Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytesâ€•

Home , Uracil

[CANCER RESEARCH 39. 2090-2095, June 1979] 0008-5472/79/0039â€”0000$02.0O Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytesâ€•

Michael A. Sirover

Fels Research Institute and the Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT induced by PHA, but nucleotide kinase activities remain con stant despite cell proliferation. The capacity of human cells to modulate the synthesis of The uracil-ONA glycosylase was induced 10-fold during lym DNA repair enzymes has been investigated by measuring the phocyte stimulation by PHA. Glycosylase stimulation was co induction of the umacil-ONAglycosylase during lymphocyte ordinate with the induction of DNA synthesis and DNA polym stimulation. Treatment of peripheral lymphocytes with phyto erase activity. Furthermore, treatment with either actinomycin hemagglutinin increased glycosylase activity 10-fold. Glyco 0 or cycloheximide at maximal stimulation diminished enzyme sylase stimulation was coordinate with the activation of DNA activity after an appreciable interval. These results suggest that synthesis and DNA polymerase activity. Two chnomatographi stable base modifications which persist in quiescent cells may cally distinct species of the glycosylase have been resolved; be miscopied during cell activation. only one species is induced during phytohemagglutinin stimu lation. The effect of actinomycin 0 and cycloheximide on gly MATERIALS AND METHODS cosylase induction was determined. Treatment with either in hibiton at 96 hr after phytohemagglutinin addition (maximal Lymphocyte Culture. Lymphocytes were isolated by sedi induction) decreased glycosylase activity after an appreciable mentation through Ficoll-Hypaque gradients (6). Final pmepa lag period. This suggested that induction of the umacil-ONA nations contained greater than 90% lymphocytes (all concen glycosylase requires transcription and translation although the trations referred to are final concentrations in the reaction enzyme may be quite stable once induced. mixture or in the buffer utilized); 99% were viable as defined by trypan blue dye exclusion. Cells were diluted to a final INTRODUCTION density of 0.75 to 1.0 x 106 cells/mI in Eagle's minimum essential medium (spinner modification) which contained 20% The excision-repair of DNA modified by chemical carcino fetal calf serum, 20 mr@i4-(2-hydroxyethyl)-1-piperazineeth gens may occur through 2 pathways: nucleotide excision-re anesulfonic acid (pH 7.4), 2 mr@iL-glutamine,100 @tgofstrep pair in which the initial event is an endonucleolytic cleavage of tomycin, and 100 units of penicillin per ml. Cells were cultured DNA (12); or base excision-repair in which the first step is the in 1-ml aliquots with 50 @lofPHA penml of cells. DNA synthesis cleavage of the base-sugar glycosyl bond producing an apu was determined by measuring the incorporation of [3H]thymi rinic or apyrimidinic site (1 7). DNA glycosylases which excise dine (6.7 Ci/mmol; 2.5 MCi/culture) for 2 hr before collection. N3-methyladenine, hypoxanthine, and uracil from DNA have These cell pellets were used to measure concurrently total been identified (9, 13, 18, 24, 27). The uracil-DNA glycosylase DNA polymenase activity as described previously (20). excises uracil residues in DNA which may be formed by the Formation of GlycosylaseSubstrates. Substrateswere pre mutagenic deamination of cytosine (3) or by the incorporation pared by copying either activated calf thymus DNA or poly of umacilduring DNA replication (5, 34). deoxyadenylate .oligodeoxythymidylate with homogeneous Although some of the enzymes comprising the excision Escherichia coli DNA polymerase I, using [3H]dUTP (15,000 repair pathways have been characterized, the mechanisms by dpm/pmol) and other appropriate deoxyribonucleoside tn which human cells regulate their synthesis and the relationship phosphates as precursors. After 30 mm incubation at 37Â°,the of such regulation to carcinogenesis remain unknown (8, 22, reaction mixture was extracted with phenol, dialyzed 4 times 23, 31). Synthesis of repair enzymes may be constitutive (10, with 3.5 liters of 1 M NaCI plus 50 m@iTnis-HCI(pH 8.0), and 33); alternatively, cells may modulate the activity of one or then dialyzed 4 times with 3.5 liters of 50 mMTmis-HCI(pH8.0). several repair enzymes (4, 25). Furthermore, the temporal Assay for Uracil-DNA Glycosylase in Cell Extracts. Uracil sequence between the induction of enzymes involved in DNA DNA glycosylase was measured by determining the release of repair and those involved in DNA replication during the cell ethanol-soluble or acid-soluble radioactivity from [3H}uracil-Ia cycle has not been established. To consider these questions, beled DNA templates. Cell pellets were freeze-thawed 3 times we have asked initially whether the uracil-DNA glycosylase is in a solution (total volume, 100 @l)whichcontained 20 mM induced in human cells which are quiescent but which can be K2PO4(pH 7.5) pIus 1 mM dipotassium EDTA, 2.5 mM OTT, stimulated to proliferate. Lymphocytes activated by PHA2were and 20% glycerol. chosen as a model system, inasmuch as PHA stimulation Uracil-DNA glycosylase activity was measured in a reaction results in a sequential activation of selective enzymatic activi mixture (total volume, 200 @sl)whichalso contained 100 mM @ ties (19, 20). For example, DNA polymerases and /@are Tnis-HCI(pH 8.0), 7.5 mM OTT, 15 mM dipotassium EDTA (pH @ 7.0), 1 of bovine serum albumin, and 3 @gof[3Hjumacil labeled DNA (19,000 dpm/pmol). Incubations were performed I This study was supported by NIH Grant ES-Ol 735 and CA-i 2227.

2 The abbreviations used are: PHA, phytohemagglutinin; OTT, dithiothreitol. at 37Â°for 60 mm. Reactions were terminated by adding se Received December 21, 1976, accepte'@March 13, 1979. quentially 300 @lofethanol, 60 @iIof2 M NaCI, and 100 @gof

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. Induction of DNA Repair Enzymes denatured calf thymus DNA (1 mg/mI in 20 mt@iKCI,pH 6.8). + After a minimum of 60 mm at â€”20Â°,themixtures were centri U' In r') @ fuged at 3000 rpm for 10 mm. The ethanol supemnatant(200 IC I.#U â€˜@

@.tl)wasremoved and counted. In some experiments, reactions E -4 were terminated by adding 10% tnichloroacetic acid (300 gil) U U C 3 and 100 i@gof denatured calf thymus DNA. These mixtures C)

were centrifuged after 20 mm at 5Â°.[3H]Uracil released in the C @ supemnatantwas identified as a reaction product by thin-layer 50 E chromatography. I- Partial Purification of the Uracil-DNA Glycosylase. Lym phocytes were cultured with PHA as described. Cells were In cultured for 4 days before collection. Cell pellets were frozen

and thawed 3 times and then suspended in 7.6 ml of 20 mt,i S Tris-HCI (pH 8.0)-i 0 mM MgCl2-1 m@OTT (Buffer 1). Triton X 3 l( 100 (0.4 ml) was added. The solution was kept at 0Â°for 30 U3 mm before homogenizing with 20 strokes in a Oounce homog 0 E enizem.The cell extract was adjusted to 20% glycerol (Buffer 2) and then centrifuged at 9000 rpm for 20 mm. The super a) 0 natant contained greater than 90% of the glycosylase activity. a) Nucleic acids were extracted by adding to the supemnatant 2.5 S ml of DEAE-cellulose previously equilibrated with 20 mr@iTris U0 HCI (pH 8.0)-i mM DTT-20% glycerol (Buffer 3). The suspen sion was stirred gently for 30 mm and then centrifuged at 3000 In rpm for 10 mm. The supemnatantwas collected and absorbed to a Sephadex G-100 column (2.5 x 42 cm) equilibrated in Buffer 3 plus 100 mM NaCI. Uracil-DNA glycosylase was as Time after PHA Stimulation (Days) sayed using 20 @slofcolumn effluent. Fractions containing Chart 1. Induction of the uracil-DNA glycosylase during lymphocyte stimula tion by PHA. Lymphocytes were isolated and cultured as previously described. glycosylase activity were pooled, dialyzed overnight against 4 DNA synthesis was determined by measuring the incorporation (Inc.) of liters of Buffer 3, and absorbed to a phosphocellulose column [3H]thymidine (6.7 Ci/mmol; 2.5 MCi/culture) for 2 hr before collection. Total DNA polymerase activity and urscil-DNA glycosylase activity were measured as (1.5 x 17 cm) equilibrated with Buffer 3. The column was previously described. Each point represents the average of quintuplicate cultures. washed with 20 ml of Buffer 3; a linear gradient of 0 to 0.5 M L@,PHA-stimulatedcells;A,unstimulatedcells. KCI plus Buffer 3 (total volume, 60 ml) was applied. Enzyme activity was monitored using 50 @lofcolumn effluent. taming [3HJdAMP,[3H]dCMP, or [3H]dGMP, increasing concen RESULTS trations of the glycosylase preparation failed to release detect able radioactivity (Chart 2). Using reaction conditions in which Lymphocyte stimulation by PHA was monitored by measuring the glycosylase released 61 .5% of umacilfrom DNA, less than [3Hjthymidine incorporation and by measuring DNA polymenase 1.3% of the other bases were rendered soluble. Using a tem activity in vitro (Chart 1A). DNA synthesis and DNA polym plate containing [3Hjuracil and [32Plphosphates, the glycosy erase activity were coordinately induced by PHA; maximal lass released 51% of uracil from DNA; less than 1% of 32Pwas activities were observed 3 to 4 days after PHA addition. Umacil solublized. Similar results were observed using cell-free ex DNA glycosylase activity was measured in parallel cultures tracts. Furthermore, the glycosylase preparation did not me (Chart 1B). In this experiment, the uracil-ONA glycosylase was lease N@-methyladeninefrom DNA (results not shown). induced 10-fold. Furthermore, glycosylase stimulation was The increase in glycosylase activity during PHA stimulation temporally coordinate to that of DNA synthesis and of DNA was also observed during enzyme purification. Low-speed su polymemaseactivity. No glycosylase induction was observed in pemnatantswere applied to a DEAE-cellulose column; umacil parallel cultures which were incubated without PHA. However, DNA glycosylase was not absorbed. However, DNA polymemase enzyme stimulation varied in different experiments from 3- to a was bound and was eluted characteristically by 0.3 M KCI, 10-fold using lymphocyte preparations from different individ yielding a separation of these 2 enzyme activities. The glyco uals. Bertazzoni et a!. (4) demonstrated that UV irradiation of sylase was then rechromatographed on Sephadex G-100 stimulated lymphocytes induced 2 peaks of DNA repair synthe (Chart 3). Two enzyme peaks were observed using either PHA sis before and after DNA replication. Maximal glycosylase stimulated on unstimulated cells. However, PHA stimulation stimulation coordinate with DNA synthesis is an initial sugges resulted in an increase in only the first glycosylase activity. The tion that base excision repair may be activated only during increase correlated with the stimulation of glycosylase activity DNA replication. observed using cell-free extracts of parallel cultures. Lymphocyte stimulation by PHA results in deoxynuclease The uracil-DNA glycosylase was further purified to determine induction (19). Therefore, to demonstrate that the enzyme whether 2 chromatognaphically distinct species could be iden activity measured in cell-free extracts (Chart 1B) was specifi tified. Using PHA-stimulated lymphocytes, chromatography on cally due to a uracil-DNA glycosylase, a series of DNA sub Sephadex G-100 again resolved 2 glycosylase species (Chart stnates were prepared, each of which was selectively labeled 4A). The 2 species were collected and were nechromato and was of comparable specific activity. Using substrates con graphed through phosphocellulose (Chart 4B). Using ion-ex

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0 E a If a) E 0 a) a) U a) C 0 z 0 C 4 a 0 C) 0 V 0 S 0a â€˜a 0 a V

Protein(@@g) Chart 2. Specificity of the uracil-DNA glycosylase. DNA substrates were prepared using E. coli DNA polymerase I; activated calf thymus DNA as the template; and [3H)dCTP,13H]dATP,[3H)dGTP,[3H]dUTP,and [a-32PJTTPasradio active precursors in separate experiments. Templates were purified as previously described. The release of acid-soluble radioactivity was measured in a mixture (total volume, 100 @d)which contained 100 mri Tris-HCI (pH 8.0), 10 m@i dipotassium EDTA (pH 7.0), 5 m@DTT, 1 @gofbovine serum albumin, and 1 @g of DNA. Uracil-DNA glycosylase from the DEAE-cellulose column of PHA-stimu lated cells was used. Reactions were incubated for 60 mm at 37Â°.Incubations without enzyme were used as controls (8 and 4 pmol for substrates containing [3H]uracll and [32Pjphosphates).Specific activities of all substrates were similar. DNA substrates contained [3H]uracil (I) and [32Pjphosphates(0), [3Hjadenine Fraction No (f.@),[3H]guanine(0), and [3H)cytosine(X). Protein was determined as described Chart 4. Purification of the uracil-DNA glycosylase from PHA-stimulated lym (21). phocytes. Cells were cultured in 30-mI aliquots as described (1 x 1 @6lympho cytes/mI; 2.18 x 108 lymphocytes). Cells were collected 96 hr after PHA addition. The glycosylase was induced 7.6-fold by PHA as measured in cell-free extracts (8.72 Â±0.38 pmol of uracil released per 10Â°cells with PHA added; 1.14 Â±0.16 pmol of uracil released per 108cells without PHA). The enzyme was isolated as described in â€˜â€˜MaterialsandMethods.â€œAfterremoval of nucleic acids by DEAE-cellulose, the supematant was chromatographed on Sephadex G-i 00 (A).TheenzymepeakselutingfromtheSephadexG-100columnwereassayed in duplicate, and the averages were determined. The 2 peaks of glycosylase activity were pooled together and reisolated on phosphocellulose (B). Uracil-DNA 0 glycosylase activity was assayed as described, using 20 @lofcolumn effluent aE from the G-i 00 column and 50 @Ifromthe phosphocellulose column. The results V from Charts 3 and 4 should not be compared with regard to the amplitude of the 2 glycosylase activities. Each chart represents experiments using lymphocytes 01#) a) from different donors. a)

0U change chromatography, the 2 species were definitely me :D solved. One species was retained on phosphocellulose but

I') eluted immediately after sample absorption. The second eluted during the gradient. This elution pattern on phosphocellulose is virtually identical to the pattern observed during purification of the glycosylase from human placenta. The sole difference is that in placenta the second species is predominant, comprising approximately 90% of the enzyme activity.3 Due to the small FRACTiON NUMBER number of lymphocytes recovered from a single donor, a Chart 3. Identification of the uracil-DNA glycosylase induced by PHA. Lym rigorous purification of the enzymes could not be attempted. phocytes were cultured with and without PHA as described. Cells were cultured However, the molecular weights of the 2 species were deter for 5 days before collection. Cell extracts were prepared as described. After centrifugation, the supernatant was applied to a 10-mI DEAE-cellulose column mined (Chart 5). The molecular weight of Peak 1 (approximately (DEAE 52) previousiy equilibrated with Buffer 2. The column was washed se 42,500) and that of Peak 2 (approximately 40,500) were quentially with 0.05 M KCI plus Buffer 2 and 0.3 N KCI plus Buffer 2 (20 ml each comparable to that of the highly purified glycosylase from wash). Uracil-DNA glycosylase activity was determined as described in the legend to Chart 2. Peak fractions from PHA-stimulated cells released 300 pmol of placenta. [3H)uracil; peak fractions from unstimulated cells released 98 pmol. In some To determine whether glycosylase induction required con preparations for PHA-stimulated cells, a minor peak of uracil-DNA glycosylase current RNA and protein synthesis, the effects of cycloheximide activity was observed in the 0.3 M KCI wash coordinate with DNA polymerase activity. Peak fraction from each column was chromatographed on a 2.5-mi and actinomycin 0 on glycosylase activity were investigated. Sephadex 0-1 00 column (0.5 x 7.5 cm) previously equilibrated with 50 [email protected] The requirement for continued RNA and protein synthesis was HCI (pH 8.0), 1 mM DTT, and 20% glycerol. Column effluents were assayed as previously described except that 0.995 @gofpolydeoxyadenylate.oligodeoxy determined at maximal stimulation (96 hr after PHA addition). thymidylate. poly[3H)deoxyuridylate (1010 dpm/pmol), and 50 @tlof column effluent were used. â€¢,PHA-stimulatedlymphocytes; 0, unstimulated lympho cytes. 3E.J. KatzandM.A. Sirover,submittedforpublication.

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80,000@

BSA 60,000

Ovalbumin PeokI ___ 40,000 Peak2 Chart 5. Molecular weight determination of the uracil-DNA L&J glycosylases. The position of the glycosylase activities relative to 4 reference proteins was determined on a Sephadex G-1 00 column (2.5 x 42 cm) equilibrated with Buffer 3 pIus I 00 mt.i ,@ NaCI. The molecular weights used for the standard are: bovine -J serum albumin (BSA), 67,000; ovalbumin, 43,000; pancreatic C-) RNase A, 13,700; cytochrome c, 12,500. The positions of the Li reference proteins were determined by measuring absorbance at @.1 0 280 nm. The elution volumes of all proteins were determined at maximal absorbance or at maximal enzyme activity. 20 ,O00

RNose A Cyfochrome C

lO ,00C 100 200 ELUTION VOLUME(ml)

inductionLymphocytesEffect of inhibitors on uracil-DNA glycosylase S 3 96hr were cultured as described. Inhibitors were added

3 comparingenzymeafter PHA addition. Percentageswere determinedby U identicaldaysactivitywithinhibitoraddedto thatwithoutinhibitoron 0 after PHAstimulation.UraclI-DNA Ea V glyco- Enzyme actlv S a) Time after ad- sylase (pmol re- lty (% of con 0 trol)NoneInhibitor dition (days) leased/culture) S S 01001 27.73 Â±393k 1002 20.08 Â±0.65 U 0 100Actlnomycln 13.61 Â±0.47 D D (0.5 @g/ml) 0781 21.62 Â±0.36 I In 502 10.08Â±1.29 26Cycloheximide 3.50Â±0.53

(5 pg/mI) 0831 23.00 Â±2.34 Time after Inhibitor Addition(hr) 612 12.29 Â±0.70 42a 5.78Â±0.71 Chart 6. Effect of actinomycin D and cycloheximide on glycosylase activity. Lymphocytes were cultured In 1-ml allquots as described. At 96 hr after PHA MeanÂ±S.D. addition, actinomycin D (0.2 gig/mI) and cycloheximide (2 @g/ml)wereadded to quintuplicate cultures. At times Indicated, cultures were collected and assayed cycloheximide induced a 21% decrease in the glycosylase as described. Cycloheximide at 2 or 5 pg/mI abolished [3H]Ieucine incorporation into protein. 0, no Inhibitor added; â€¢,cycloheximide(2 pg/mI); X , actinomycin activity; at 24 hr, the decrease was 41 %. Similar results were D (0.2 @g/ml). observed using cycloheximide (5 j@g/ml)(results not shown). In contrast, uracil-DNA glycosylase activity was resistant to As shown in Chart 6, cycloheximide (2 @sg/ml)hadno effect on actinomycin D (0.2 @sg/mI)for7 hr. However, at 24 hr, enzyme glycosylase activity until 5 hr after inhibitor addition. Corn activity in actinomycin D-treated cultures had decreased by mencing at 5 hr, there was a steady decrease in glycosylase 27%. As the enzyme is apparently quite stable, the effects of activity as compared to the simultaneous control. At 6 hr, actinomycin 0 and cycloheximide were monitored for longer

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. M. A. Sirover intervals (Table 1). Addition of cycloheximide or actinomycin 0 during lymphocyte stimulation (20). Thus, the glycosylase is inhibited glycosylase synthesis 6 days after PHA stimulation. fairly stable but may have to be synthesized continually. Be In control cultures, glycosylase activity had decreased to 49% cause quiescent lymphocytes synthesize little DNA, RNA, or of enzyme activity at maximal stimulation. In inhibitor-treated protein, replenishment of the repair enzyme may be unlikely. cultures, enzyme activity had decreased by 84 and 75%, respectively, for actinomycin 0- and cycloheximide-treated ACKNOWLEDGMENTS cultures. The counsel of Dr. S. Kim, Dr. 0. Paul, and Dr. I. Winicov is greatly appreciated. DISCUSSION

In this report, the induction of the uracil-DNA glycosylase REFERENCES during lymphocyte stimulation by PHA has been determined. 1. Ahmed, F. E., and Setlow, R. B. Different rate-limiting steps in excision The glycosylase was induced 10-fold during lymphocyte pro repair of ultraviolet- and N-acetoxy-2-acetylaminofiuorene-damaged DNA in normal human fibroblasts. Proc. NatI. Acad. Sci. U. S. A., 74: 1548â€”1552, lifemation.Induction was coordinate with that of DNA polymer 1977. ase and DNA synthesis, reaching a maximum 4 days after PHA 2. Amacher, D. E., Elliott, J. A., and Lieberman, M. W. Differences in removal addition. Glycosylase induction required transcription and of acetylaminofiuorene and pyrimidine dimers from the DNA of cultured mammalian cells. Proc. NatI. Acad. Sci. U. S. A., 74: 1553â€”1557,1977. translation although the enzyme appeared to be quite stable. 3. Baltz, R. H., Bingham, P. M., and Drake, J. W. Heat mutagenesis in Unstimulated lymphocytes have a limited capacity to repair bacteriophage T4: the transition pathway. Proc. NatI. Acad. Sd. U. S. A., 73: 1269â€”1273,1976. DNA (7, 16). Stimulation by concanavalin A increases by 10- 4. Bertazzoni, U., Stefanini, M., Pedrali Noy, G., Giulotto, E., Nuzzo, F., fold their capacity to initiate repair synthesis induced by N- Falaschi, A., and Spadari, S. Variations of DNA polymerases-a-and-$ during acetoxy-2-acetylaminofluorene (26). Similarly, PHA stimulation prolonged stimulation of human lymphocytes. Proc. NatI. Acad. Sci. U. S. A., 73: 785-789, 1976. increases repair synthesis by 8-fold after UV irradiation (4) and 5. Bessman, M. J., Lehman, I. R., Adler, J., Zimmerman, S. B., Simms, E. S., by 20-fold after ionizing radiation (15). Although adducts and Kornberg, A. Enzymatic synthesis of DNA. Ill. The incorporation of pyrimidine and purine analogues into DNA. Proc. NatI. Acad. Sd. U. S. A., formed by these agents may not necessarily be repaired by 44:633-640,1958. base excision, the 10-fold induction of the uracil-ONA glyco 6. Boyum, A. Isolation of leukocytes from whole blood. Scand. J. Clin. Lab. sylase is compatible with those results. Furthermore, the uracil Invest., 2 1: 31â€”50,1968. 7. Clarkson. J. M., and Evans, H. J. Unscheduled DNA synthesis in human DNA glycosylase is induced coordinate with the induction of leucocytes after exposure to UV light, y-rays and chemical mutagens. Mutat. DNA synthesis. This is in accord with previous results demon Res., 14: 413-430, 1972. strating that repair synthesis after exposure to N-acetoxy-2- 8. Cleaver, J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature (Lond.), 218: 652â€”656,1968. acetylaminofluorene or ionizing radiation is also induced con 9. Cone, R., Duncan, J., Hamilton, L., and Friedberg, E. Partial purification and comitant with DNA synthesis (15, 26). However, Bertazzoni et characterization of a uracil DNA N-glycosidase from Bacillus subtilis. Bio a!. (4) have demonstrated that repair of UV-induced lesions is chemistry,16:3194â€”3201,1977. 10. Gautschi,J.A.,Young, B. A.,and Cleaver,J.E. Repairofdamaged DNA in activated in a biphasic manner before and after DNA synthesis. the absence of protein synthesis in mammalian cells. Exp. Cell Res., 76: This difference may reflect the activation of distinct DNA repair 87-94, 1973. 11. Goth, R., and Rajewsky, M. F. Persistence of 06-ethylguanine in rat-brain pathways (1, 2). DNA: correlation with nervous system-specific carcinogenesis by ethyl-nitro The limited capacity of nondividing cells to repair DNA poses sourea. Proc. NatI. Acad. Sci. U. S. A., 71: 639-643, 1974. a central problem when cell activation occurs. As DNA lesions 12. Grossman, L., Braun, A., Feldberg, A., and Mahler, I. Enzymatic repair of DNA. Annu. Rev. Biochem., 44: 19-43, 1975. that are quite stable may remain in the cell genome, the 13. Karran, P., and Lindahl, T. Enzymatic excision of free hypoxanthine from temporal sequence with which DNA repair is activated relative polydeoxynucleotides and DNA containing deoxyinosine monophosphate to that of DNA replication may be critical. If DNA repair is residues. J. Biol. Chem., 253: 5877â€”5879,1978. 14. Kleihues, P., and Margison, G. P. Exhaustion and recovery of repair excision induced coordinate with DNA replication, persistent lesions of 06-methylguanine from rat liver DNA. Nature (Lond.), 259: 153.-i 55, may be copied (28, 29) before excision. Thus, recent evidence 1976. suggests that DNA replication before DNA repair may be im 15. Lavin, M. F., and Kidson, C. Repair of ionizing radiation induced DNA damage in human lymphocytes. Nucleic Acid Res., 4: 401 5â€”4022,1977. plicated in the induction of kidney tumors by dimethylnitrosa 16. Lieberman, M. W., and Dipple, A. Removalof bound carcinogen during DNA mine (30, 32). repair in nondividing human lymphocytes. Cancer Res., 32: 1855â€”1860, 1972. It may be argued that the limited capacity of quiescent cells 17. Lindahl, T. New class of enzymes acting on damaged DNA. Nature (Lond.), to repair DNA is sufficient to remove all stable lesions. In this 259: 64-66, 1976. regard, unstimulated lymphocytes do contain detectable levels 18. Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B., and Sperens, B. DNA N- glycosidases. Properties of uracil-DNA glycosidase from Escherichia coli. J. of glycosylase activity. Furthermore, they repair UV-induced Biol. Chem., 252: 3286â€”3294,1977. lesions (7) and remove 15 to 17% of 7-bmomomethylbenz(a)- 19. Loeb, L. A. Molecular analysis of lymphocyte transformation. In: A. A. anthracene adducts from DNA at saturation (16). However, Gottleib(ed.), Biology of Lymphoid Cells, pp. 103-1 32. New York: Academic Press, Inc., 1974. DNA adducts may persist for appreciable intervals. Goth and 20. Loeb, L. A., Ewald, J. L., and Agarwal, S. S. DNA polymerase and DNA Rajewsky (1 1) demonstrated that the half-life of Oâ€•-ethylguan replication during lymphocyte transformation. Cancer Res., 30: 2514â€”2520, 1970. me is 220 hr in rat brain DNA. In matliver, repair capacities can 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, A. J. Protein be exhausted and require several days to recover (14). Pre measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265â€”275, sumably, this may reflect a requirement for enzyme synthesis. 1951. 22. Miller, E. C., and Miller, J. A. Biochemical mechanisms of chemical carci Inhibitor studies presented in this report demonstrate that nogenesis. In: H. Busch (ed.) The Biology of Cancer, pp. 377â€”402.New transcription and translation may be required for glycosylase York: Academic Press, Inc., 1974. induction. The uracil-DNA glycosylase activity started to de 23. Nicoll, J., Swann, P., and Pegg. A. Effect of dimethylnitrosamine on persist ence of methylated guanines in rat liver and kidney DNA. Nature (Lond.). crease 5 hr after cycloheximide addition. This is in contrast to 254: 261â€”262.1975. the immediate effect of puromycin on DNA polymerase activity 24. Riazuddin, S., and Lindahl, T. Properties of 3-methyladenine-DNA glycosy

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lase from Escherichia coli. Biochemistry, 17: 2110â€”2111, 1978. 30. Stewart, B. W., and Magee, P. N. Effect of single dose of dimethylnitrosamine 25. Samson, L, and Cairns, J. A new pathway for DNA repair in Escherichia on biosynthesis of nucleic acid and protein in rat liver and kidney. Biochem. coli. Nature (Lond.), 267: 281â€”283,1977. J., 125:943â€”952,1971. 26. Scudiero, D., Norm, A., Karran, P., and Strauss, B. DNA excision-repair 31. Swann, P. F., and Magee, P. N. Nitrosamine-induced carcinogenesis. Bio deficiency of human peripheral blood lymphocytes treated with chemical chem.J., 125:841â€”847,1971. carcinogens. Cancer Res., 36: 1397-1 403, 1976. 32. Swann, P. F., Magee, P. N., Mohr, U., Reznick, G., Green, U., and Kaufman, 27. Sekiguchi, M., Hayakawa, H., Makino, F., Tanaka, K., and Ohada, Y. A D. G. Possible repair of carcinogenic damage caused by dimethylnitrosamine human enzyme that liberates uracil from DNA. Biochem. Biophys. Res. in rat kidney. Nature (Lond.). 263: 134â€”136.1976. Commun., 73: 293-299, 1976. 33. Teebor, G. W., Duker, N. J., and Becker, F. F. Normal endonuclease 28. Sirover, M. A., and Loeb, L. A. Erroneous base-pairing induced by a activities for damaged DNA during hepatocarcinogenesis. Biochim. Biophys. chemical carcinogen during DNA synthesis. Nature (Lond.), 252: 414â€”416, Acts, 477: 125â€”131,1977. 1974. 34. Tye, B-K., Nyman, P-O., Lehman, I. R., Hochhauser, S., and Weiss, B. 29. Sirover, M. A., and Loeb, L. A. Restriction of carcinogen-induced error Transient accumulation of Okazaki fragments as a result of uracil incorpo incorporation during in vitro DNA synthesis. Cancer Res., 36: 516â€”523, ration into nascent DNA. Proc. NatI. Acad. Sci. U. S. A., 74: 154â€”157,1977. 1976.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytes

Michael A. Sirover

Cancer Res 1979;39:2090-2095.

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