwas 3,000 ergs/mm2, there are approximately 6x 109 dimers per competition assay sample inthe transforming DNA. Based on the abovecalculations, there are approximately 3 x 104dimers induced per yeast cell by an exposureof 1,500 ergs/mm2, which corresponds to 1 to3% survival. For the two strains used in thepresent study, there is a large difference insurvival after UV treatment (Fig. 6). The av-erage dose required to kill a wild-type cell inthe dose range where exponential inactivationof the cell population occurs is 290 ergs/mm2or about 6,000 dimers, whereas for the UV-sensitive strain it is 10 ergs/mm2 or about 200dimers per cell (Fig. 6). A double mutant ob-tained from uvs9-2 and uxsl parents [14; corre-sponds to rad2-16 and radl8-2 (4), respec-tively] has been shown by Khan et al. (9) to beeven 20 to 30 times more sensitive than thecorresponding uvs9-2 strain. Cells of thedouble mutant strain therefore could be ex-pected to tolerate only approximately 10 di-mers.By analogy with results in bacteria, it has

been suggested by several authors that thelarge differences in the capacity of mutantsand wild-type cells to tolerate UV damage areattributable to differences in ability to repairpyrimidine dimers in DNA. That this is thecase for exponentially growing cells is seen inFig. 5 and 7. Wild-type cells alter pyrimidinedimers so as to render them noncompeting inthe in vitro competition assay, whereas in theUV-sensitive cells the dimers remain un-changed, as measured by ability to be photo-reactivated in vivo or in vitro. It is concludedthat a mutation in the rad2 gene (11) causes adeficiency in an early step of a repair mecha-nism that specifically acts on pyrimidinedimer regions. We suggest that the rad2 strainmay lack an endonuclease which is similar tothat characterized in Micrococcus luteus (3, 8).

Although competition by extracts of sta-tionary-phase cells incubated in growth me-

dium for 220 min after irradiation does notchange, there is a significant loss of ability toremove the competition by photoreactivationof the cells (Fig. 8). This loss of photoreac-tivability of competition is considered to beattributable at least to the steps in repairwhich are lacking in the rad2 strain, sincethere is only a small loss in the UV-sensitivestrain. Similarly, for the wild-type cells thereis a concomitant loss in ability to photoreacti-vate survival.The difference in behavior of repair mecha-

nisms of wild-type stationary and exponentialcells may be attributable to secondary effectsof repair. We tested the possibility that in sta-tionary-phase cells the dimers are excised aspart of polynucleotides that are longer thanthe minimum size of 18 bases required forbinding photoreactivating enzyme. Since re-sults with Sepharose 4B columns indicate thatdimers appear in polynucleotides that are atleast 600 bases long (Fig. 10), the dimers pre-sumably remain in the chromosomal DNA. Itis therefore concluded that, in wild-type sta-tionary and exponential cells, there is repair ofpyrimidine dimer regions, presumably re-quiring an endonuclease in the early step. Instationary-phase cells, however, the subse-quent repair processes (or even the action ofother enzymes) render the dimer regions lesssusceptible to in vivo photoreactivation whileleaving them capable of in vitro competitionfor photoreactivating enzyme. The differencebetween the two types of cells may result fromdifferences in amounts of repair enzymes, ashas already been found for the photoreacti-vating enzyme (2).

ACKNOWLEDGMENTS

We thank K. Beattie for competence in early experi-ments.

M. Resnick was a University of Tennessee PostdoctoralTrainee under contract no. 3322 with the Biology Division,Oak Ridge National Laboratory, during the course of thesestudies. 0

The Oak Ridge National Laboratory is operated by UnionCarbide Corporation for the U.S. Atomic Energy Commis-sion.

LITERATURE CITED1. Belozersky, A. N., and A. S. Spirin. 1960. Chemistry of

the nucleic acids of microorganisms, p. 147-185. In E.Chargaff and J. N. Davidson (ed.), The nucleic acids,vol. 3. Academic Press Inc., New York.

2. Boling, M. E., and J. K. Setlow. 1967. Photoreactivatingenzyme in logarithmic-phase and stationary-phaseyeast cells. Biochim. Biophys. Acta 145:502-505.

3. Carrier, W. L., and R. B. Setlow. 1970. Endonucleasefrom Micrococcus luteus which has activity towardultraviolet-irradiated deoxyribonucleic acid: purifica-tion and properties. J. Bacteriol. 102:178-186.

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4. Game, J. C., and B. S. Cox. 1971. Allelism tests of mu-tants affecting sensitivity to radiation in yeast, andproposed nomenclature. Mutat. Res. 12:328-333.

5. Grivell, A. R., and J. F. Jackson. 1968. Thymidine ki-nase: evidence for its absence from Neurospora crassa

and some other micro-organisms, and the relevance ofthis to the specific labelling of deoxyribonucleic acid.J. Gen. Microbiol. 54:307-317.

6. Howard-Flanders, P. 1968. DNA repair. Annu. Rev.Biochem. 37:175-200.

7. Jannsen, S., E. Lochman, and R. Megnet. 1970. Specificincorporation of exogenous thymidine monophosphateinto DNA in Saccharomyces cerevisiae. Fed. Eur.Biochem. Soc. Lett. 8:113-115.

8. Kaplan, J. C., S. R. Kushner, and L. Grossman. 1969.Enzymatic repair of DNA. I. Purification of two en-

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