Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated

Harrison, Gail Singer

Karrer, Kathleen M.

English

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Marquette Universitye-Publications@MarquetteBiological Sciences Faculty Research andPublications Biological Sciences, Department of2-1-1989Methylation of Replicating and NonreplicatingDNA in the Ciliate Tetrahymena thermophilaGail Singer HarrisonBrandeis UniversityKathleen M. KarrerMarquette University, kathleen.karrer@marquette.eduPublished version. Molecular and Cellular Biology, Vol. 9, No. 2 (February 1989): 828-830.Permalink. © 1989 American Society for Microbiology. Used with permission.Kathleen Karrer was affiliated with Brandeis University at the time of publication.MOLECULAR AND CELLULAR BIOLOGY, Feb. 1989, p. 828-830 Vol. 9, No. 20270-7306/89/020828-03$02.00/0Copyright C 1989, American Society for MicrobiologyMethylation of Replicating and Nonreplicating DNA in the CiliateTetrahymena thermophilaGAIL SINGER HARRISONt AND KATHLEEN M. KARRER*Brandeis University, Department of Biology, Waltham, Massachusetts 02254Received 5 August 1988/Accepted 27 October 1988Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila wasexamined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, butmethylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cellswas demonstrated.The modified base N6-methyladenine (N6MeA) is presentas a minor component in DNA from several unicellulareucaryotes, including members of the genera Chlamydomo-nqs (9), Chlorella (16), Oxytricha (15), Paramecium (2), andTetrahymena (9). DNA in the germ line micronucleus inTetrahymena thermophila is unmethylated, while 0.8% ofadenines in DNA of the somatic, polyploid macronucleus aremodified to N6MeA (3). The function of N6MeA in T.thermophila is not known.During sexual reproduction, the old macronucleus is de-stroyed and a new macronucleus develops from a mitoticproduct of the zygotic micronucleus. De novo methylation ofDNA in the developing macronucleus is not random butoccurs according to a specific pattern. Some GATC sites, forexample, are unmethylated while others are methylated in allmacronuclear DNA molecules (6). Still others are methyl-ated in a fraction of the molecules that is characteristic of aparticular site and that is consistent between cell lines (1, 17;Capowski et al., submitted for publication). Because T.thermophila undergoes phenotypic assortment (for a review,see reference 13), the existence of partially methylated sitesimplies that there is a methylase in vegetative cells whichrecognizes unmethylated DNA as a substrate.Methylation of replicating DNA. If de novo methylationcontributes to maintenance of the methylation pattern, thenboth the newly synthesized strand and, to a lesser extent, theparental strand are expected to undergo methylation inreplicating DNA. Vegetatively growing cells were culturedin axenic medium containing 1% proteose peptone (4) to adensity of 3 x 105 cells per ml. Bromodeoxyuridine (BUdR;50 ,ug/ml) and [methyl-3H]thymidine (55 to 80 Ci/mmol;concentration, 0.5 to 1.0 mCi/ml) were added to the medium,and the cells were grown for an additional 270 min (1.5generations). Nuclear DNA was purified as described previ-ously (6). The parental and daughter strands of the newlyreplicated DNA were separated on alkaline CsCl2-Cs2SO4gradients (12) with 3.9 g of CsCl2 in each 4.8-ml gradient.After BUdR was incorporated into the DNA and the DNAwas labeled with [3H]thymidine, approximately 40% of theDNA, which was assayed by A260 determination, migratedas a broad peak with a heavy density (Fig. la). The majority(92%) of newly replicated DNA, which was labeled with[3H]thymidine, migrated in the gradient as a heavy peak.Thus, replicating DNA was efficiently labeled and there was* Corresponding author.t Present address: University of Colorado Health Sciences Cen-ter, Division of Medical Oncology, Denver, CO 80262.good separation of newly replicated daughter strands fromunreplicated parental strand DNA.Since T. thermophila does not synthesize purines andpyrimidines (11), DNA was labeled with methionine only bypostreplicative methylation of adenine (14) (see Table 2). Agradient ofDNA from cells dividing in the presence of BUdRand L-[methyl-3H]methionine (12 to 80 Ci/mmol) at 8 to 25Ci/ml is shown in Fig. lb. The majority of counts (87%)comigrated with newly synthesized (heavy) DNA.In contrast to DNA labeled with [3H]thymidine, [3H]me-thionine-labeled DNA produced a small but distinct peak of0.40.30toCN440.20.13.02.00CY441.00.00 1 0 20Fraction No.E20..U3 02000EzC.100 0.u00 1 0 20 30Fraction No.FIG. 1. Alkaline CsCl2-Cs2SO4 gradients of [methyl-3H]thy-midine- (a) and L-[methyl-3H]methionine- (b) labeled DNA fromcells cultured vegetatively in the presence of BUdR. Density de-creased as the fraction number increased. Symbols: WH, A260; *,counts per minute per 100 ,ul of sample.828NOTES 829sE.CIU0 1 0 20 30Fraction No.3.0 -2.0 -01.0 -0.000CDI1 0 2 0Fraction No.0 1 0 20 3'Fraction No.FIG. 2. Alkaline CsCl2-Cs2SO4 gradients of [ndine- (a) and L-[methyl-3H]methionine- (b and c) lalcells starved in 10 mM Tris. Cells were cultured irand b) or absence (c) of BUdR. Gradients in panelsin a separate experiment from the gradient inaccounts for the difference in elution of the unDNA. Symbols: [D, A260; *, counts per minutesample.radioactivity comigrating with unreplicatedAbout 13% of incorporated counts were in Igesting that a significant fraction of methylating DNA occurs on the parental strand.Methylation of unreplicated DNA. Autorcells starved in the presence of [3H]thymidindicated that starved cells do not synthesizewas confirmed by density gradient analysisTABLE 1. DNase I digestion of L-[methyl-3H]methionine- and[8-3H]adenine-labeled DNAcpm (% recovery) of DNAslabeled with:DNA treatmentL-[methyl-3H] [8-3HladeninemethionineMinus DNase IBefore filtration 100 192After filtration (bound to filter) 56 (56) 151 (79)Plus DNase IBefore filtration 110 135After filtration (bound to filter) 9 (8) 8 (6)200 starved cells (Fig. 2a). Log-phase T. thermophila was trans-ferred to 10 mM Tris and starved for 22 to 24 h, BUdR and[3H]thymidine were added to the medium, and the cells weremaintained for an additional 18 h before DNA extraction.When the DNA was run on an alkaline CsCl2-Cs2SO4gradient, there was a single peak, as measured by determin--100 s ing the A26, and there was no evidence for the incorporationE of [3H]thymidine.Although DNA synthesis was below detectable levels instarved cells (Fig. 2a), DNA methylation occurred nonethe-less (Fig. 2b and c). The addition of [3H]methionine tomedium containing starved cells resulted in the incorpora-0 tion of label into DNA both in the presence (Fig. 2b) and theo absence (Fig. 2c) of BUdR. Since we demonstrated thatstarved cells do not synthesize DNA, incorporation of[3H]methionine must have occurred in the absence of DNAreplication.200 We showed that radioactivity incorporated by labelingwith [3H]methionine was in DNA and not protein by twocriteria. The first was that the counts were sensitive todigestion with DNase I. Table 1 shows the result of digesting0.5 ,ug of DNA from cells which were labeled in vivo witheither [3H]methionine or, as a control, [8-3H]adenine. Fol--100 El lowing DNase I treatment, DNA was coprecipitated from10% trichloroacetic acid with 50 ,ug of calf thymus DNA.The majority of counts were released by DNase I digestion,with only 6 to 8% of the radioactivity remaining bound to thefilter.0 Thin-layer chromatography of bases was done to show0 that the DNA was labeled in methyladenine residues. Acidhydrolysis ofDNA was performed as described by Hattman(8). The hydrolyzed DNA was spotted, along with standards,tehyl-3H]thymi- onto a silica thin.layer chromatographic sheet containing abeled DNA from UV fluorescence indicator (J. T. Baker Chemical Co., Phill-i the presence (a ipsburg, N.J.). The difference in migration between adenineb and c were run and methyladenine was maximized by using an ascending,panel a, which one-dimensional, two-step solvent system (ethyl acetate-replicated (light) methanol; [70:30] with overnight drying, followed by chlo-per 100 ,ul of roform-methanol [40:60]). The spots were visualized withUV light, cut out, and counted. Results from one experimentare shown in Table 2. Guanine and cytosine residues ran asa smear near the origin in this system and were not mea-(light) DNA. sured. A total of 58 cpm (94% of the total counts per minutethis peak, sug- measured for the bases adenine, methyladenine, and thy--ion in replicat- mine) was detected in the spot corresponding to N6MeA,compared with 4 cpm (6%) for adenine and 0 cpm forradiography of thymine. The 21 cpm measured at the origin probablyline previously corresponded to partially hydrolyzed DNA. Thus, labelDNA (7). This incorporated into the DNA of starved cells was the result ofof DNA from adenine methylation.bVOL. 9, 1989I3MOL. CELL. BIOL.TABLE 2. Thin-layer chromatography of hydrolyzedL-[methyl-3H]methionine-labeled DNADistance (cm)Base migrated from RI cpm in spotoriginThymine 12.5 0.39 0N6MeA 11.0 0.34 58Adenine 10.0 0.31 4Origin 0 0 21a The Rf was determined as follows: distance migrated by base/distancemigrated by solvent.We showed that the majority of methylation occurs on thedaughter strand during DNA replication in T. thermophila.In addition, there was significant incorporation of label onthe unreplicated parental strand in DNA from dividing cellsand methylation of nonreplicating DNA in starved cells. Oneexplanation for the methylation of nonreplicating DNAcould be the turnover of methyl groups, with no change insteady-state levels. Another possible explanation, at least ingrowing cells, is delayed methylation, as has been describedpreviously for cytosine in mammalian systems (5, 10, 12, 18).It is unlikely, however, that delayed methylation accountsfor methylation ofDNA in starved cells, which was synthe-sized 22 to 24 h before the addition of the radioisotope.Repair synthesis cannot readily account for methylation ofnonreplicating DNA in starved cells. The assay for DNAreplication in growing cells by labeling with [3H]thymidine is100-fold more sensitive than labeling with [3H]methionine(Fig. 1), yet no DNA synthesis was detected in starved cells.It is unlikely that DNA synthesis was masked in starved cellsby a large pool of unlabeled thymidine, since replicatingDNA is readily labeled by thymidine incorporation whenstarved cells with complementary mating types are mixed toinduce conjugation (7). These results indicate that DNAmethylation occurs in the absence of DNA synthesis in T.thermophila and are consistent with the hypothesis thatunmethylated sites are a substrate for a maintenance meth-ylase.This work was supported by Public Health Service grants GM32989 and GM39890 from the National Institutes of Health.LITERATURE CITED1. Blackburn, E. H., W.-C. Pan, and C. C. Johnson. 1983. Meth-ylation of ribosomal RNA genes in the macronucleus of Tetra-hymena thermophila. Nucleic Acids Res. 11:5131-5145.2. Cummings, D. J., A. Tait, and J. M. Goddard. 1974. Methylatedbases in DNA from Paramecium aurelia. Biochim. Biophys.Acta 374:1-11.3. Gorovsky, M. A., S. Hattman, and G. L. Pleger. 1973.[6N]methyl adenine in the nuclear DNA of a eucaryote, Tetra-hymena pyriformis. J. Cell. Biol. 56:697-701.4. Gorovsky, M. A., M.-C. Yao, J. B. Keevert, and G. L. Pleger.1975. Isolation of micro- and macronuclei in Tetrahymenapyriformis. Methods Cell Biol. 9:311-327.5. Gruenbaum, Y., M. Azyf, H. Cedar, and A. Razin. 1983.Methylation of replicating and post-replicated mouse L-cellDNA. Proc. Natl. Acad. Sci. USA 80:4919-4921.6. Harrison, G. S., R. C. Findly, and K. M. Karrer. 1986. Site-specific methylation of adenine in the nuclear genome of aeucaryote, Tetrahymena thermophila. Mol. Cell. Biol. 6:2364-2370.7. Harrison, G. S., and K. M. Karrer. 1985. DNA synthesis,methylation and degradation during conjugation in Tetrahymenathermophila. Nucleic Acids Res. 13:73-87.8. Hattman, S. 1970. DNA methylation of T-even bacteriophagesand of their nonglycosylated mutants: its role in Pl-directedrestriction. Virology 42:359-367.9. Hattman, S., C. Kenny, L. Berger, and K. Pratt. 1978. Compar-ative study ofDNA methylation in three unicellular eucaryotes.J. Bacteriol. 135:1156-1157.10. Hilliard, J. K., and T. W. Sneider. 1975. Repair methylation ofparental DNA in synchronized cultures of Novikoff hepatomacells. Nucleic Acids Res. 2:809-819.11. Kidder, G. W., and V. C. Dewey. 1945. Studies on the biochem-istry of Tetrahymena. Arch. Biochem. 8:293-320.12. Kowalski, J., and W. P. Cheevers. 1976. Synthesis of highmolecular weight DNA strands during S phase. J. Mol. Biol.104:603-615.13. Nanney, D. L., and R. M. Preparata. 1979. Genetic evidenceconcerning the structure of the Tetrahymena thermophila mac-ronucleus. J. Protozool. 26:2-9.14. Pratt, K., and S. Hattman. 1981. Deoxyribonucleic acid meth-ylation and chromatin organization in Tetrahymena thermo-phila. Mol. Cell. Biol. 1:600-608.15. Rae, P. M. M., and B. B. Spear. 1978. Macronuclear DNA of thehypotrichous ciliate Oxytricha fallax. Proc. Natl. Acad. Sci.USA 75:4992-4996.16. Van Etten, J. L., A. M. Schuster, L. Girton, D. E. Burbank, D.Swinton, and S. Hattman. 1985. DNA methylation of virusesinfecting a Chlorella-like green alga. Nucleic Acids Res. 13:3471-3478.17. White, T. C., N. C. McLaren, and S. A. Alien. Methylation sitewithin a facultatively persistent sequence in the macronucleusof Tetrahymena thermophila. Mol. Cell. Biol. 6:4742-4744.18. Woodcock, D. M., D. L. Simmons, P. J. Crowther, I. A. Cooper,K. J. Trainor, and A. A. Morley. Delayed DNA methylation isan integral feature ofDNA replication in mammalian cells. Exp.Cell Res. 166:103-112.830 NOTES

Methylation of Replicating and Nonreplicating DNA in the Ciliate \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

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Methylation of Replicating and Nonreplicating DNA in the Ciliate \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

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