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The relationship between synthesis and methylation of DNA in mouse fibroblasts
BIOCHIMICA ET BIOPHYSICA ACTA
205
BBA 97083
THE RELATIONSHIP BETWEEN DNA I N MOUSE F I B R O B L A S T S
S Y N T H E S I S AND lV[ETHYLATION OF
R. L. P. ADAMS
Department o] Biochemistry, University o/Glasgow, Glasgow (Great Britain) (Received J u l y Ist, 1971)
SUMMARY
Studies on the in vivo methylation of DNA in cultured mouse L cells confirm the presence of a distinct lag between synthesis of DNA and its methylation. This lag is most pronounced for the DNA made in late S phase and the DNA is methylated to only 5 ° % the extent of that DNA made in early S phase. Methylation of DNA does not occur in stationary L cell cultures nor prior to DNA synthesis when such cultures are stimulated to grow. Neither does methylation occur on the DNA of phytohaemagglutinin stimulated horse lymphocytes prior to DNA synthesis.
INTRODUCTION
Only a single radioactive base, namely 5-methylcytosine, has been found in DNA from mouse L929 cells following short periods of incubation in culture medium containing L-EMe-ZHlmethionine 1. DNA methylase activity, which results in the transfer of methyl groups from S-adenosyl-L-methionine to 5-methylcytosine has been found in the chromatin fraction of mouse ascites tumour cell nuclei 2. Although BILLEN8 demonstrated that in Escherichia coli methylation of DNA normally occurs only on nascent DNA we have b~en unable to inhibit DNA methylation b y more than 70 % in mouse L cells when synthesis of DNA was inhibited b y treatment with hydroxyurea to levels less than 6 % of the control x. Preincubation with the drug for 2 h did reduce methylation to 15 % of control values. In chemically synchronised mouse L cells the rate of DNA methylation is maximal in mid S phase. However the rate of methylation of DNA does not fall in G~ to the levels expected if only nascent DNA were being methylated 1. 1V[ethylation of DNA in G2 cells has also been reported in Physarum polycephalum 4. These results have been confirmed and elaborated to show that the DNA made in early S phase is rapidly and highly methylated, but that DNA made in late S phase continues to be methylated for several hours after synthesis is complete. The evidence does not support a role for DNA methylation in the control of I ranscription. MATERIALS AND METHODS
Materials L-EMe-3H]lV[ethionine (7.6 C/mmole) and [~Plorthophosphate were purchased from the Radiochemical Centre, Amersham, Bucks., England. Biochim. Biophys. Acta, 254 (I97 I) 2o5-212
206
R.L.P.
ADAMS
Aminopterin, L-methionine, 2'-deoxycytidine and adenine were obtained from Sigma London Chemical Co. Ltd., London, S.W. 6; glycine, sodium dodecyl sulphate, sodium p-amlnosalicylate, and 8-hydroxyquinoline from British Drug Houses Ltd., Poole, Dorset; 5-bromodeoxyuridine, thymine and 5-methylcytosine from Calbiochem Ltd., London, W.I. and hydroxyurea from Nutritional Biochenlicals Corporation, Cleveland, Ohio, U.S.A. The caesium chloride used was tile Analar grade sold b y Hopkin and Williams Ltd., Chadwell Heath, Essex, England.
Cell culture Mouse fibroblasts (L929) cell cultures were routinely subcultured in minimum essential medimn (Eagle) supplemented with IO % (v/v) calf serum (ECIo). Cultures and media were obtained from Flow Laboratories Ltd., Irvine, U.K. or from BioCult Laboratories, Glasgow, U.K. Cells were grown to stationary phase in Roux bottles as described previously a. Stationary cells were subcultured and synchronised b y treatment with aminopterin (0.2 #M), adenosine (56/~lV[), glycine (80/~l~I) and deoxycytidine (20/~M) from 6-22 h after subculture. The block in DNA synthesis was reversed b y addition of thymidine (or 5-bromodeoxyuridine) at the concentrations cited in the text. When cells were to be labelled for short periods with L-[Me-3H~methionine this was carried out in EC10 containing one-tenth the normal methionine concentration and supplemented with 20 mM sodium formate to prevent equilibration of lab311ed methyl groups with the CI unit pool 6. Lymphocytes were purified from horse blood b y fractionation on a density gradient (B. S. gAIN et al.l°). They were grown in minimum essential medium (Eagle) supplemented with IO °/o (v/v) autologous horse plasma and stimulated with phytohemagglutinin M (Difco Laboratories, Detroit, Mich., U.S.A.). DNA preparation and CsCl centri[ugation DNA was prepared b y the method of KIRBY7. After radioactive labelling cell cultures were washed twice with Earle's balanced salt solution (minus glucose) and then, if necessary, scraped from the glass with a rubber spatala. Following sedimentation the pellet (about lO T cells) was resuspended in 2 ml of the same salt solution containing 6 °/o sodium p-aminosalicylate and added to 2 ml of 2 °/o sodium dodecyl sulphate, 4 mM versene at 7 °0 and maintained at 7 °0 with occasional shaking for 30 rain. After returning to room temperature 4 ml of a mixture of 88 °/o phenol, 12 °/o m-cresol, o.I ~o 8-hydroxyquinoline was added and the tube shaken for 30 rain before centrifugation for 20 min at IO ° and IO ooo rev./min in an MSE 18 centrifuge. The upper layer was removed and the DNA spooled from it after overlayering with 2 vol. cold ethanol. The DNA was dissolved in o.15 M KC1 0.02 M tris HC1 p H 7.5 and respooled. For equiliblium centrifugation the DNA was dissolved in CsC1 solution density 1.815 g/ml containing o.I l~I NaOH. Centrifugation was in the SW 65 rotor of the Spinco model L2 65 B ultracentrifuge at 30 ooo rev./min at 20 ° for 4 ° h. The tubes were punctured at the bottom and two drop fractions collected. These were diluted with I ml 0.3 M N a O H and the optical density profile was measured. Samples (either before or after centrifugation) were incubated at 37 ° in 0.3 M N a O H for 2 h to degrade any RNA which m a y remain 8. Bovine selum albumin (i mg Fraction V, Armour Pharmaceuticals, Eastbourne) was then added and DNA and Biochim. Biophys. Acta, 254 (1971) 2o5-212
DNA METHYLATIONin vivo
207
protein precipitated b y adjusting to 5 % (w/v) trichloroacetic acid. The precipitate was washed twice with 5-ml portions 5 % trichloroacetic acid and finally the DNA was extracted into I ml 0.5 M perchloric acid b y incubating for 30 rain at 7 o°. The radioactivity in 0.5 ml of the supernatant fluid was estimated in IO ml dioxane containing 0. 7 % 2,5-diphenyloxazole plus IO % naphthalene. To check the location of the radioactivity some of the perchloric acid extract was evaporated to give an approximately 12 M solution. The DNA was then hydrolysed to the bases b y incubation at IOO° for 60 min. The hydrolysate was chromatographed using the solvent b u t a n o l - w a t e r - a m m o n i a (87 : 13 : I, b y vol.) ~ as previously described ~. Under these conditions 95-99 % of the tritium radioactivity co-chromatographed with 5-methylcytosine and all the a2p radioactivity remained at the origin P
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16 24 32 Tube number Fig. I. Methylation of newly synthesised and older DNA. Mouse L cells {io ~ cells) were grown in ]~oux bottles in ECIo. I n order to increase the incorporation of tritiated methionine the m e d i u m was replaced w i t h 50 ml w a r m E C I o containing only o n e - t e n t h the n o r m a l methionine concent r a t i o n a n d s u p p l e m e n t e d w i t h 2o rrul~I s o d i u m formate. After three h o u r s incubation in this m e d i u m the following were added: a m i n o p t e r i n (o.2 ~M), adenosine I56/~M), glycine (8o ~M), deoxycytidine (2o /~M), 5-bromodeoxyuridine (2o /~M), [32P]o~hophosphate (Soo ~C) and L-E3//e-3I-I]methionine (5o0/~C). 3 h after addition of r a d i o a c t i v i t y the cells were h a r v e s t e d a n d t h e D N A e x t r a c t e d and centrifuged to equilibrium on gradients of alkaline CsCl, as described in MATI~RIALSAND METHODS; O, 82p)< i o - l ; (~, 3H counts/ruin. The c o n t i n u o u s lines r e p r e s e n t the +3260 nm and t h e d e n s i t y of the gradient.
Biochim. Biophys. Acta, 254 (1971) 2o5-212
20~
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RESULTS
When mouse L cells are grown in the presence of aminopterin and 5-brom~deoxyuridine the newly synthesised daughter strand can be separated from the remainder of the DNA b y centrifugation to equilibrium in alkaline CsC1. If L-FMe-'~Hq methionine is present during the incubation then tlitium is associated with both the newly synthesised, heavy DNA and the older, light fraction (Fig. I). During a 3 h incubation 17 °/o of the methylation occurs on light DNA and 83 o~ on newly ,~\'nthesised DNA. If the production of heavy DNA is allowed to proceed for 3 h and then methylation studied in the subsequent 3 h period in the presence of hydroxyurea (2 raM) to inhibit synthesis of DNA then 30 % of methylation now occurs on light DNA (i.e. DNA more than 3 h old) and 7 ° O~/ooccurs on DNA made in the previous 3 h. This raises the possibility that methylation of new and older DNA represent two distinct processes and it was considered that the latter m a y be related to cellular differentiation. Mouse L cells will enter a stationary phase after growth for several days on a restricted area under conditions of frequent medium replenishment 5. However the rate of DNA methylation falls in an approximately parallel fashion to the rate of DNA synthesis (Fig. 2). When such stationary cells are subcultured they
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Fig. 2. Methylation of D~IA d u r i n g g r o w t h and s t a t i o n a r y phase in mouse L ceils. Cells were established in E C I o in R o u x bottles (lO 7 cells/bottle) and the m e d i u m was changed after 3 days a n d after 5 days. The cells were i n c u b a t e d with L-[Me-3H]methionine (ioo/~C) and [32P] orthop h o s p h a t e (5 ° #C) for 6-h periods on successive d a y s and the D N A extracted and purified as described in MATERIALS AND METHODS. The results are expressed as lO -3 × c o u n t s / m i n per u n i t of a b s o r b a n c e (260 rim); O , sip; O , 3H.
Biochim. Biophys. Acta, 254 (1971) 2o5-212
DNA METHYLATIONin
vivo
2o 9
do not start to make DNA for at least 12 h. The growth period before DNA synthesis starts (referred to as the first Gl-phase) can be extended by incubation in the presence of aminopterin. No DNA methylation occurs during this lag phase (see Fig. 4). A more suitable system for studying DNA methylation in the absence of DNA synthesis is the phytohaemagglutinin stimulated lymphocyte. Following stimulation horse lymphocytes show a lag period of from one to two days before initiation of DNA synthesis 1°. During this time there are marked changes in RNA and protein metabolism yet there is no incorporation of radioactivity from L-EMe-3H~methionine into the 5-methylcytosine of DNA (Fig. 3)specific activity
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Fig. 3. L a c k of m e t h y l a t i o n of t h e I ) N A of p h y t o h a e m a g g l u t i n i n ( P H A ) s t i m u l a t e d h o r s e l y m p h o c y t e s in t h e period prior to D N A s y n t h e s i s . Purified h o r s e l y m p h o c y t e s were s t i m u l a t e d b y a d d i t i o n of p h y t o h a e m a g g l u t i n i n a t zero time. T h e y were i n c u b a t e d w i t h ~32P]orthophosphate (ioo pC) a n d L - [ M e - S H ] m e t h i o n i n e (ioo/~C) for 16 h periods e n d i n g a t t h e t i m e s s h o w n . T h e zero t i m e figure w a s o b t a i n e d u s i n g u n s t i m u l a t e d cells. D N A was e x t r a c t e d a n d purified as described in MATERIALS AND METHODS. T h e r e s u l t s are e x p r e s s e d as io -2 × c o u n t s / m i n per u n i t of a b s o r b a n c e (26o n m ) : 0 , 82p; O,SH.
To determine the lag period between DNA synthesis and methylation mouse L cells subcultured from stationary phase were synehronised by treatment with aminopterin for 16 h 5. As mentioned above there is no methylation of DNA during the first GI-phase. Synthesis and methylation of DNA occur when the aminopterin block is reversed by addition of thymidine when more than 9 ° 5/o of the cells enter S phase together (Fig. 4). DNA synthesis is normally complete within 6-8 h, but to remove any complication arising from a second wave of DNA synthesis the aminopterin block can Biochim. Biophys. Acta, 254 (1971) 2 o 5 - 2 1 2
210
R.L.P.
ADAMS
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Time (h) after thymtdine addition Fig. 4. Time course of m e t h y l a t i o n of D N A t h r o u g h o u t S phase. S t a t i o n a r y cells were subcult u r e d into E C I o containing o n e - t e n t h the n o r m a l methionine concentration and 20 mM sodium formate. After 6 h of g r o w t h a m i n o p t e r i n , adenosine, glycine and deoxycytidine were added as described in MATERIALS AND METHODS along w i t h L-EMe-SH]methionine (IOOttC) and [82p]_ o r t h o p h o s p h a t e (2o/~C). After 16 h the block in D N A synthesis was reversed b y addition of t h y m i d i n e . Bottles were h a r v e s t e d at the times shown. The a m i n o p t e r i n block was reimposed after 8 h b y replacing the m e d i u m w i t h fresh radioactive m e d i u m containing a m i n o p t e r i n b u t no thymidine. D N A was extracted f r o m the cells a n d purified as described in MATERIALS AND 3IETHODS. The results are expressed as a precentage of the 24-h figure: O, s~P; O , 3H.
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Fig. 5. Lag in Inethylation of DINA m a d e at different periods of S phase. ~ t a t i o n a r y cells were s u b c u l t u r e d into E C I o containing o n e - t e n t h the n o r m a l m e t h i o n i n e concentration and 2o mM sodium formate. After 6 h of g r o w t h aminopterin, adenosine, glycine and deoxycytidine were added as described in MATERIALS ASD METHODS along w i t h L-[Me-3Hlmethionine (ioo/~C) and E3~P]orthophosphate (2o #C). After 16 h t h e block in D N A synthesis was reversed b y addition <)f t h y m i d i n e (2 #M). Cells were i n c u b a t e d for I - h periods in the presence of 5-bromodeoxyuridine b y r e m o v i n g the initial m e d i u m and replacing w i t h identical m e d i u m b u t containing 5-bromodeoxyuridine (20/~M) instead of thymidine. This i n c u b a t i o n was carried out from o - i h, ( 0 ) ; 2-3 h ( O ) ; or 4-5 h, ( A ) . At the end of the h o u r in 5-bromodeoxyuridine some bottles were h a r v e s t e d a n d the r e m a i n d e r h a d t h e b r o m o d e o x y u r i d i n e m e d i u m replaced w i t h the initial m e d i u m n o w c o n t a i n i n g ioo FeM thymidine. Cells were h a r v e s t e d at the times shown. The 24th p o i n t w a s obt a i n e d w i t h o u t reimposing the a m i n o p t e r i n block. DlqA was extracted from the cells and purified as described in MATERIALS AND METHODS. I t WaS subjected to centrifugation to equilibrium in CsCl and the figure gives the ratio of SH to 3~p radioactivity in the h e a v y 5 - b r o m o d e o x y u r i d i n e peak.
I)NA
METHYLATION i~ vivo
2II
be reimposed after 8 h. Although this effectively stops DNA synthesis methylation of DNA continues and the incorporation into 5-methylcytosine rises b y 15 % within the next 16 h. The DNA made at different points in S phase was investigated by incubating synchronised cells with 5-bromodeox)mridine for different I h intervals. At various times after returning the cells to growth on thymidine the heavy DNA was isolated on gradients of alkaline CsC1. Fig. 5 shows that the DNA made in the first hour of S phase (5 % of the total DNA) is more extensively methylated than that made later in S phase. Moreover methylation of this 'early DNA' is complete very soon after synthesis (the extent of methylation rises by less than 5 % on continued incubation). This is in contrast to DNA made between 2 h and 3 h of S phase (24 % of the total DNA) and particularly to DNA made between 4 h and 5 h of S phase (13 % of the total DNA). The extent of immediate methylation of this latter is less than half that of 'early DNA' and methylation continues for several hours after synthesis is complete.
DISCUSSION
The function of methyl groups in 5-methylcytosine in DNA is unknown but the lack of DNA methylation at all times except during and soon after DNA synthesis does not support a role of methylation on the determination of which genes are to be transcribed. In particular the DNA of the phytohaemagglutinin stimulated horse lymphocyte is not methylated at a time when cellular metabolism is undergoing dramatic changes in the absence of DNA syalthesis. We cannot rule out the possibility however, that demethylation is occurring at this time, although we have demonstrated the stability of 5-methylcytosine in mouse L cell DNA 1. LARK11 has shown that bacteria deprived of methionine can complete one round of DNA replication to produced methyl deficient DNA, but this DNA cannot act as a template for further DNA synthesis. Similar experiments were tried with mouse L cells but total deprivation of methionine rapidly lead to cell death. However, the results in Fig. 5 suggest that there is no immediate function for the methyl groups in DNA with the possible exception of those added to the DNA made at the beginning of S phase. The significance of the observation that the extent of methylation varies over S phase is unclear. It has, however, been established 14 that the DNA made at a particular time in S phase is a specific portion of the DNA which, in subsequent S phases will always replicate at the same time. An integral part of the mechanism of DNA synthesis may be the nicking of newly synthesised DNA and the subsequent rejoining of the pieces using polynucleotide ligase. Subsequent methylation of the DNA may protect it from endonucleolytic cleavage and a parallel situation has been observed in bacteriophage DNA 12. In Lilium 13 the very newly synthesised DNA, i.e. that which can be recovered from the interphase on phenol extraction, is not methylated. This appears to exclude a role of methylation in DNA synthesis per se and to indicate a function in the stages immediately following release of DNA from the membrane. Biochim. Biophys. Acla, 254 (1971) 2o5-2Iz
212
R. L. P. ADAMS
ACKNOWLEDGEMENTS
Thanks are due to Professor J. N. DAVIDSON, F. R. S. and to Professor R. lV[. S. Smellie for their interest and for providing the necessary facilities with the aid of grants from the Cancer Research Campaign and the Welcome Research Foundation. Thanks are also due to D. P. Donnelly for skilled technical assistance. REFERENCES i 2 3 4 5 6 7 8 9 lO II 12 13 14
R. H. ]3URDON AND R. L. P. ADAMS, Biochim. Biophys. Acta, 174 (1969) 322. R. H. BURDON, Biochim. Biophys. Acta, 232 (1971) 359. D. BILLEN, J. Mol. Biol. 31 (1968) 477. H. H. EVANS AND T. E. EVANS, J. Biol. Chem., 245 (197 o) 6436. R. L. I ). ADAMS, Expt. Cell Res., 56 ti969) 55. E. WINOCOUR, A. M. KAYE AND V. STOLLAR, Virology, 27 ti965) 156. K. S. KIRBY, Progr. Nucleic Acad Res. Mol. Biol., 3 (1964) I. H. •. MUNRO AND A. FLECK, in D. GLICK, Methods o/ Biochemical Analysis, Vol. 14, New York, Interscience, 1966, p. 113. J. DOSKOCIL AND Z. SORMOVA,Biochim. Biophys. Aeta, 95 (1965) 513 . ]~. S. ZAIN, R. IMRIE AND R. L. t 3. ADAMS, results to be published. C. LARK, J. Mol. Biol., 31 (1968) 4Ol. S. LINN AND W'. ARBER, Proc. Natl. Acad. Sci. U.S., 59 11968) ~3 oo. Y. HOTTA AND N. HECHT, Biochim. Biophys. Acta, 238 (1971) 5 o. C. C. ~,{UELLER AND K. KAJIWARA, Biochim. Biophys. Acta, 114 (1966) lO8
Biochim. Biophys. Hcta, 254 (1971) 2o5-212
205
BBA 97083
THE RELATIONSHIP BETWEEN DNA I N MOUSE F I B R O B L A S T S
S Y N T H E S I S AND lV[ETHYLATION OF
R. L. P. ADAMS
Department o] Biochemistry, University o/Glasgow, Glasgow (Great Britain) (Received J u l y Ist, 1971)
SUMMARY
Studies on the in vivo methylation of DNA in cultured mouse L cells confirm the presence of a distinct lag between synthesis of DNA and its methylation. This lag is most pronounced for the DNA made in late S phase and the DNA is methylated to only 5 ° % the extent of that DNA made in early S phase. Methylation of DNA does not occur in stationary L cell cultures nor prior to DNA synthesis when such cultures are stimulated to grow. Neither does methylation occur on the DNA of phytohaemagglutinin stimulated horse lymphocytes prior to DNA synthesis.
INTRODUCTION
Only a single radioactive base, namely 5-methylcytosine, has been found in DNA from mouse L929 cells following short periods of incubation in culture medium containing L-EMe-ZHlmethionine 1. DNA methylase activity, which results in the transfer of methyl groups from S-adenosyl-L-methionine to 5-methylcytosine has been found in the chromatin fraction of mouse ascites tumour cell nuclei 2. Although BILLEN8 demonstrated that in Escherichia coli methylation of DNA normally occurs only on nascent DNA we have b~en unable to inhibit DNA methylation b y more than 70 % in mouse L cells when synthesis of DNA was inhibited b y treatment with hydroxyurea to levels less than 6 % of the control x. Preincubation with the drug for 2 h did reduce methylation to 15 % of control values. In chemically synchronised mouse L cells the rate of DNA methylation is maximal in mid S phase. However the rate of methylation of DNA does not fall in G~ to the levels expected if only nascent DNA were being methylated 1. 1V[ethylation of DNA in G2 cells has also been reported in Physarum polycephalum 4. These results have been confirmed and elaborated to show that the DNA made in early S phase is rapidly and highly methylated, but that DNA made in late S phase continues to be methylated for several hours after synthesis is complete. The evidence does not support a role for DNA methylation in the control of I ranscription. MATERIALS AND METHODS
Materials L-EMe-3H]lV[ethionine (7.6 C/mmole) and [~Plorthophosphate were purchased from the Radiochemical Centre, Amersham, Bucks., England. Biochim. Biophys. Acta, 254 (I97 I) 2o5-212
206
R.L.P.
ADAMS
Aminopterin, L-methionine, 2'-deoxycytidine and adenine were obtained from Sigma London Chemical Co. Ltd., London, S.W. 6; glycine, sodium dodecyl sulphate, sodium p-amlnosalicylate, and 8-hydroxyquinoline from British Drug Houses Ltd., Poole, Dorset; 5-bromodeoxyuridine, thymine and 5-methylcytosine from Calbiochem Ltd., London, W.I. and hydroxyurea from Nutritional Biochenlicals Corporation, Cleveland, Ohio, U.S.A. The caesium chloride used was tile Analar grade sold b y Hopkin and Williams Ltd., Chadwell Heath, Essex, England.
Cell culture Mouse fibroblasts (L929) cell cultures were routinely subcultured in minimum essential medimn (Eagle) supplemented with IO % (v/v) calf serum (ECIo). Cultures and media were obtained from Flow Laboratories Ltd., Irvine, U.K. or from BioCult Laboratories, Glasgow, U.K. Cells were grown to stationary phase in Roux bottles as described previously a. Stationary cells were subcultured and synchronised b y treatment with aminopterin (0.2 #M), adenosine (56/~lV[), glycine (80/~l~I) and deoxycytidine (20/~M) from 6-22 h after subculture. The block in DNA synthesis was reversed b y addition of thymidine (or 5-bromodeoxyuridine) at the concentrations cited in the text. When cells were to be labelled for short periods with L-[Me-3H~methionine this was carried out in EC10 containing one-tenth the normal methionine concentration and supplemented with 20 mM sodium formate to prevent equilibration of lab311ed methyl groups with the CI unit pool 6. Lymphocytes were purified from horse blood b y fractionation on a density gradient (B. S. gAIN et al.l°). They were grown in minimum essential medium (Eagle) supplemented with IO °/o (v/v) autologous horse plasma and stimulated with phytohemagglutinin M (Difco Laboratories, Detroit, Mich., U.S.A.). DNA preparation and CsCl centri[ugation DNA was prepared b y the method of KIRBY7. After radioactive labelling cell cultures were washed twice with Earle's balanced salt solution (minus glucose) and then, if necessary, scraped from the glass with a rubber spatala. Following sedimentation the pellet (about lO T cells) was resuspended in 2 ml of the same salt solution containing 6 °/o sodium p-aminosalicylate and added to 2 ml of 2 °/o sodium dodecyl sulphate, 4 mM versene at 7 °0 and maintained at 7 °0 with occasional shaking for 30 rain. After returning to room temperature 4 ml of a mixture of 88 °/o phenol, 12 °/o m-cresol, o.I ~o 8-hydroxyquinoline was added and the tube shaken for 30 rain before centrifugation for 20 min at IO ° and IO ooo rev./min in an MSE 18 centrifuge. The upper layer was removed and the DNA spooled from it after overlayering with 2 vol. cold ethanol. The DNA was dissolved in o.15 M KC1 0.02 M tris HC1 p H 7.5 and respooled. For equiliblium centrifugation the DNA was dissolved in CsC1 solution density 1.815 g/ml containing o.I l~I NaOH. Centrifugation was in the SW 65 rotor of the Spinco model L2 65 B ultracentrifuge at 30 ooo rev./min at 20 ° for 4 ° h. The tubes were punctured at the bottom and two drop fractions collected. These were diluted with I ml 0.3 M N a O H and the optical density profile was measured. Samples (either before or after centrifugation) were incubated at 37 ° in 0.3 M N a O H for 2 h to degrade any RNA which m a y remain 8. Bovine selum albumin (i mg Fraction V, Armour Pharmaceuticals, Eastbourne) was then added and DNA and Biochim. Biophys. Acta, 254 (1971) 2o5-212
DNA METHYLATIONin vivo
207
protein precipitated b y adjusting to 5 % (w/v) trichloroacetic acid. The precipitate was washed twice with 5-ml portions 5 % trichloroacetic acid and finally the DNA was extracted into I ml 0.5 M perchloric acid b y incubating for 30 rain at 7 o°. The radioactivity in 0.5 ml of the supernatant fluid was estimated in IO ml dioxane containing 0. 7 % 2,5-diphenyloxazole plus IO % naphthalene. To check the location of the radioactivity some of the perchloric acid extract was evaporated to give an approximately 12 M solution. The DNA was then hydrolysed to the bases b y incubation at IOO° for 60 min. The hydrolysate was chromatographed using the solvent b u t a n o l - w a t e r - a m m o n i a (87 : 13 : I, b y vol.) ~ as previously described ~. Under these conditions 95-99 % of the tritium radioactivity co-chromatographed with 5-methylcytosine and all the a2p radioactivity remained at the origin P
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16 24 32 Tube number Fig. I. Methylation of newly synthesised and older DNA. Mouse L cells {io ~ cells) were grown in ]~oux bottles in ECIo. I n order to increase the incorporation of tritiated methionine the m e d i u m was replaced w i t h 50 ml w a r m E C I o containing only o n e - t e n t h the n o r m a l methionine concent r a t i o n a n d s u p p l e m e n t e d w i t h 2o rrul~I s o d i u m formate. After three h o u r s incubation in this m e d i u m the following were added: a m i n o p t e r i n (o.2 ~M), adenosine I56/~M), glycine (8o ~M), deoxycytidine (2o /~M), 5-bromodeoxyuridine (2o /~M), [32P]o~hophosphate (Soo ~C) and L-E3//e-3I-I]methionine (5o0/~C). 3 h after addition of r a d i o a c t i v i t y the cells were h a r v e s t e d a n d t h e D N A e x t r a c t e d and centrifuged to equilibrium on gradients of alkaline CsCl, as described in MATI~RIALSAND METHODS; O, 82p)< i o - l ; (~, 3H counts/ruin. The c o n t i n u o u s lines r e p r e s e n t the +3260 nm and t h e d e n s i t y of the gradient.
Biochim. Biophys. Acta, 254 (1971) 2o5-212
20~
R.L.P.
AI):kMS
RESULTS
When mouse L cells are grown in the presence of aminopterin and 5-brom~deoxyuridine the newly synthesised daughter strand can be separated from the remainder of the DNA b y centrifugation to equilibrium in alkaline CsC1. If L-FMe-'~Hq methionine is present during the incubation then tlitium is associated with both the newly synthesised, heavy DNA and the older, light fraction (Fig. I). During a 3 h incubation 17 °/o of the methylation occurs on light DNA and 83 o~ on newly ,~\'nthesised DNA. If the production of heavy DNA is allowed to proceed for 3 h and then methylation studied in the subsequent 3 h period in the presence of hydroxyurea (2 raM) to inhibit synthesis of DNA then 30 % of methylation now occurs on light DNA (i.e. DNA more than 3 h old) and 7 ° O~/ooccurs on DNA made in the previous 3 h. This raises the possibility that methylation of new and older DNA represent two distinct processes and it was considered that the latter m a y be related to cellular differentiation. Mouse L cells will enter a stationary phase after growth for several days on a restricted area under conditions of frequent medium replenishment 5. However the rate of DNA methylation falls in an approximately parallel fashion to the rate of DNA synthesis (Fig. 2). When such stationary cells are subcultured they
specific
activity 3H 32p
14 - 70 F
L I I-
7
o
35
[
I
1
2
I I 3 4 Days after subculture
I
I
I
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6
7
Fig. 2. Methylation of D~IA d u r i n g g r o w t h and s t a t i o n a r y phase in mouse L ceils. Cells were established in E C I o in R o u x bottles (lO 7 cells/bottle) and the m e d i u m was changed after 3 days a n d after 5 days. The cells were i n c u b a t e d with L-[Me-3H]methionine (ioo/~C) and [32P] orthop h o s p h a t e (5 ° #C) for 6-h periods on successive d a y s and the D N A extracted and purified as described in MATERIALS AND METHODS. The results are expressed as lO -3 × c o u n t s / m i n per u n i t of a b s o r b a n c e (260 rim); O , sip; O , 3H.
Biochim. Biophys. Acta, 254 (1971) 2o5-212
DNA METHYLATIONin
vivo
2o 9
do not start to make DNA for at least 12 h. The growth period before DNA synthesis starts (referred to as the first Gl-phase) can be extended by incubation in the presence of aminopterin. No DNA methylation occurs during this lag phase (see Fig. 4). A more suitable system for studying DNA methylation in the absence of DNA synthesis is the phytohaemagglutinin stimulated lymphocyte. Following stimulation horse lymphocytes show a lag period of from one to two days before initiation of DNA synthesis 1°. During this time there are marked changes in RNA and protein metabolism yet there is no incorporation of radioactivity from L-EMe-3H~methionine into the 5-methylcytosine of DNA (Fig. 3)specific activity
16
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./o
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/
/ /
b ~ _ ~
_ _
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60
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Time (h) a/ter P H A addLticm
Fig. 3. L a c k of m e t h y l a t i o n of t h e I ) N A of p h y t o h a e m a g g l u t i n i n ( P H A ) s t i m u l a t e d h o r s e l y m p h o c y t e s in t h e period prior to D N A s y n t h e s i s . Purified h o r s e l y m p h o c y t e s were s t i m u l a t e d b y a d d i t i o n of p h y t o h a e m a g g l u t i n i n a t zero time. T h e y were i n c u b a t e d w i t h ~32P]orthophosphate (ioo pC) a n d L - [ M e - S H ] m e t h i o n i n e (ioo/~C) for 16 h periods e n d i n g a t t h e t i m e s s h o w n . T h e zero t i m e figure w a s o b t a i n e d u s i n g u n s t i m u l a t e d cells. D N A was e x t r a c t e d a n d purified as described in MATERIALS AND METHODS. T h e r e s u l t s are e x p r e s s e d as io -2 × c o u n t s / m i n per u n i t of a b s o r b a n c e (26o n m ) : 0 , 82p; O,SH.
To determine the lag period between DNA synthesis and methylation mouse L cells subcultured from stationary phase were synehronised by treatment with aminopterin for 16 h 5. As mentioned above there is no methylation of DNA during the first GI-phase. Synthesis and methylation of DNA occur when the aminopterin block is reversed by addition of thymidine when more than 9 ° 5/o of the cells enter S phase together (Fig. 4). DNA synthesis is normally complete within 6-8 h, but to remove any complication arising from a second wave of DNA synthesis the aminopterin block can Biochim. Biophys. Acta, 254 (1971) 2 o 5 - 2 1 2
210
R.L.P.
ADAMS
aminopter i/~ block reimposed 100 i
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8O
60 24h
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40
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Time (h) after thymtdine addition Fig. 4. Time course of m e t h y l a t i o n of D N A t h r o u g h o u t S phase. S t a t i o n a r y cells were subcult u r e d into E C I o containing o n e - t e n t h the n o r m a l methionine concentration and 20 mM sodium formate. After 6 h of g r o w t h a m i n o p t e r i n , adenosine, glycine and deoxycytidine were added as described in MATERIALS AND METHODS along w i t h L-EMe-SH]methionine (IOOttC) and [82p]_ o r t h o p h o s p h a t e (2o/~C). After 16 h the block in D N A synthesis was reversed b y addition of t h y m i d i n e . Bottles were h a r v e s t e d at the times shown. The a m i n o p t e r i n block was reimposed after 8 h b y replacing the m e d i u m w i t h fresh radioactive m e d i u m containing a m i n o p t e r i n b u t no thymidine. D N A was extracted f r o m the cells a n d purified as described in MATERIALS AND 3IETHODS. The results are expressed as a precentage of the 24-h figure: O, s~P; O , 3H.
-O
I
0
I
l
12 18 6 Time (11)~ter reversal of aminopterin block
l
24
Fig. 5. Lag in Inethylation of DINA m a d e at different periods of S phase. ~ t a t i o n a r y cells were s u b c u l t u r e d into E C I o containing o n e - t e n t h the n o r m a l m e t h i o n i n e concentration and 2o mM sodium formate. After 6 h of g r o w t h aminopterin, adenosine, glycine and deoxycytidine were added as described in MATERIALS ASD METHODS along w i t h L-[Me-3Hlmethionine (ioo/~C) and E3~P]orthophosphate (2o #C). After 16 h t h e block in D N A synthesis was reversed b y addition <)f t h y m i d i n e (2 #M). Cells were i n c u b a t e d for I - h periods in the presence of 5-bromodeoxyuridine b y r e m o v i n g the initial m e d i u m and replacing w i t h identical m e d i u m b u t containing 5-bromodeoxyuridine (20/~M) instead of thymidine. This i n c u b a t i o n was carried out from o - i h, ( 0 ) ; 2-3 h ( O ) ; or 4-5 h, ( A ) . At the end of the h o u r in 5-bromodeoxyuridine some bottles were h a r v e s t e d a n d the r e m a i n d e r h a d t h e b r o m o d e o x y u r i d i n e m e d i u m replaced w i t h the initial m e d i u m n o w c o n t a i n i n g ioo FeM thymidine. Cells were h a r v e s t e d at the times shown. The 24th p o i n t w a s obt a i n e d w i t h o u t reimposing the a m i n o p t e r i n block. DlqA was extracted from the cells and purified as described in MATERIALS AND METHODS. I t WaS subjected to centrifugation to equilibrium in CsCl and the figure gives the ratio of SH to 3~p radioactivity in the h e a v y 5 - b r o m o d e o x y u r i d i n e peak.
I)NA
METHYLATION i~ vivo
2II
be reimposed after 8 h. Although this effectively stops DNA synthesis methylation of DNA continues and the incorporation into 5-methylcytosine rises b y 15 % within the next 16 h. The DNA made at different points in S phase was investigated by incubating synchronised cells with 5-bromodeox)mridine for different I h intervals. At various times after returning the cells to growth on thymidine the heavy DNA was isolated on gradients of alkaline CsC1. Fig. 5 shows that the DNA made in the first hour of S phase (5 % of the total DNA) is more extensively methylated than that made later in S phase. Moreover methylation of this 'early DNA' is complete very soon after synthesis (the extent of methylation rises by less than 5 % on continued incubation). This is in contrast to DNA made between 2 h and 3 h of S phase (24 % of the total DNA) and particularly to DNA made between 4 h and 5 h of S phase (13 % of the total DNA). The extent of immediate methylation of this latter is less than half that of 'early DNA' and methylation continues for several hours after synthesis is complete.
DISCUSSION
The function of methyl groups in 5-methylcytosine in DNA is unknown but the lack of DNA methylation at all times except during and soon after DNA synthesis does not support a role of methylation on the determination of which genes are to be transcribed. In particular the DNA of the phytohaemagglutinin stimulated horse lymphocyte is not methylated at a time when cellular metabolism is undergoing dramatic changes in the absence of DNA syalthesis. We cannot rule out the possibility however, that demethylation is occurring at this time, although we have demonstrated the stability of 5-methylcytosine in mouse L cell DNA 1. LARK11 has shown that bacteria deprived of methionine can complete one round of DNA replication to produced methyl deficient DNA, but this DNA cannot act as a template for further DNA synthesis. Similar experiments were tried with mouse L cells but total deprivation of methionine rapidly lead to cell death. However, the results in Fig. 5 suggest that there is no immediate function for the methyl groups in DNA with the possible exception of those added to the DNA made at the beginning of S phase. The significance of the observation that the extent of methylation varies over S phase is unclear. It has, however, been established 14 that the DNA made at a particular time in S phase is a specific portion of the DNA which, in subsequent S phases will always replicate at the same time. An integral part of the mechanism of DNA synthesis may be the nicking of newly synthesised DNA and the subsequent rejoining of the pieces using polynucleotide ligase. Subsequent methylation of the DNA may protect it from endonucleolytic cleavage and a parallel situation has been observed in bacteriophage DNA 12. In Lilium 13 the very newly synthesised DNA, i.e. that which can be recovered from the interphase on phenol extraction, is not methylated. This appears to exclude a role of methylation in DNA synthesis per se and to indicate a function in the stages immediately following release of DNA from the membrane. Biochim. Biophys. Acla, 254 (1971) 2o5-2Iz
212
R. L. P. ADAMS
ACKNOWLEDGEMENTS
Thanks are due to Professor J. N. DAVIDSON, F. R. S. and to Professor R. lV[. S. Smellie for their interest and for providing the necessary facilities with the aid of grants from the Cancer Research Campaign and the Welcome Research Foundation. Thanks are also due to D. P. Donnelly for skilled technical assistance. REFERENCES i 2 3 4 5 6 7 8 9 lO II 12 13 14
R. H. ]3URDON AND R. L. P. ADAMS, Biochim. Biophys. Acta, 174 (1969) 322. R. H. BURDON, Biochim. Biophys. Acta, 232 (1971) 359. D. BILLEN, J. Mol. Biol. 31 (1968) 477. H. H. EVANS AND T. E. EVANS, J. Biol. Chem., 245 (197 o) 6436. R. L. I ). ADAMS, Expt. Cell Res., 56 ti969) 55. E. WINOCOUR, A. M. KAYE AND V. STOLLAR, Virology, 27 ti965) 156. K. S. KIRBY, Progr. Nucleic Acad Res. Mol. Biol., 3 (1964) I. H. •. MUNRO AND A. FLECK, in D. GLICK, Methods o/ Biochemical Analysis, Vol. 14, New York, Interscience, 1966, p. 113. J. DOSKOCIL AND Z. SORMOVA,Biochim. Biophys. Aeta, 95 (1965) 513 . ]~. S. ZAIN, R. IMRIE AND R. L. t 3. ADAMS, results to be published. C. LARK, J. Mol. Biol., 31 (1968) 4Ol. S. LINN AND W'. ARBER, Proc. Natl. Acad. Sci. U.S., 59 11968) ~3 oo. Y. HOTTA AND N. HECHT, Biochim. Biophys. Acta, 238 (1971) 5 o. C. C. ~,{UELLER AND K. KAJIWARA, Biochim. Biophys. Acta, 114 (1966) lO8
Biochim. Biophys. Hcta, 254 (1971) 2o5-212