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Chemistry Faculty Publications Chemistry

1971

Ultraviolet-stimulated thymidine incorporation inxeroderma pigmentosum lymphocytes / Peter G.Burk Marvin A. Lutzner Donald D. Clarke and JayH. Robbins Bethesda, Md and Bronx, N.Y. From theDermatology Branch, National Cancer Institute,National Institutes of Health and the ChemistryDepartment, Fordham UniversityPeter G. BurkNational Cancer Institute (U.S.). Dermatology Branch

Marvin A. LutznerNational Cancer Institute (U.S.). Dermatology Branch

Donald Dudley Clarke PhDFordham University, clarke@fordham.edu

Jay H. RobbinsNational Cancer Institute (U.S.). Dermatology BranchFollow this and additional works at: https://fordham.bepress.com/chem_facultypubs

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Recommended CitationBurk, Peter G.; Lutzner, Marvin A.; Clarke, Donald Dudley PhD; and Robbins, Jay H., "Ultraviolet-stimulated thymidineincorporation in xeroderma pigmentosum lymphocytes / Peter G. Burk Marvin A. Lutzner Donald D. Clarke and Jay H. RobbinsBethesda, Md and Bronx, N.Y. From the Dermatology Branch, National Cancer Institute, National Institutes of Health and theChemistry Department, Fordham University" (1971). Chemistry Faculty Publications. 72.https://fordham.bepress.com/chem_facultypubs/72

Ultraviolet-stimulated thymidine

incorporation in xeroderma pigmentosum lymphocytes

PETER G. BURK, MARVIN A. LUTZNER, DONALD D. CLARKE, and JAY H. ROBBINS Bethesda, Md., and Bronx, N.Y.

Autoradiographic and l iquid scintillation counting techniques were utilized to measure the ultraviolet (UV)-stimulated incorporation of radioactive thymidine during repair replication of the UV-damaged deoxyribonucleic acid (DNA) of irradiated peripheral blood lymphocytes from 4 patients w ith xeroderma pigmentosum (XDP) and from normal donors. lymphocytes from 3 of the 4 patients had a markedly decreased rate of incorporation soon after irradiation in comparison with the rate obtained with normal donors' lymphocytes. However, the duration of incorporation into these 3 patients' lymphocytes was prolonged, so that XDP lymphocytes appear eventually to incorporate as much total thymidine as normal lymphocytes. Lymphocytes from the fourth patient had a rate and duration of thymidine incorporation after irradiation simi lar to normal lymphocytes. This patient, therefore, may have a d ifferent genetic and biochemical defect from the other XDP patients.

X eroderma pigmentosum (XDP) is a rare autosomal recessive disease in which the patients develop actinic damage, abnormalities in pigmentation, and malignancies in areas of skin exposed to sunlight. 2- 5 These cutaneous lesions sometin1es occur in association ·with abnormalities of other organ systems, including the central nervous system.3 ' ~ Cleaver6 was the first to report that ultraviolet (UV) -stimulated thymidine uptake was absent or reduced in cul­tured fibroblasts from patients with XDP in contrast to the uptake in cultured fibroblasts from normal individuals. Similar results were obtained subsequently by Epstein and associates7 from autoradiographic studies of epidermal cells

From the Dermatology Branch, National Cancer Institute, National Institutes of Health, and the Chemistry Department, Fordham University.

A preliminary report of these studies was published in abstract form1 and was presented at a joint meeting sponsored by The American Federation for Clinical Research and The American Society for Clinical Investigation, May 1970.

ReceiYed for publication Sept. 30, 1970. Accepted for publication Feb. 1, 1971. RePrint requests: Dr. Peter G. Burk. Dermatology Bca.nch, Bldg. 10, Rm. 12N238, National

Institutes of Health, Bethesda. Md. 20014.

759

760 Burk et al. ] . L:tb. Clin. Med. May, 1971

from patients 'vith XDP after UV irradiation in vivo. Uptake of labeled thymidine into nuclear deoxyribonucleic acid (DNA) following UV irradiation has been demonstrated in several mammalian cell types,s-u including normal human lymphocytes.12

-14 As this uptake occurs during phases of the cell cycle

other than the normal DNA synthetic phase, it is referred to as "unscheduled" uptake. This unscheduled uptake may represent a step in the repair of the UV­induced thymine dimer damage produced in DNA, and such repair n1ay be analogous to dark repair of UV damage in bacteria.15- 21 Cleaver6• 22' 23 has postulated that patients with XDP may develop their cutaneous malignancies after exposure to the UV radiation in sunlight because of a defective repair mechanism.

We have reported brie:fiy1 3 results of autoradiographic studies in 'vhich peripheral blood lymphocytes from a patient with XDP were found to haYe a slower rate of UV-stimulated tritiated thymidine uptake shortly after l:""V ir­radiation than cells from normal individuals. More recently, we performed similar autoradiographic studies on lymphocytes from 3 more patients and, in addition, have used a liquid scintillation assay24 modified after the procedure of Evans and Nonnan.12 This assay has enabled us to demonstrate1 that XDP cells which have a decreased rate of UV-stimulated thymidine uptake soon after irradiation continue incorporating thymidine for a longer tin1e than norn1al cells. In this paper we present these findings, as well as studies of a patient 'vith XDP ·whose lymphocytes do not have any detectable impairment in the rate of UV­stimulated thymidine incorporation.

Materials and methods

Patients. In early childhood, each of the 4 XDP patients was noted to have freckles in areas of the skin exposed to sunlight. Subsequently, these areas were found to contain telangiectasia, hypopigmentation, and atrophy. Each patient has had many cutaneous malignancies, including at least one malignant melanoma. They are all healthy and of normal intelligence otherwise. The patients are unrelated, and there is no known con­sangujnity in their families.

Patient 1 is a 25-year-old woman; her 2 siblings do not ha:ve XDP. Patient 2 is a 20-year-old man who has congenital i chthyosis in addition to XDP; 4 of his 6 siblings have clinical evidence of XDP. A clinical and genetic study of this family, with this patient as proband, has been reported by Lynch and co-workers.25 Patients 3 and 4 are 21- and 26-year-old men, respectively; neither has siblings.

Auto'radiography. Leukocytes for autoradiographic studies were obtained from heparinized periphe1·al blood. In each experiment with XDP cells, a normal donor's cells were treated simultaneously in a similar manner. Leukocytes were adjusted to a concentration of 1.2 x 10s cells per milliliter of a culture fluid consisting of 1 part of autologous plasma and 4 parts of Difco medium 199 (Difco Laboratories, Detroit, 1.fich.) containing bica1·bonate, phenol red, penicillin, and streptomycin. The leukocyte suspensions were placed in open petri dishes to a depth of 1 mm. and were irradiated at room temperature for 20 seconds at a distance of 75 em. from a 15 watt 2,537 .A germicidal lamp (General Electric lamp, No. G15T8). The incident flux was approximately 8 ergs per square millimeter per second at the surface of the culture fluid as measured by a Blak-Ray J225 UV intensity meter (Ultra­Violet Products, Inc., San Gabriel, Calif.). Thymidine-methyl-3H (BHTdR; .concentration, 1 mCi. per milliliter; specific activity, 17 to 22 Ci per millimole) was then added to a final concentration of 10 p.Ci per milliliter of culture fluid. Following incubation at 38° C. for 3 or 6 hours, the cells were washed 3 times with a mixture of plasma and medium 199,

Volume 77 Number 5 Thyrnidine incorporation in lymphocytes 7 61

smeared on coverslips, and coated with gelatin and NTB 3 nuclear emulsion (Eastman I{odak, Rochester, N . Y.). The emulsion was exposed at 4° C. for 4 to 8 weeks and then developed. The cells were then stained with Giemsa stain.

Liq1Jid scintillation spectrometry. Liquid scintillation studies were conducted on suspen­sions of leukocytes of which at least 98 per cent were lymphocytes. These lymphocytes, obtained by passing peripheral blood leukocyte suspensions through nylon fiber columns (Fenwal Laboratories, Morton Grove, Ill.) 126 were adjusted to a concentration of 106 cells per milliliter of a culture :fluicl composed of 1 part each of XDP and normal plasma and 8 parts of the medium 199. The lymphocytes were irradiated in the manner described pre­viously for the autoradiographic studies. Immediately after irradiation, hydroxyurea was added to a :final concentration of 10-a:M to retardll, 12, 21, 28 the semiconservativo (i.e., scheduled) S-phase) DNA synthesis associated with normal preparation for mitosis occurring in a sma11 percentage of cells.12, 14, 29 3HTdR (10 p.Ci) was added to each 1 ml. aliquot of culture fluid before incubation at 38° C. for 1 or 3 hours. When incorporation was to be measured during successive 3 hour periods after irradiation, the sHTdR was added 3 hours prior to harvesting of the cells. The cells were harvested by filtering the culture fluid through a ).fillipore filter which, with the entrapped cells, was washed with saline, 5 per cent trichloroacetic acid, and ethanol. The radioactivity retained on the filters was determined as describ(\d previously.24 At least 95 per cent o.r this incorporated radioactivity trapped from UV-irradiated cells could be removed from the filters by treatment with deoxyribonuclease I (Worthington Biochemical Corp., Freehold, N . J .).

Results from this liquid scintillation assay procedure are expressed as counts per minute per 106 lymphocytes and 1·cpresent either the "obligatory UV-stimulated incorporation" or the "maximum possible UV-stimulated incorporation." The former is the amount by which the mean of the gross counts per minute obtained from duplicate incubated i1-radiated cultures exceeds the m(\an of the gross counts per minute obtained from duplicate incubated 'Unir­'radiatecl eultures. The "maximum possible UV-stimulated incorporation" is the amount by which the mean of the gross counts per minute obtained from the duplicate incubated ir­radiated cultures exceeds the gross counts per minute in an unincubuted unirradiated sHTdR­containing culture. This difference between the obligatory and the maximum possible in­corporation is called the "possible additional UV-stimulatcd incorporation" and can be equal to, but not less than, the semiconscrvative synthesis not stopped by the hydroxyurea. If an unknown amount of this semiconservative synthesis were stopped by the UV radiation, then the actual UV-stimuluted incorporation would exceed the obligatory incorporation by that unknown amount, but in no case could the UV-stimulated incorporation be greater than the maximum possible UV-stimulated incorporation.

Results

Autorctdiog'raphic data. Results from 3 autoradiographic experiments are presented in Table I. As sho\vn in column A, there was no significant difference in the average nun1ber o.f grains per cell for each person's unirradiated cells. As shovrn in column B, irradiated lymphocytes from the normal donors and fron1 patient 4 had a marked increase in grains, while those from patients 1, 2, and 3 had definite, but considerably smaller, increases. The UV-stimulated incorporation of column C '\Yas calculated by subtracting the values obtained for the unirradiated lymphocytes (column A) .from the values obtained for the irracliateclly1nphocytes (column B) . It is apparent from experiment C that there was no significant difference between the UV-stimulated 3HTdR incorporation into lymphocytes from patient 4 and the incorporation into ly1nphocytes fro1n the no1mal donor (P == 0.18) . Incorporation into lymphocytes from patients 1, 2, and 3 differed significantly (P < 0.001) from the incorporation into lymphoc-ytes from the normal donors.

762 Burk et al. J. Lab. Clin. Med. May, 1971

Table I. Autoradiographic data on UV -stimulated, 3HTdR incorporation into normal and XDP lymphocytes

Lymphocyte donor

Experiment A

Normal Patient 1

Experiment B

Normal Patient 3

Experiment C

Normal Patient 2 Patient 4

Average grain count p~r lymphocyte*

U nir1·adiated (A)

1.15 ± 0.12 1.02 ± 0.11

1.28 ± 0.12 1.06 ± 0.12

1.65 ± 0.14 1.37 ± 0.13 1.42 ± 0.15

Irradiated (B)

6.46 ± 0.31 1.64 ± 0.14

7.{)1 ± 0.38 1.71 ± 0.15

7.75 ± 0.39 2.07 ± 0.16 8.05 ± 0.38

U V -stimulated incorporation

(C)

5.31 ± 0.33 0.62 ± 0.18

5.73 ± 0.40 0.65 ± 0.20

6.10 ± 0.41 0.70 ± 0.21 6.63 ± 0.41

P val·uet

< 0.001

< 0.001

< O.UOl 0.18

•Each value represents the average grain count ± S.E. per lymphocyte and was determined from 100 cells evaluated in each case. Adjacent noncellular background < 0.7 grain per cell area.

tPatient's UV -stimulated incorporation compared to that of the normal of the same ex­periment.

Scintillation spectrontetric studies. Results of scintillation spectrometric studies agreed 'vith the results obtained by autoradiography. Fig. 1 shows the incorporation of 3HTdR into 106 lymphocytes from normal donors and from each of the 4 patients during the first hour of incubation following irradiation. Patients 1, 2, and 3 had reduced incorporation, and patient 4 had normal amounts.

Incorporation of 3HTdR into the DNA of lymphocytes from normal donors and fro1n each of the 4 patients was studied during 4 successive 3 hour incubation periods following irradiation (Fig. 2, A to C). Incorporation into normal lymphocytes decreased rapidly during these successiye time periods so that the obligatory incorporation during the 6 to 9 and 9 to 12 hour periods amounted to less than 5 per cent of the obligatory incorporation which occurred during the 6 hours immediately following irradiation. Similar results have been reported previously for normallymphocytes.12 However, in contrast to this rapid decrease of incorporation into the normal lymphocytes, incorporation into lymphocytes from patients 1 and 2 (Fig. 2, A) and patient .3 (Fig. 2, B) decreased 1nuch more slowly, so that the obligatory incorporation into their lymphocytes 'vas actually greater than either the obligatory or maximum possible incorporation into the normal lymphocytes during the 6 to 9 and 9 to 12 hour periods. However, the rate of decrease in the incorporation of 3HTdR into irradiated lymphocytes from patient 4 (Fig. 2, C) was essentially the same as that oc­curring into lymphocytes from nonnal donors.

In Fig. 2, A and B it can be seen that lymphocytes from patients 1, 2, and 3 'vere still incorporating significant amounts of thymidine in the 9 to 12 hour period after irradiation when the controls had virtually stopped incorporating.

Volume 77 Number 5

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Thyntidine incorpot·ation in lymphocytes 7 6 3

Obligatory incorp.

I Pt. I I Pt. 3 I Normals Pt. 2 Pt. 4

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Fi g. 1. UV-stim.ulated, aHTdR incorporation into 10s lymphocytes from normal donors and from XDP patients during the first hour of incubation after irradiation. The counts per minute (c. p.m.) per lOa cells represent the mean of the values obtained in the replicate expe1·iments performed. The S.E . of each of these means was less than 56 c.p.m.

From the rate of decline or these patients' incorporation during the first 12 hours, it was projected that their incorporation would continue well beyond the 12 hour period. Studies to confirm this extrapolation were perfonned on lynlpho­cytes from patient 1 (Fig. 3). This patient's irradiated lymphocytes continued to incorporate significant amounts of 8HTdR during each of the 10 successive 3 hour time periods, so that by 30 hours after irradiation they had incorporated approximately 100 per cent of the amount incorporated by lymphocytes fron1 the normal donor whose lymphocytes were tested simultaneously.

Discussion

Unscheduled 3HTdR incorporation into UV-irradiated mammalian cells n1ay reflect one step in the repair o£ DNA damaged by UV-induced intrastrand thymine dimers. s-u This repair process, which resembles dark repair of U\T damage in bacteria, 15- 21 is postulated generally to involve: ( 1) an endonucleolytic incision to one side of the dimer; ( 2) dimer excision by an exonuclease which makes a "cut" some,vhere on the other side of the dimer; (3) insertion of ap-

7 64 Burk et al.

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Possible odd1tional

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8

Obligatory incorp. ~ ~

Poss1ble oddillonol

incorp

D -E 0.. <..>

- 900 ·

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6-9 9 12 0-3 3-6 6-9 9-12 0-3 TIME PERIOD AFTER IRRADIATION {hrs)

]. Lab. Clin. Med.

c

I[] I Pt4

Normal

May, 1971

Poss1ble odd1l1ono l mcorp

D

[QDJ 3-6 6-9 9-12

Fig. !!. UV-stimulated, aHTdR incorporation into lOG lymphocytes from normal donors and from patients 1 to 4 duTing 4 successive 3 hour incubations after irradiation. The counts per minute per 106 cells for the obligatory incorporation represent the mean difference ± S.E. of durlicate cultmes.

propriate nucleotides, i.e., repair replication by a DNA polymerase (resulting in unscheduled incorporation of 3liTdR when the latter is exogenously sup­plied ) ; and ( 4) linking, by polynucleotide ligate, of the free end of the inserted nucleotide chain to the remaining part of the DNA strand. Cleaver22 was the first to report that patients with XDP might lacl~ an endonuclease or have a functionally deficient enzyme so that their cells could not perform the initial incision adequately and, therefore, the unscheduled synthesis \vhich can normally occur only thereafter. :M~ore recent studies have supported this hypothesis by dc1nonstrating that XDP fibroblasts performed unscheduled synthesis if single­strand breaks w·ere made in the DNA by X-irradiation22

• 30 and that loss or UV­

induccd thymine din1ers from the DNA of XDP patients' cells \vas either reduced n1arkedly31 or not detectable.2 3

• 31 The defect in repair of UV-induced damage,

therefore, n1ay be silnilar to that found in a bacterial mutant \Vhich cannot perforrn excisional repair or UV-induced thymine dimers because it lacks the required endonuclease activity. 32 This endonuclease hypothesis, ho,vever, ·while the 1nost likely, is not the only tenable hypothesis; for example, decreased rates of unscheduled thymidine incorporation and of djmer excision might also result i.f the XDP cells were able to close endonuclcolytic incisions before unscheduled sYnthesis and dimer excision occurred.

'" Our studies sho\v that peripheral blood lymphocytes from 3 of our 4 XDP

Volume 77 Number 5

z 0

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900

~ 300

0-3 3-6 6-9

Thyntidine incorporation in lymphocytes 7 6 5

Obi igotory i ncorp -I Pt.l

Norma I

9-12 12-15 15-18 18-21 21-24 24-27 27-30

Tl ME PER 100 AFTER IRRADIATION ( hrs)

Fig. 3. UV-sUmulated, obligatory incorporation of aii'l'dR into l-OG lymphocytes from a normal donor and from patient 1 during 10 successive 3 hour incubations a.fter irradiation. The counts per minute per 106 cells represent the mean difference± S.E. of duplicate cultures. The absence of incorporation during the incubation period indicated by the astedsk is presumed to be the result of an experimental error.

patients have a Inarkedly decreased rate of unscheduled 3HTdR incorporation shortly after UV irradiation in vitro. Ilowever, incorporation into these cells continued for a longer time than it did into normal cells, so that over a uf­ficiently extended period XDP lymphocytes appeared to incorporate at least as much thymidine as normal cells. It should be noted, ho·wever, that demonstration of unscheduled incorporation of 3HTdR, ·which reflects an essential step in cxcisional dimer repair, docs not necessarily indicate that a functionally adequate repair has occurred.

It is also not known ·whether the decreased rate of UV-stimulated incorpora­tion found shortly after irradiation in lymphocytes from 3 o.f our 4 patient"' an<.l in skin cells from all the previously reported patients is due to a primary defect in a repair enzyme or to an effect on the repair mechanism transmitted indirectly through a chain of steps !rom a primary UV-sensitive XDP-specific defect far removed from the repair events. This question might be resolved by direct measurement of the activity of the endonuclease after its isolation fron1 unir­radiated XDP cells or by testing the ability of unirradiated XDP cells to repair a UV-irradiated "substrate." One such substrate \vhich has been tried by Rabson and colleagues33 is UV-irradiatcd herpes simplex virus. These authors reported that UV-irradiated virus "gre·w" less efficiently in cultured XDP "kin fibroblasts than in cultured non-XDP ce]Js. If it can be shown that these dif­ferences actually result :from different rates of repair of the irradiated Yirus, the results \vill support strongly the l1ypothesis that the decreased rate of G\T­stimulated thymidine incorporation into XDP cells represents a highly specific defect in the DNA repair process.

766 Burk et al. J. Lab. Clin. Med. May, 1971

Patient 4 was unique in that his lymphocytes appeared to have a normal rate of UV-stimulated thymidine incorporation shortly after irradiation. 1-Io\v­eYer, Cleaver23 has sho,vn that the degree of impairment of UV-stimulated thyn1iclinc incorporation into XDP skin fibroblasts varied from patient to patient. Confirmatory evidence of such a spectrmn of activity among XDP pa­tients has been reported recently by Bootsma and associates34 who showed by autoradiography that the 3 hour incorporation into XDP skin fibroblasts after u·v irradiation in vitro ranged from 10 to 79 per cent of the incorporation into nor1nal donors' cells. It is possible, therefore, that our patient 4 represents a case of XDP at the extreme end of such a spectrum of activity and that his cells 1nay haYe a small defect in the rate of incorporation which we have been unable to detect. Further speculation on this patient must a·wait results of experiments on his epider1nal cells to determine whether the observed response is unique to l~'"n1phocytes.

\Ye are indebted to Professor Leonard Ornstein for helpful discussion and a critical reading of the manuscript, to Miss Rosemary Cuddy for editing the manuscript, and to Dr. John Gart for performing the statistical analyses on the autoradiographic data.

REFERENCES

1. Burk, P. G., Lutzner, M. A., and Robbins, J. H.: Decreased rate of thymidine incorporation into DNA of some xeroderma pigmentosum patients' lymphocytes after ultraviolet ir­radiation in vitro, Clin. Res. 18: 346, 1970. (Abst.)

2. :Montgomery, H.: Dermatopathology 1, New York, 1967, Harper & Row, Publishers, pp. 123-127.

3. Reed, \V. B., May, S. B., and Nickel, \V. B.: Xeroderma pigmentosum with neurological complications, Arch. Derm. 91: 224, 1965.

4. Reed, W. B ., Landing, B., Sugarman, G., Cleaver, J. E., and Melnyk, J.: Xeroderma pigmentosum: Clinical and laboratory investigation of its basic defect, J. A. M. A . 207: 2073, 1969.

5. El-Hefnawi, H ., Smith, S. ~f . , and Penrose, L. S. : Xeroderma pigmcntosum-its h1herltance and 1·elationship to the ABO blood-group system, Ann. Hum. Genet. 28: 273, 1965.

6. Cleaver, J. E . : Defective repair replication of DNA in xeroderma pigmentosum, Nature 218 : 652, 1968.

7. Epstein, J. H ., Fukuyama, K ., Reed, W. B., and Epstein, vV. L.: Defect in DNA synthesis in skin of patients with xeroderma pigmeniosum demonstrated in vivo, Science 168: 1477, 1970.

8. Rasmussen, R. E ., and Painter, R. B.: Radiation-stimulated DNA synthesis in cultured mammalian cells, J. Cell Blol. 29 : 11, 1966.

9. Djordjevic, B., and Tolmach, L . J. : Responses of synchronous populations of Hela cells to ultraviolet inadiation at selected stages of the generation cycle, Rad. Res. 32: 327, 1967.

10. Painter, R. B., and Cleaver, J. E . : Repair replication, unscheduled DNA synthesis, and the repah of mammalian DNA, Rad. Res. 37: 451, 1969.

11. Cleaver, J. E.: Repair replication of mammalian cell DNA: Effects of compounds that inhibit DNA synthesis or dark repair, Rad. Res. 37: 334, 1969.

12. E"Vans, R. G., and Norman, A . : Unscheduled incorporation of thymidine in ultraviolet­in·adiated human lymphocytes, Rad. Res. 36: 287, 1968.

13. Burk, P. G., Levis, W. R., Lutzner, M. A., and Robbins, J. H.: Thymidine incorporation into DNA of lymphocytes and monocytes after ultraviolet irradiation in vitro, Fed. Proc. 28: 295, 1969. ( Abst.)

14. Frey-Wettstein, M., Longmire, R., and Craddock, C. G.: Deoxyribonucleic acid (DNA)

Volume 77 ~umber 5

Thymidine incorporation in lymphocytes 7 6 7

repair replication of ultraviolet (UV) irradiated normal and leukemic leulwcytes, J. LAB. CLIN. MED. 74: 109, 1969.

15. Setlow, R. B., and Carrier, W. L. : The disappearance of thymine dimers from DNA: An en-or-correcting mechanism, Proc. Nat. Acad. Sci. 51: 226, 1964.

16. Boyce, R. P., and Howard-Flanders, P.: Release of ultraviolet light-induced thymine dimers from DNA in E. Coli K-12, Proc. Nat. Acad. Sci. 51: 293, 1964.

17. Pettijohn, D., and llanawalt, P. C.: Evidence for repair-replication of ultraviolet uamaged DNA in bacteria, J. Molec. Bioi. 9: 395, 1964.

18. Setlow, R. B.: The photochemistry, photobiology, and repair of polynucleotiues, Prog. Nucl. Acid Res. 8: 257, 1968.

19. Strauss, B. S.: DNA repair mechanisms and their relation to mutation and recombina­tion, Curr. Top. Microbiol. Immun. 44 : 1, 1968.

20. Kelly, R. B., Atkinson, M. R., Huberman, J. A., and Kornberg, A.: Excision of thymine dimers and other mismatched sequences by DNA polymerase of Esche'richia coli, Nature 224: 495, 1969.

21. Smith, K. C., and Hanawalt, P. C.: Molecular photobiology: Inactivation and recovery, New York, 1969, Academic Press, Inc., pp. 132-164.

22. Cleaver, J . E .: Xeroderma pigmentosum: A human disease in which an initial stnge of DNA repair is defective, Proc. Nat. Acad. Sci. 63: 428, 1969.

23. Cleaver, J. E.: DNA damage and repair in light-sensitive human skin disease, J. Tnvest. Derm. 54: 181, 1970.

24. Robbins, J. H., Burk, P. G., and Levis, W. R. : Rapid and sensitive assay for DNA synthesis in leucocyte cultures, Lancet 2: 98, 1970.

25. Lynch, H. T., Anderson, D. E., Smith, J. L ., Jr., !Iowell, J. L., and Krush, A. J.: Xeroderma pigmentosum, malignant melanoma, and congenital ichthyosis: A famHy stutly, Arch. Dermatol. 96: 625, 1967.

26. Levis, W. R., and Robbins, J. H.: Antigen-induced blastogenesis: The human cell deter­mining the specificity of response in vitro, J. Immun. 104: 1295, 1970.

27. Sinclair, W. K.: Hydroxyurea: Differential lethal effects on cultured mammalian cells during the cell cycle, Science 150: 1729, 1965.

28. Pfeiffer, S. E., and Tolmach, L. J.: Inhibition of DNA synthesis in HeLa. coils by hydroxyurea, Cancer Res. 27: 124, 1967.

29. Bond, V. P., Cronkite, E. P., Fliedner, E. M., and Schork, P.: Deoxyribonucleic nciu­synthesizing cells in peripheral blood of normal human beings, Science 128: 202, 1958.

30. Kleijer, W. J., Lohman, P. H. M., Mulder, M. P ., and Bootsma, D.: Repair of x-ray damage in DNA o£ cultivated cells from patients having xeroderma pigmentosum, ~utat. Res. 9: 517, 1970.

31. Setlow, R. B., Regan, J . D., German, J., and Carrier, W. L.: Evidence that xeroderma pigmentosum cells do not perform the fhst step in the repair of ultraviolet damage to their DNA, Proc. Nat. Acad. Sci. 64: 1035, 1969.

32. Grossman, L., Kaplan, J. C., Kushner, S. R., and Mahler, I.: Enzymes involved in the early stages o:E repair o£ ultraviolet-irradiated DNA, Cold Spring Harbor Symposia on Quantitative Biology 33: 229, 1968.

33. Rabson, A. S., Tyrrell, S. A., and Legallais, F. Y.: Growth of ultraviolet-damaged herpes virus in xeroderma pigmentosum cells, Proc. Soc. Exp. Biol. Med. 132: 802, 1969.

34. Bootsma, D., Mulder, M. P., Pot, F., and Cohen, J. A.: Different inherited levels o:f DN.A repair in xeroderma pigmentosu1n cell strains after exposure to ultraviolet irradiation, Mutat. Res. 9: 507, 1970.