28
Biochirnica et Biophysica Acta, 563 ( 1 9 7 9 ) 2 8 - - 3 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99464
DISTRIBUTION PATTERN AND ENZYMIC HYPERMETHYLATION OF INVERTED REPETITIVE DNA SEQUENCES IN P815 MASTOCYTOMA CELLS *
D U S A N D R A H O V S K Y a, T H O M A S L.J. B O E H M a a n d WILLI K R E I S
b
a Zentrum der Biologischen Chemie der Universitb't Frankfurt am Main, D-6000 Frankfurt~Main , F.R.G. and b Memorial Sloan-Kettering Cancer Center, N e w York, N Y 10021 (U.S.A.) (Received October 9th, 1978)
Key words: Repetitive DATA; DNA rnethylation; (Mastocytoma cell)
Summary A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L- [Me-aH]methionine and [ 14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.
Introduction
During replication the role of enzymic methylation of DNA is still unknown. This reaction was shown to take place after the ligation of the shortest replication intermediates [1,2] and the only product of this reaction, 5-methylcytosine, was found by means of pyrimidine isoplith analysis in a large variety of nucleotide sequences [3--5]. The methyl group of 5-methylcytosine origi* D e d i c a t e d t o P r o f e s s o r A. Wacker on his 60th birthday.
29 nates from activated L-methionine, and enzymes catalyzing DNA methylation have been isolated from different mammalian tissues and cells [6--10]. The high fidelity by which some of these enzymes select the nucleotide sequencespecific methylation sites [11--13] may be compatible with the original suggestions of Borek and Srinivasan [14] that the enzymic methylation of nucleic acids may play a role in cell differentiation and that abberant process may be involved in oncogenesis. In the eucaryotic genome, certain DNA sequences are present in a large number of copies and after denaturation, they reassociate in a rate proportional to the degree of their repetitiveness [15]. The repetitive sequences can be divided into so-called 'ordinary' repetitive ones of type ABC ... ABC which reassociate with a bimolecular second-order kinetic (C/Co = 1/(1 + kC0t)) and the inverted or foldback sequences of the type ABC ... CBA. The last ones form a double-stranded structure within one polynucleotide chain and, hence, reassociate very fast with a unimolecular first-order kinetic (C/Co = e - k t ) . The inverted repetitive sequences have been found in a variety of higher organisms [16--20]; their biological function is, however, still unknown. We found that the inverted repetitive sequences were methylated significantly higher than other DNA sequence classes. Materials and Methods
Mouse P815 mastocytoma cells isolated from ascites fluid on the 7th day after intraperitoneal inoculation of about 106 cells in BD2F1 ([C57B1/6 × DBA/2]F~) w e r e washed three times in physiological saline and resuspended in McCoy's 5a medium supplemented with 20% fetal calf serum (1--2 • l 0 s cells per ml). The following precursors were added: 5 pCi [~4C]deoxycytidine (429 Ci/mol. The Radiochemical Centre, Amersham, U.K.) and 150 pCi L-[Me-3H]methionine (50 Ci/mmol, New England Nuclear, Dreieich, F.R.G.) per 100 ml medium. To prevent incorporation of the methyl groups from L-methionine via the 'one-carbon' pool, the medium was supplemented with 0.02M sodium formate [21--23]. Under these labeling conditions, all pyrimidine residues of the DNA became labeled with the 14C radioactivity of [14C]deoxycytidine, whereas the 3H radioactivity of L-[Me-3H]methionine was incorporated solely into 5-methylcytosine. A distribution pattern of these two radioactivities after chromatographic separation of DNA bases is presented in Fig. 1. After 48-h cultivation (2--3 cell divisions), DNA was isolated by a modification of the method of Marmur [24], including repeated RNAase and pronase treatments and sheared in a Branson (Branson Sonic Power, Dunbury, T., U.S.A.) sonifier (Model $125, power setting 4) for various times. The fragment size was analyzed by acrylamide gel electrophoresis (7% acrylamide, 0.35% bisacrylamide) in 1.2 g Tris, 5.7 g glycine/1 (pH 8.3). The electrophoresis was performed with 40 mA, 20 V/cm for 3--4 h. After the run gels were stained with 40 mg/1 toluidine blue and destained in distilled water. DNA length standards (Boehringer, Mannheim, F.R.G.) were used. After heat denaturation, the DNA was allowed to reassociate at 60°C for
30 5O
D > I-
5-Methylcytosine
4o
U 0
IQ "Q 30 la i.
Thymine
~_ 20
Cytosine
r" "~ 10
15
2O
25
30
Distance f r o m the start (cm)
Fig. 1. D i s t r i b u t i o n of r a d i o a c t i v i t y i n u n i q u e D N A s e q u e n c e s o f P S l 5 m u t o c y t o m a cells ] J b e l e d w i t h L - [ M e - 3 H ] m e t h i o n i n e a n d [ 1 4 C ] d e o x y c y t i d i n e in p r e s e n c e of 0 . 0 2 M s o d i u m f o r m a t e . The D N A o f these cells was i s o l a t e d b y t h e m e t h o d o f M a r m u r . T h e i n d i v i d u a l D N A f r a c t i o n s w e r e h y d r o l y z e d i n 96% f o r m i c a c i d a t 1 7 5 ° C for 9 0 r a i n and chrornatographed in i s o p r o p a n o l / H C l / H 2 0 ( 6 8 : 6 : 26, v/v) for 3 0 h. A b o u t 96% of 3H r a d i o a c t i v i t y was r e c o v e r e d as 5 - m e t h y l c y t o s i n e , a n d a b o u t 93% of t h e 14C r a d i o a c t i v i t y as t h y m i n e a n d c y t o s i n e r e s i d u e s , r e s p e c t i v e l y . T o t a l 3H r a d i o a c t i v i t y (o o) was 5 0 0 0 cprn, t o t a l 14C r a d i o a c t i v i t y ( , A) was 1 0 8 8 2 0 c p m .
periods of time corresponding to different Cot values ( t o o l ' s " 1-1) [15,25]. The reassociation was carried out in phosphate buffers of different concentrations and the Cot values were then corrected for a reassociation in 0.12 M sodium phosphate as suggested by Britten et al. [25]. Single-stranded and reassociated DNA structures were isolated by chromatography on hydroxyapatite (Bio-Gel, Bio Rad Laboratories, Miinchen, F.R.G.) at 60°C. The single-stranded structures were eluted with 0.18 M, and the double-stranded structures with 0.4 M sodium phosphate buffer (pH 6.8). For base analysis, DNA was hydrolyzed in the presence of 1 mg unlabeled carrier DNA in 96% formic acid at 170°C for 90 rain. Individual bases were separated by paper chromatography in isopropanol/HC1/H20 (68 : 6 : 26, v/v) on MN 214 paper (Machery and Nagel, Diiren, F.R.G.) for 30 h. The dried paper was cut into 1-cm strips, the radioactivity eluted with H20 and measured in dioxan-based scintillation cocktail. Treatment of DNA with single-strand specific nuclease S1 from Aspergillus oryzae (Type III, Sigma, Miinchen, F.R.G.) was carried out as follows: 5 units enzyme/pg DNA in 10 pl 0.01 M NaCl/0.03 M sodium acetate/0.003 M ZnSO4 (pH 4.6) were incubated at 37°C for 45 min. The reaction was terminated by buffering to pH 6.8 with 0.4 M sodium phosphate buffer. Carrier DNA was added and the sample was precipitated at 0°C with HC104 to a final concentration of 1.5 M. After centrifugation at 10 000 × g for 20 rain. The sample was washed twice with ice-cold 0.5 M HCIO4, the precipitate dissolved in 0.5 ml Soluene (Packard Instruments GmbH, Frankfurt, F.R.G.) and the radioactivity determined in a toluene-based scintillation fluid. To control whether the amount of enzyme was sufficient to digest all single-stranded DNA, 1 #g denatured unique DNA sequences of P815 cells was treated with 5 units nuclease S1 under the same conditions. No acid-precipitable material was found.
31 CsC1 gradient centrifugation was performed at neutral pH by the method of Flamm et al. [26] and the buoyant density was calculated [27]. Results
After the labeling of P815 cells with [14C]deoxycytidine and L-[Me-3H] methionine for two cell cycles, DNA was isolated and fractionated as following. When the sonicated DNA, with an average length of 500 nucleotides, was denatured and reassociated to a Cot value of 0.1, about 12% of the starting material reassociated and was retained on hydroxyapatite in 0.18 M sodium phosphate buffer (pH 6.8). This fraction was eluted with 0.4 M buffer, diluted and carried through the second step, which consisted of heat denaturation of 10 min, adjustment to 60°C and immediate chromatography on hydroxyapatite [25]. Taking the dilution and the time between denaturation and loading on the hydroxyapatite column into account, the Cot of the second step is below 0.001. About 33% of DNA which reassociated at Cot 0.1 was retained on the hydroxyapatite in the second step. This double-stranded DNA represents about 4% of the total genome. Since the rate of its reassociation is very fast, it is likely that these sequences represent the inverted repetitive ones of the type ABC ... CBA. Using this method, Dott et al. [17] isolated inverted repetitive sequences from a human genome and found that about 4.7% of the placental DNA represents inverted repetitive sequences. This correlates well with the amount of this DNA in P815 cells. The Sl
Biochirnica et Biophysica Acta, 563 ( 1 9 7 9 ) 2 8 - - 3 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99464
DISTRIBUTION PATTERN AND ENZYMIC HYPERMETHYLATION OF INVERTED REPETITIVE DNA SEQUENCES IN P815 MASTOCYTOMA CELLS *
D U S A N D R A H O V S K Y a, T H O M A S L.J. B O E H M a a n d WILLI K R E I S
b
a Zentrum der Biologischen Chemie der Universitb't Frankfurt am Main, D-6000 Frankfurt~Main , F.R.G. and b Memorial Sloan-Kettering Cancer Center, N e w York, N Y 10021 (U.S.A.) (Received October 9th, 1978)
Key words: Repetitive DATA; DNA rnethylation; (Mastocytoma cell)
Summary A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L- [Me-aH]methionine and [ 14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.
Introduction
During replication the role of enzymic methylation of DNA is still unknown. This reaction was shown to take place after the ligation of the shortest replication intermediates [1,2] and the only product of this reaction, 5-methylcytosine, was found by means of pyrimidine isoplith analysis in a large variety of nucleotide sequences [3--5]. The methyl group of 5-methylcytosine origi* D e d i c a t e d t o P r o f e s s o r A. Wacker on his 60th birthday.
29 nates from activated L-methionine, and enzymes catalyzing DNA methylation have been isolated from different mammalian tissues and cells [6--10]. The high fidelity by which some of these enzymes select the nucleotide sequencespecific methylation sites [11--13] may be compatible with the original suggestions of Borek and Srinivasan [14] that the enzymic methylation of nucleic acids may play a role in cell differentiation and that abberant process may be involved in oncogenesis. In the eucaryotic genome, certain DNA sequences are present in a large number of copies and after denaturation, they reassociate in a rate proportional to the degree of their repetitiveness [15]. The repetitive sequences can be divided into so-called 'ordinary' repetitive ones of type ABC ... ABC which reassociate with a bimolecular second-order kinetic (C/Co = 1/(1 + kC0t)) and the inverted or foldback sequences of the type ABC ... CBA. The last ones form a double-stranded structure within one polynucleotide chain and, hence, reassociate very fast with a unimolecular first-order kinetic (C/Co = e - k t ) . The inverted repetitive sequences have been found in a variety of higher organisms [16--20]; their biological function is, however, still unknown. We found that the inverted repetitive sequences were methylated significantly higher than other DNA sequence classes. Materials and Methods
Mouse P815 mastocytoma cells isolated from ascites fluid on the 7th day after intraperitoneal inoculation of about 106 cells in BD2F1 ([C57B1/6 × DBA/2]F~) w e r e washed three times in physiological saline and resuspended in McCoy's 5a medium supplemented with 20% fetal calf serum (1--2 • l 0 s cells per ml). The following precursors were added: 5 pCi [~4C]deoxycytidine (429 Ci/mol. The Radiochemical Centre, Amersham, U.K.) and 150 pCi L-[Me-3H]methionine (50 Ci/mmol, New England Nuclear, Dreieich, F.R.G.) per 100 ml medium. To prevent incorporation of the methyl groups from L-methionine via the 'one-carbon' pool, the medium was supplemented with 0.02M sodium formate [21--23]. Under these labeling conditions, all pyrimidine residues of the DNA became labeled with the 14C radioactivity of [14C]deoxycytidine, whereas the 3H radioactivity of L-[Me-3H]methionine was incorporated solely into 5-methylcytosine. A distribution pattern of these two radioactivities after chromatographic separation of DNA bases is presented in Fig. 1. After 48-h cultivation (2--3 cell divisions), DNA was isolated by a modification of the method of Marmur [24], including repeated RNAase and pronase treatments and sheared in a Branson (Branson Sonic Power, Dunbury, T., U.S.A.) sonifier (Model $125, power setting 4) for various times. The fragment size was analyzed by acrylamide gel electrophoresis (7% acrylamide, 0.35% bisacrylamide) in 1.2 g Tris, 5.7 g glycine/1 (pH 8.3). The electrophoresis was performed with 40 mA, 20 V/cm for 3--4 h. After the run gels were stained with 40 mg/1 toluidine blue and destained in distilled water. DNA length standards (Boehringer, Mannheim, F.R.G.) were used. After heat denaturation, the DNA was allowed to reassociate at 60°C for
30 5O
D > I-
5-Methylcytosine
4o
U 0
IQ "Q 30 la i.
Thymine
~_ 20
Cytosine
r" "~ 10
15
2O
25
30
Distance f r o m the start (cm)
Fig. 1. D i s t r i b u t i o n of r a d i o a c t i v i t y i n u n i q u e D N A s e q u e n c e s o f P S l 5 m u t o c y t o m a cells ] J b e l e d w i t h L - [ M e - 3 H ] m e t h i o n i n e a n d [ 1 4 C ] d e o x y c y t i d i n e in p r e s e n c e of 0 . 0 2 M s o d i u m f o r m a t e . The D N A o f these cells was i s o l a t e d b y t h e m e t h o d o f M a r m u r . T h e i n d i v i d u a l D N A f r a c t i o n s w e r e h y d r o l y z e d i n 96% f o r m i c a c i d a t 1 7 5 ° C for 9 0 r a i n and chrornatographed in i s o p r o p a n o l / H C l / H 2 0 ( 6 8 : 6 : 26, v/v) for 3 0 h. A b o u t 96% of 3H r a d i o a c t i v i t y was r e c o v e r e d as 5 - m e t h y l c y t o s i n e , a n d a b o u t 93% of t h e 14C r a d i o a c t i v i t y as t h y m i n e a n d c y t o s i n e r e s i d u e s , r e s p e c t i v e l y . T o t a l 3H r a d i o a c t i v i t y (o o) was 5 0 0 0 cprn, t o t a l 14C r a d i o a c t i v i t y ( , A) was 1 0 8 8 2 0 c p m .
periods of time corresponding to different Cot values ( t o o l ' s " 1-1) [15,25]. The reassociation was carried out in phosphate buffers of different concentrations and the Cot values were then corrected for a reassociation in 0.12 M sodium phosphate as suggested by Britten et al. [25]. Single-stranded and reassociated DNA structures were isolated by chromatography on hydroxyapatite (Bio-Gel, Bio Rad Laboratories, Miinchen, F.R.G.) at 60°C. The single-stranded structures were eluted with 0.18 M, and the double-stranded structures with 0.4 M sodium phosphate buffer (pH 6.8). For base analysis, DNA was hydrolyzed in the presence of 1 mg unlabeled carrier DNA in 96% formic acid at 170°C for 90 rain. Individual bases were separated by paper chromatography in isopropanol/HC1/H20 (68 : 6 : 26, v/v) on MN 214 paper (Machery and Nagel, Diiren, F.R.G.) for 30 h. The dried paper was cut into 1-cm strips, the radioactivity eluted with H20 and measured in dioxan-based scintillation cocktail. Treatment of DNA with single-strand specific nuclease S1 from Aspergillus oryzae (Type III, Sigma, Miinchen, F.R.G.) was carried out as follows: 5 units enzyme/pg DNA in 10 pl 0.01 M NaCl/0.03 M sodium acetate/0.003 M ZnSO4 (pH 4.6) were incubated at 37°C for 45 min. The reaction was terminated by buffering to pH 6.8 with 0.4 M sodium phosphate buffer. Carrier DNA was added and the sample was precipitated at 0°C with HC104 to a final concentration of 1.5 M. After centrifugation at 10 000 × g for 20 rain. The sample was washed twice with ice-cold 0.5 M HCIO4, the precipitate dissolved in 0.5 ml Soluene (Packard Instruments GmbH, Frankfurt, F.R.G.) and the radioactivity determined in a toluene-based scintillation fluid. To control whether the amount of enzyme was sufficient to digest all single-stranded DNA, 1 #g denatured unique DNA sequences of P815 cells was treated with 5 units nuclease S1 under the same conditions. No acid-precipitable material was found.
31 CsC1 gradient centrifugation was performed at neutral pH by the method of Flamm et al. [26] and the buoyant density was calculated [27]. Results
After the labeling of P815 cells with [14C]deoxycytidine and L-[Me-3H] methionine for two cell cycles, DNA was isolated and fractionated as following. When the sonicated DNA, with an average length of 500 nucleotides, was denatured and reassociated to a Cot value of 0.1, about 12% of the starting material reassociated and was retained on hydroxyapatite in 0.18 M sodium phosphate buffer (pH 6.8). This fraction was eluted with 0.4 M buffer, diluted and carried through the second step, which consisted of heat denaturation of 10 min, adjustment to 60°C and immediate chromatography on hydroxyapatite [25]. Taking the dilution and the time between denaturation and loading on the hydroxyapatite column into account, the Cot of the second step is below 0.001. About 33% of DNA which reassociated at Cot 0.1 was retained on the hydroxyapatite in the second step. This double-stranded DNA represents about 4% of the total genome. Since the rate of its reassociation is very fast, it is likely that these sequences represent the inverted repetitive ones of the type ABC ... CBA. Using this method, Dott et al. [17] isolated inverted repetitive sequences from a human genome and found that about 4.7% of the placental DNA represents inverted repetitive sequences. This correlates well with the amount of this DNA in P815 cells. The Sl
