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In vivo methylation of Escherichia coli DNA following ultraviolet and X-irradiation
J. Mol. Bid.
(1972) 63, 363-372
In viva Methylation of Escherichia coli DNA Ultraviolet and X-irradiation BRADFORD
L.
WHITnELDt
AND
DANIEL
following
BILLEN
Radiation Biology Laboratory ati Departmentsof Immunology and Medid Microbiology and Radiology, Collegeof Medicine University of Florida Gainesville, Flu., U.S.A. (Received4 May 1971, and in revisedform 17 September1971) E&e&Ku coli 15T(555-7) w&8 irradiated with ultraviolet light and X-rays and the DNA examined for quantitative and qualitative changes in the methylation pattern. DNA synthesized following ultraviolet irradiation was under-methylated by approximately 25% as compared to DNA from u&radiated cells. The ratio of 6-methyladenine to 5-methylcytosine was unchanged. DNA synthesized following X-irradiation was methylated about 15% more than normal with a slight increase in the 6-methyladenine to 5-methylcytosine
ratio. No additional methyl&ion or turnover of methyl groups could be detected in parental DNA present at the time of ultraviolet or X-irradiation. E. coli 15T- (555-7) synthesized “unmethylated DNA” by growth in the absence of methionine. Ultraviolet irradiation of cells containing unmethylated DNA did not quantitatively change the ability of the cells subsequently to methylate this DNA when methionine was added to the culture. The ratio of 6-methyladenine to 5-methylcytosine in both control and ultraviolet-irradiated cells changed from 1.8 -f 0.2 when examined 15 minutes after addition of methionine to 1.4 5 0.1, the normal value, when examined one hour after methionine was added.
1. Introduction The specific methylation of basesin DNA is a well-established phenomenon ocurring in virtually all organisms examined. It involves the enzymic transfer of the methyl group of X-adenosylmethionine to specific basesin DNA (Mandel & Borek, 1963; Srinivasan & Borek, 1964). This occurs at the polynucleotide level in bacteria either at or near the replication point (Gold & Hurwitz, 1964; Fleissner & Borek, 1962; Billen, 1968). The methylation pattern is a unique feature of DNA from different, speciesand is the basis of restriction-modification systemsin bacteria (for a current’ review see Arber & Linn, 1969). Methylation of cytosine on the 5 position followed. by a specific deamination may be involved in differentiation in developing seaurchin embryos (Grippo, Parisi, Carestia & Scarano, 1970) and has been suggestedto occur t Present
address:
Biology
Division,
Oak
Ridge
National
363
Laboratories,
OakRidge,
Term.,
U.S.A.
364
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L.
WHITFIELD
AND
D.
BILLEN
in Novikoff hepatoma cells (Sneider & Potter, 1969). Other possible functions of methylation have been proposed such as a punctuation system for transcription or a method of regulating the number of times a message is translated (Griffith & Mahler, 1969). Increased transfer RNA methylase activities have been reported in tumor cells (Bergquist & Matthews, 1962; Tsutsui, Srinivasan & Borek, 1966; Burdon, 1966). Radiation alters the pattern of DNA replication with respect to sequence (Billen, 1969). In this report we have examined the methylation of Escherichia coli DNA following ultraviolet and X-irradiation to determine whether radiation alters this aspect of DNA metabolism.
2. Materials
and Methods
(a) Bacteria
E. coli 15T-
(555-7) is a multiauxotroph methionine. Complete growth medium 2 pg thymine/ml., 14 pg tryptophan/ml., 2% glucose as a carbon source. (b)
Radiosotopic
used
requiring thymine, consists of minimal 38 pg arginine/ml.,
arginino, tryptophan salts (MS-7, Billen, 30 pg methionine/ml.
and 1959), and
and density labeling of DNA
Bulk labeling of DNA was accomplished by growth in the presence of 0.1 &!i/2pg per ml. [2- 14C]thymine. n-[methyZ-3H]methionine at specific activity of 5.0 #%/lo pg per ml. was used as a source of labeled methyl groups. When density labeling was required, thymine was replaced by 5-bromouracil (10 pg/ml.). (c) Equilibrium
density
analysis
Cells were harvested by centrifugation, washed in 0.1 M-Tris buffer (pH 8.1) and resuspended in 0.1 M-Tris-(ethylenedinitrilo) . tetra-acetic acid buffer (pH 8.1). Lysates were prepared by adding lysozyme (100 pg/ml.) to the cell suspensions, freezing in liquid nitrogen and incubating at 37°C for 10 min. Pronase (100 pg/ml.) was added and the samples incubated 15 mm at 37°C. Portions were mixed with CsCl to a density of l-710 g/ ml. and the DNA banded by centrifugation at 33,000 rev./min for 45 hr in an SW39 or SW65 rotor in a Spinco model L2-65 or L2-65B ultracentrifuge. After centrifugation, the bottoms of the centrifuge tubes were punctured and fractions collected by alternating 1 drop on Whatman no. 1 paper disks and 9 drops in tubes containing 2 drops TEP buffer (0.1 M Tris*HCl, 0.02 M-EDTA, 0.05 M-sodium pyrophosphate, pH 8.1). The disks were washed in cold 5% trichloracetic acid, rinsed in cold 95% ethanol, dried and counted in a model 3375 Tricarb liquid-scintillation counter. The fractions containing the desired DNA, either parental or newly synthesized, were pooled and centrifuged as before. Five-drop fractions were collected on paper disks and counted as described above. (d)
DNA
puri$cation
Cells were lysed as described above and the volume brought to 5 ml. by addition of Tris-EDTA buffer. DNA was purified by a modified Marmur procedure (Marmur, 1961). Samples were digested with pronase for 1 hr at 37°C and then shaken with cold phenol for 10 mm. The aqueous layer was removed and extracted several times with ether. 95% ethanol was added and the DNA spooled from the interface with a glass rod, resuspended in O-1 x SSC (0.015 Ma-Nacl, 0.0015 M-sodium citrate), and brought to 1 x SSC when the DNA had dissolved. Samples were then shaken with chloroform-isoamylalcohol (24:l v/v) for 10 min and the aqueous layer extracted with ether until the solution was clear. Pancreatic RNase A (100 pg/ml.) was added and the samples incubated for 1 hr at 37OC, brought to 0.1 N-NaOH and incubated 1 hr. Finally, tho DNA was precipitated with cold 10% trichloracetic acid, washed in ethanol and dried.
DNA
METHYLATION
FOLLOWING
(e) DNA
hydrolysis
365
IRRADIATION
and base analysis
The purified DNA plus 50 pg unlabeled E. coli carrier DNA was hydrolyzed to free bases with 70% perchloric acid at 100°C for 1 hr. The hydrolysate was neutralized with KOH and centrifuged to remove the precipited potassium perchlorate and charred sugars. The second and preferred method of hydrolysis was to treat the DNA with 90% formic acid for 30 min at 175°C in sealed Pyrex tubes (Wyatt & Cohen, 1953). The formic acid and volatile salts were removed by evaporation and the residue resuspended in 0.25 ml. 0.1 N-HCl. The clear sample was mixed with known bases and co-chromatographed on Whatman no. 1 paper in a descending direction using n-butyl alcohol-water-NH,OH (86 : 14 : 2 by vol.) as the solvent. The bases were located by U.V. absorption and the chromatogram then cut into l-cm strips and counted in a liquid-scintillation counter as described. Labeled methylated bases were those derived from the hydrolyzed DNA. The relative extent of methylation of the newly formed DNA was determined as the ratio of 14C-counts in thymine to 3H-counts in 6-MeAde plus 5-MeCyti. A given 14C-count represents the same amount of DNA in the control and irradiated cells, and comparison of the 14C/3H ratios in the two samples is a measure of the relative extent of methylation of the new strand. (f) Irradiation Cells were placed 200 to 600 ergs/mm2 cells were irradiated
on a cold shallow watch glass, stirred constantly and U.V. at 7.4 ergs/mm2 per set or 10,000 r X-irradiation. in non-supplemented minimal salts medium.
irradiated In all
with cases
3. Results (a) Lack of additional methylation DNA following
or turnover of methyl groups ultraviolet irradiation
in parental
An inoculum of exponentially growing E. coli 15T- (555-7) was added to 10 ml. of fully supplemented MS-7 medium containing [2-14C]thymine as a bulk label. At an optical density of 0.4 the cells were harvested on a Millipore filter and resuspended in 10 ml. ice-cold unsupplemented MS-7 medium. Half the sample was irradiated as described. The other half-sample was held in an ice water bath and served as the unirradiated control. Each samplewasthen added to 5 ml. of MS-7 containing the necessarysupplements with 5-bromouracil and [methyL3H]methionine. The cells were incubated at 37°C for 30 to 90 minutes and collected in centrifuge tubes containing 3 ml. frozen saline. After harvesting by centrifugation and washing in 0.1 M-Tris buffer, the cells were lysed and centrifuged as described in Materials and Methods. Parental DNA synthesized before irradiation bands at a density of 1.710 g/ml., as shown in Figure 1. The DNA banding at a density of 1.755 g/ml. is hybrid DNA containing one strand synthesized following irradiation and one parental strand. The high 3H-labeled background results from the distribution of methylated RNA through the gradient. Fractions under the peak at 1.710 g/ml. were pooled and the DNA rebanded to determine if any 3H-label was present in the unreplicated parental DNA. Fractions under the peak at 1.755 g/ml. were pooled and the DNA denatured and rebanded to determine if any 3H-label was present in the parental strand in hybrid DNA. No 3H-label was found in either the rebanded unreplicated parental DNA or in the rebanded parental strand from denatured hybrid DNA. We concluded that there t Abbreviations
used:
6-MeAde,
6-methyladenine;
5MeCyt,
Smethylcytosine
366
B.
L.
WHITFIELD
AND
Fraction
D.
BILLEN
no
FIG. 1. CsCl density profile of DNA from cells labeled with [2-r4C]thymine and [naethyZ-3H] methionine. The experimental procedures were as described in the text. The profile shown is of DNA from [14C]thymine-labeled cells exposed to 200 ergs/mm2 followed by 90 mm incubation in the presence of 5bromonracil and [methyZ-3H]methionine. The DNA banding at a density of 1.710 g/ml. is parental DNA synthesized before irradiation. The DNA banding at a density of 1.755 g/ml. is a hybrid containing one strand synthesized following irradiation and one parental strand. The 3H-label background is a result of methylation of RNA. The small 3H-label peak at a density of 1.798 g/ml, is the beginning of second rounds of DNA replication. -a-.--, W; ---A--A--, 3H. was no turnover of methyl groups or additional methylation of parental DNA following u.v. irradiation. The failure to find 3H-label in the parental DNA also indicated no new methylase activity as a result of defective prophage induction. Up to a 20-fold increase in 6-MeAde methylase activity has been reported in other derivatives of E. coli 15T- following prophage induction by a variety of treatments including U.V. irradiation (Dunn & Smith, 1958; Yudelevich & Gold, 1969).
(b) Fate of 3H-label from [methyl-3H]methionine When [methyZ-3H]methionine is used as a labeled methyl donor, the 3H-label can be incorporated into positions 2 and 8 of purine rings and into the methyl group of thymine through one-carbon metabolism as shown in Figure 2(a). The addition of sodium formate to the growth medium effectively eliminated the incorporation of label into the ring structure of adenine and guanine by diluting the radioactivity in the one-carbon pool (Kit, Beck, Graham & Gross, 1958) (see Fig. 2(b)). Label was still found in thymine. E. coli 15T- (555-7) is blocked in thymidylate synthetase and like most similar thymine-requiring strains, the block may not be complete (Barner & Cohen, 1954; Seno & Melechen, 1964). It was also possible that the label in thymine was due in part to an enzymic deamination of 5-MeCyt at the polymer level as reported by Grippo et al. (1970) in sea urchin embryos, or to a chemical deamination of 5-MeCyt during acid hydrolysis of DNA. An enzymic deamination of 5-MeCyt was eliminated by the following experiment. Methionine-requiring cells can synthesize unmethylated DNA by growth in the absence of methionine (Billen, 1968; Lark, 1968aJ). During amino acid starvation, the round of DNA replication in progress goes to completion and new rounds of replication are not initiated (Maalse & Hanawalt, 1961). When methionine is supplied,
DNA
METHYLATION
FOLLOWING
Distance FIG.
2. Incorporation
of 3H-label
from
IRRADIATION
367
from origin km1
[m&hyZ-3H]methionine
into
bases in DNA.
Plots are shown of the distribution of 3H-oounts on chromatograms of formic acid-hydrolyzed DNA from cells grown in the presence of [methyl-sH]methionine. Procedures are described in Materials and Methods. The samples chromatographed are: (a) hydrolyzed DNA from an exponential cell culture without sodium formate; and (b) with 20 mra-sodium formate. Abbreviations: guanine (Gus). 5-methylcytosine (5MeCyt), thymine (Thy), 6.methyladenine (6-MeAde) and adenine (Ade).
the DNA synthesized during the starvation period is methyl&ted. A log-phase culture of cells was transferred to medium lacking methionine, arginine s,nd tryptophan and incubated 90 minutes. The cells were then shifted to medium containing [mmethyL3H] methionine but lacking arginine, tryptophan and thymine and incubated 15 minutes. This permitted methylation of the unmethylated DNA while continuing the inhibition of DNA and protein synthesis. DNA from half the sample was analyzed as a control. Methyl&ion was 70% complete after 15 minutes, with 98% of the 3H-label in 6-MeAde and 5-MeCyt as shown in Figure 3(a). The remaining 2% of the label was in thymine. The other half-sample was harvested on a Millipore filter, washed with MS-7 salts and transferred to complete medium with ten times the normal concentration cd unlabeled methionine to dilute out any remaining labeled methionine in the pool. The cells were incubated one hour to permit deamination to occur. If an enzymic deamination of B-MeCyt occurred during this time, there should be an increase of 3H-label in thymine and a decrease in 5-MeCyt. There was no change in the distribu.tion of 3H-label its shown in Figure 3(b). It is, therefore, likely that the label appearing in thymine is a result of an incomplete block in thymidylate synthetase with a small amount of chemical deamination during hydrolysis. (c) Hype-methyl&ion of newly synthesized DNA following ultraviolet irradiation An exponential culture of E. coli at 3 x lo8 cells per ml. was harvested on a Millipore filter and resuspended in cold MS-7, divided and treated as in the previous experiment. Following 400 ergs/mm2 U.V. irradiation, each sample was added to complete medium containing [14C]thymine and [methyL3H]methionine with 20 mM-SOdhm formate and incubated 90 minutes. DNA was purified and hydrolyzed as described. As shown in Table 1, DNA synthesized after U.V. irradiation was under-methylated by 25% as compared to DNA from unirradiated cells. The ratio of 6-MeAde to 5-MeCyt remained unchanged at 1.4.
368
B.
L.
WHITFIELD
5
AND
IO
I5 20
Dstance
FIG.
3. Methyl&ion
of DNA
D.
BILLEN
25 30
from oqn
following
35
40
(cm)
methionine
starvation.
Following 90 min of methionine, arginine and tryptophan starvation the cells were shifted to medium containing [methyZ-3H]methionine, but lacking arginine, tryptophan and thymine, and incubated 15 min. (a) Is a plot of the distribution of 3H-counts on a chromcttogram of hydrolyzed DNA extracted immediately after the 15.min methyl&ion period. (b) The chromatogrem is of hydrolyzed DNA from cells that were incubated 60 min in complete medium with no labels after the 15-min methyl&ion period. Abbreviations are the s&me as in Fig. 2.
TABLE
Methylation
of DNA
synthesized Methyl&ion (%)
Sample
Control u.v. (400 ergs/mm2) X-rays (10,000 r)
1 after irradiation Ratio (6.MeAde/5-MeCyt)
100 75 115
1.4kO.l 1*4+0.1 1.5*0.1
The relative extent of methyl&ion w&s determined by dividing the amount of DNA synthesized (as determined by [l%]thymine counts) by the amount of methyl&ion (as determined by sH-counts in methylated bases). A comparison of the 14C/3H ratios in the irradiated samples to the ratio in the control gave the percentage methyl&ion. The ratio of 6-MeAde to 5-MeCyt was determined by the distribution of 3H-label on chrometograms of hydrolyzed DNA.
(d) Does pool dilution
contribute to hype-methyl&on following ultraviolet irradiation
of DNA
synthesized
?
This possibility was eliminated by first labeling cells with [methyL3H]methionine and determining if any label appeared in DNA subsequently synthesized. The cells were grown to an optical density of 0.4 in the presence of labeled methionine and [14C]thymine. They were harvested and irradiated with 400 ergs/mm2 as in previous experiments with post-irradiation incubation in fully supplemented MS-7 medium containing Ekbromouracil. Cells were lysed and DNA banded as before. Hybrid DNA was collected and denatured by heating for 10 minutes at 100°C followed by rapid cooling. The denatured DNA was rebanded in CsCl to separate the old and newly
DNA
METHYLATION
FOLLOWING
IRRADIATION
369
made DNA strands. No 3H-label was found in the newly made strand, and we concluded that pool dilution did not influence the observed methylation after U.V. irradiation. (e) Is hype-methyl&on
a result of recall of the chromosomul origin following ultraviolet irra4Sation ?
Following U.V. irradiation of bacteria, the DNA replication fork in progress either stops or is greatly slowed. When replication resumes, it starts from the genetic origin (Billen, 1969). If the first half of the chromosome normally contains fewer methylated bases than the terminal half, hypo-methylation could be expected in our experiments. To determine if this was the explanation, a log-phase culture of cells was deprived of arginine and tryptophan for 90 minutes to align the chromosome. One-half of the sample was added to complete medium containing [methyL3H]methionine and [14C]thymine and incubated 35 minutes, then transferred to complete medium with no radioisotopic label for 20 minutes more. The other half-sample was added to medium containing no radioisotopic label for the first 35 minutes, then transferred to medium containing [methyL3H]methionine and [14C]thymine for the last 20 minutes. This permitted us to examine DNA selectively labeled, on the average, in the first half and last half of the chromosome. There was no difference found in either extent of methylation or ratio of methylated bases in the two samples. Recall of the origin is unlikely to be responsible for the hype-methylation observed after U.V. irradiation. (f) Methyl&ion
of unmethylated DNA
after ultraviolet irradiation
Cells were starved for methionine and other required amino acids to permit the synthesis of unmethylated DNA as described in an earlier section. [14C]Thymine was present during starvation to measure DNA synthesis. The culture was divided and one half irradiated with 400 ergs/mm2 U.V. Each sample was allowed to methylate the unmethylated DNA by addition of [methyL3H]methionine in the absence of additional DNA or protein synthesis. There was no difference in the ability of irradiated and control cells subsequently to methylate the unmethylated DNA, indicating no detectable damage to the methylating system. When examined 15 minutes after re-addition of methionine, the ratio of 6-MeAde to 5-MeCyt in DNA was 1.8 & 0.2 in both control and irradiated cells. The ratio decreased to the log-phase value when methylation was allowed to continue one hour (see Table 2). The decrease primarily reflected an increase in 5-MeCyt content. TABLE
2
DNA methyl&ion followins methionine starvation Time after methionine addition (mb)
Relative methyl&ion (%)
10 30 60 The relative methylation described in Table 1.
percentages
Ratio (B-MeAde/5-MeCyt)
60 90 100 and
ratios
l+Jf0.2 1.5*0.1 1.4,tO.l of 6.MeAde
to 5-MeCyt
were
determined
aa
370
B.
L.
WHITFIELD
(g) Methyl&on
AND
D.
BILLEN
of DNA following X-irradiation
To determine the extent of methylation and ratio of methylated bases in DNA synthesized after X-irradiation, the experimental procedures were as described for the ultraviolet studies. After 10,000 r, the newly synthesized DNA was methylated approximately 15% more than control DNA, as shown in Table 1. The ratio of 6-MeAde t,o 5-MeCyt was slightly increased. Parental DNA did not undergo additional methylation after X-irradiation.
4. Discussion It is apparent that when methylation occurs on DNA being synthesized from an ultraviolet damaged template, the newly made DNA does not contain the full complement of methylated bases. Hypo-methylation is not the only abnormality of this DNA. It has been shown in E. coli K12 (Rupp & Howard-Flanders, 1968; Smith & Meun, 1970) and E. coli B/r (Billen & Carreira, 1971) that the DNA synthesized following U.V. irradiation exists in smaller pieces than normal for a time after synthesis. In Her+ cells the small pieces become larger with time; however, in Hercells the DNA remains in small pieces. Billen & Bruns (1970) have reported that the DNA synthesized after U.V. irradiation is less likely to be replicated in each succeeding replication cycle than normal DNA. Finally, the DNA replication fork present at the time of U.V. irradiation is known to be slowed or stopped, with most new synthesis occurring at a new replication fork starting at the replicative origin (Billen, 1969). The possibility that the DNA synthesized after U.V. irradiation is viral or episomal in origin rather than cellular, is eliminated by earlier published studies with E. coli 15T- (555-7). Hewitt & Billen (1965) using density-shift techniques showed that post-irradiation DNA synthesis after U.V. irradiation primarily reflects replication of the E. coli DNA. A similar study with X-rays leading to the same conclusion was reported by Billen, Hewitt, Lapthisophon & Achey (1967). In contrast to methylation of DNA synthesized after U.V. irradiation, the methylation of previously synthesized unmethylated DNA does not appear to be affected by ultraviolet light. Since DNA is normally methylated close to or at the time of synthesis, it is possible that the methylases and any required base sequence recognition proteins are part of a larger DNA replication complex. The different response to U.V. irradiation of methylation of DNA synthesized post-irradiation versus methylation of previously synthesized DNA may indicate some difference in the methylation process under the two conditions. Several investigators have reported that unmethylated DNA synthesized in t,he absence of methionine was normally methylated when methionine was supplied (Billen, 1968; Lark, 1968a$). Billen & Hewitt (1966) reported that this DNA subsequently replicated slower than normal. Data in the present paper show that methylation of the unmethylated DNA is not normal for at least one hour after methionine is added in either control or u.v.-irradiated cells. It is obvious that methionine starvation is an abnormal condition and can induce significant physiological changes. The changing 6-Megde to 5-MeCyt ratio with time after addition of methionine may be due either to selective turnover of methylases during starvation or to an inherent difference in rate of act)ion of the two met.hylases. However, the results may not
DNA
METHYLATION
FOLLOWING
IRRADIATION
371
have been affected by starvation but may simply reflect the normal operation of methylases not involved in semi-conservative replication at the replication fork. The presence of unmethylated DNA at sites other than the immediate vicinity of the replication fork is not a usual condition of a cell, except for small segments synthesized by the repair system. It doesnot necessarily follow that a methylase system free to methylate any region of the chromosome such as a repaired segment should be the samein all respects to the system used at the time of semi-conservative synthesis. The most likely explanation for hypo-methylation of the DNA synthesized postu.v.-irradiation is that the u.v.-damaged DNA cannot serve as a proper template, at least with respect to methylation. For example, the damaged template might lead to the failure to methylate an adenine or cytosine residue in someof the basesequences normally lesspreferred for methylation in the new strand. If damage to the DNA template is responsiblefor the hypo-methylation observed in our experiments, other types of damage might affect methylation differently. X-irradiation produces single- and double-strand breaks in DNA, interstrand crosslinks, depurination and other effects, as opposedto pyrimidine dimers as the primary effect of U.V. irradiation. DNA synthesized after 10,000 r X-rays, a dose giving survival comparable to the U.V. studies, was not hypo-methylated and was in fact methylated about 15% more than normal, with a slight increase in the 6-MeAde to 5MeCyt rat,io. No attempt was made to explore the X-ray data further, and no significance is being given to the data beyond the fact that hypo-methylation does not occur in DNA synthesized post-X-irradiation. This may indicate that the type of damage the DNA sustainsis important in subsequent methylation. This investigation AT-(4&l)-3596.
was supported
in part by the U.S. Atomic Energy Commission
REFERENCES Rev. Biochem. 38, 467.
Arber, W. & Linn, S. (1969). Ann. Barrier, H. D. & Cohen, S. S. (1954). J. Bact. 68, 280. Bergquist, P. L. & Matthews, R. E. F. (1962). B&hem J. 85, 305. Billen, D. (1959). Biochim. biophys. Acta, 34, 110. Billen, D. (1968). J. Mol. Biol. 31, 477. Billen, D. (1969). J. Bad. 97, 1169. Billen, D. & Bruns, L. (1970). Biophye. J. 10, 509. Billen, D. & Carreira, L. (1971). Microbios, 3, 153. Billen, D. & Hewitt, R. (1966). J. Bact. 92, 609. Billen, D., Hewitt, R., Lapthisophon, T. & Achey, P. M. (1967). J. Bact. 94, 1538. Burdon, R. H. (1966). Nature, 210, 797. Dunn, D. B. & Smith, J. D. (1958). Biochem. J. 68, 627. Fleissner, N. & Borek, E. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1199. Gold, M. & Hurwitz, J. (1964). J. Biol. Chem. 239, 3858. Griffith, J. S. & Mahler, H. R. (1969). Nature, 223, 580. Grippo, P., Parisi, E., Carestia, C. & Scarano, E. (1970). Biochemktry, 9, 2605. Hewitt, R. & Billen, D. (1965). J. MoZ. Biol. 13, 40. Kit, S., Beck, C., Graham, 0. L. & Gross, A. (1958). J. BioZ. Chem. 233, 944. Lark, C. (1968a). J. Mol. BioZ. 31, 389. Lark, C. (19688). J. MOE. BioZ. 31, 301. Maalae, 0. & Hanawalt, P. C. (1961). J. Mol. BioZ. 3, 144. Mandel, L. R. & Borek, E. (1963). Biochemistq, 2, 555. Marmur, J. (1961). J. Mol. BioZ. 3, 208.
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Rupp, W. D. & Howard-Flanders, P. (1968). J. Mol. Biol. 31, 291. Seno, T. & Melechen, N. E. (1964). J. Mol. Biol. 9, 340. Smith, K. C. & Meun, H. C. (1970). J. Mol. BioZ. 51, 459. Sneider, T. W. & Potter, V. R. (1969). J. Mol. BioZ. 42, 271. Srinivasan, R. P. & Borek, E. (1964). Science, 145, 548. Tsutsui, E., Srinivasan, P. R. & Borek, E. (1966). Proc. Nat. Acad. Sci., Wyatt, G. R. & Cohen, S. S. (1953). Biochem. J. 55, 774. Yudelevich, A. & Gold, M. (1969). J. Mol. BioZ. 40, 77.
Wash.
56,
1003.
(1972) 63, 363-372
In viva Methylation of Escherichia coli DNA Ultraviolet and X-irradiation BRADFORD
L.
WHITnELDt
AND
DANIEL
following
BILLEN
Radiation Biology Laboratory ati Departmentsof Immunology and Medid Microbiology and Radiology, Collegeof Medicine University of Florida Gainesville, Flu., U.S.A. (Received4 May 1971, and in revisedform 17 September1971) E&e&Ku coli 15T(555-7) w&8 irradiated with ultraviolet light and X-rays and the DNA examined for quantitative and qualitative changes in the methylation pattern. DNA synthesized following ultraviolet irradiation was under-methylated by approximately 25% as compared to DNA from u&radiated cells. The ratio of 6-methyladenine to 5-methylcytosine was unchanged. DNA synthesized following X-irradiation was methylated about 15% more than normal with a slight increase in the 6-methyladenine to 5-methylcytosine
ratio. No additional methyl&ion or turnover of methyl groups could be detected in parental DNA present at the time of ultraviolet or X-irradiation. E. coli 15T- (555-7) synthesized “unmethylated DNA” by growth in the absence of methionine. Ultraviolet irradiation of cells containing unmethylated DNA did not quantitatively change the ability of the cells subsequently to methylate this DNA when methionine was added to the culture. The ratio of 6-methyladenine to 5-methylcytosine in both control and ultraviolet-irradiated cells changed from 1.8 -f 0.2 when examined 15 minutes after addition of methionine to 1.4 5 0.1, the normal value, when examined one hour after methionine was added.
1. Introduction The specific methylation of basesin DNA is a well-established phenomenon ocurring in virtually all organisms examined. It involves the enzymic transfer of the methyl group of X-adenosylmethionine to specific basesin DNA (Mandel & Borek, 1963; Srinivasan & Borek, 1964). This occurs at the polynucleotide level in bacteria either at or near the replication point (Gold & Hurwitz, 1964; Fleissner & Borek, 1962; Billen, 1968). The methylation pattern is a unique feature of DNA from different, speciesand is the basis of restriction-modification systemsin bacteria (for a current’ review see Arber & Linn, 1969). Methylation of cytosine on the 5 position followed. by a specific deamination may be involved in differentiation in developing seaurchin embryos (Grippo, Parisi, Carestia & Scarano, 1970) and has been suggestedto occur t Present
address:
Biology
Division,
Oak
Ridge
National
363
Laboratories,
OakRidge,
Term.,
U.S.A.
364
B.
L.
WHITFIELD
AND
D.
BILLEN
in Novikoff hepatoma cells (Sneider & Potter, 1969). Other possible functions of methylation have been proposed such as a punctuation system for transcription or a method of regulating the number of times a message is translated (Griffith & Mahler, 1969). Increased transfer RNA methylase activities have been reported in tumor cells (Bergquist & Matthews, 1962; Tsutsui, Srinivasan & Borek, 1966; Burdon, 1966). Radiation alters the pattern of DNA replication with respect to sequence (Billen, 1969). In this report we have examined the methylation of Escherichia coli DNA following ultraviolet and X-irradiation to determine whether radiation alters this aspect of DNA metabolism.
2. Materials
and Methods
(a) Bacteria
E. coli 15T-
(555-7) is a multiauxotroph methionine. Complete growth medium 2 pg thymine/ml., 14 pg tryptophan/ml., 2% glucose as a carbon source. (b)
Radiosotopic
used
requiring thymine, consists of minimal 38 pg arginine/ml.,
arginino, tryptophan salts (MS-7, Billen, 30 pg methionine/ml.
and 1959), and
and density labeling of DNA
Bulk labeling of DNA was accomplished by growth in the presence of 0.1 &!i/2pg per ml. [2- 14C]thymine. n-[methyZ-3H]methionine at specific activity of 5.0 #%/lo pg per ml. was used as a source of labeled methyl groups. When density labeling was required, thymine was replaced by 5-bromouracil (10 pg/ml.). (c) Equilibrium
density
analysis
Cells were harvested by centrifugation, washed in 0.1 M-Tris buffer (pH 8.1) and resuspended in 0.1 M-Tris-(ethylenedinitrilo) . tetra-acetic acid buffer (pH 8.1). Lysates were prepared by adding lysozyme (100 pg/ml.) to the cell suspensions, freezing in liquid nitrogen and incubating at 37°C for 10 min. Pronase (100 pg/ml.) was added and the samples incubated 15 mm at 37°C. Portions were mixed with CsCl to a density of l-710 g/ ml. and the DNA banded by centrifugation at 33,000 rev./min for 45 hr in an SW39 or SW65 rotor in a Spinco model L2-65 or L2-65B ultracentrifuge. After centrifugation, the bottoms of the centrifuge tubes were punctured and fractions collected by alternating 1 drop on Whatman no. 1 paper disks and 9 drops in tubes containing 2 drops TEP buffer (0.1 M Tris*HCl, 0.02 M-EDTA, 0.05 M-sodium pyrophosphate, pH 8.1). The disks were washed in cold 5% trichloracetic acid, rinsed in cold 95% ethanol, dried and counted in a model 3375 Tricarb liquid-scintillation counter. The fractions containing the desired DNA, either parental or newly synthesized, were pooled and centrifuged as before. Five-drop fractions were collected on paper disks and counted as described above. (d)
DNA
puri$cation
Cells were lysed as described above and the volume brought to 5 ml. by addition of Tris-EDTA buffer. DNA was purified by a modified Marmur procedure (Marmur, 1961). Samples were digested with pronase for 1 hr at 37°C and then shaken with cold phenol for 10 mm. The aqueous layer was removed and extracted several times with ether. 95% ethanol was added and the DNA spooled from the interface with a glass rod, resuspended in O-1 x SSC (0.015 Ma-Nacl, 0.0015 M-sodium citrate), and brought to 1 x SSC when the DNA had dissolved. Samples were then shaken with chloroform-isoamylalcohol (24:l v/v) for 10 min and the aqueous layer extracted with ether until the solution was clear. Pancreatic RNase A (100 pg/ml.) was added and the samples incubated for 1 hr at 37OC, brought to 0.1 N-NaOH and incubated 1 hr. Finally, tho DNA was precipitated with cold 10% trichloracetic acid, washed in ethanol and dried.
DNA
METHYLATION
FOLLOWING
(e) DNA
hydrolysis
365
IRRADIATION
and base analysis
The purified DNA plus 50 pg unlabeled E. coli carrier DNA was hydrolyzed to free bases with 70% perchloric acid at 100°C for 1 hr. The hydrolysate was neutralized with KOH and centrifuged to remove the precipited potassium perchlorate and charred sugars. The second and preferred method of hydrolysis was to treat the DNA with 90% formic acid for 30 min at 175°C in sealed Pyrex tubes (Wyatt & Cohen, 1953). The formic acid and volatile salts were removed by evaporation and the residue resuspended in 0.25 ml. 0.1 N-HCl. The clear sample was mixed with known bases and co-chromatographed on Whatman no. 1 paper in a descending direction using n-butyl alcohol-water-NH,OH (86 : 14 : 2 by vol.) as the solvent. The bases were located by U.V. absorption and the chromatogram then cut into l-cm strips and counted in a liquid-scintillation counter as described. Labeled methylated bases were those derived from the hydrolyzed DNA. The relative extent of methylation of the newly formed DNA was determined as the ratio of 14C-counts in thymine to 3H-counts in 6-MeAde plus 5-MeCyti. A given 14C-count represents the same amount of DNA in the control and irradiated cells, and comparison of the 14C/3H ratios in the two samples is a measure of the relative extent of methylation of the new strand. (f) Irradiation Cells were placed 200 to 600 ergs/mm2 cells were irradiated
on a cold shallow watch glass, stirred constantly and U.V. at 7.4 ergs/mm2 per set or 10,000 r X-irradiation. in non-supplemented minimal salts medium.
irradiated In all
with cases
3. Results (a) Lack of additional methylation DNA following
or turnover of methyl groups ultraviolet irradiation
in parental
An inoculum of exponentially growing E. coli 15T- (555-7) was added to 10 ml. of fully supplemented MS-7 medium containing [2-14C]thymine as a bulk label. At an optical density of 0.4 the cells were harvested on a Millipore filter and resuspended in 10 ml. ice-cold unsupplemented MS-7 medium. Half the sample was irradiated as described. The other half-sample was held in an ice water bath and served as the unirradiated control. Each samplewasthen added to 5 ml. of MS-7 containing the necessarysupplements with 5-bromouracil and [methyL3H]methionine. The cells were incubated at 37°C for 30 to 90 minutes and collected in centrifuge tubes containing 3 ml. frozen saline. After harvesting by centrifugation and washing in 0.1 M-Tris buffer, the cells were lysed and centrifuged as described in Materials and Methods. Parental DNA synthesized before irradiation bands at a density of 1.710 g/ml., as shown in Figure 1. The DNA banding at a density of 1.755 g/ml. is hybrid DNA containing one strand synthesized following irradiation and one parental strand. The high 3H-labeled background results from the distribution of methylated RNA through the gradient. Fractions under the peak at 1.710 g/ml. were pooled and the DNA rebanded to determine if any 3H-label was present in the unreplicated parental DNA. Fractions under the peak at 1.755 g/ml. were pooled and the DNA denatured and rebanded to determine if any 3H-label was present in the parental strand in hybrid DNA. No 3H-label was found in either the rebanded unreplicated parental DNA or in the rebanded parental strand from denatured hybrid DNA. We concluded that there t Abbreviations
used:
6-MeAde,
6-methyladenine;
5MeCyt,
Smethylcytosine
366
B.
L.
WHITFIELD
AND
Fraction
D.
BILLEN
no
FIG. 1. CsCl density profile of DNA from cells labeled with [2-r4C]thymine and [naethyZ-3H] methionine. The experimental procedures were as described in the text. The profile shown is of DNA from [14C]thymine-labeled cells exposed to 200 ergs/mm2 followed by 90 mm incubation in the presence of 5bromonracil and [methyZ-3H]methionine. The DNA banding at a density of 1.710 g/ml. is parental DNA synthesized before irradiation. The DNA banding at a density of 1.755 g/ml. is a hybrid containing one strand synthesized following irradiation and one parental strand. The 3H-label background is a result of methylation of RNA. The small 3H-label peak at a density of 1.798 g/ml, is the beginning of second rounds of DNA replication. -a-.--, W; ---A--A--, 3H. was no turnover of methyl groups or additional methylation of parental DNA following u.v. irradiation. The failure to find 3H-label in the parental DNA also indicated no new methylase activity as a result of defective prophage induction. Up to a 20-fold increase in 6-MeAde methylase activity has been reported in other derivatives of E. coli 15T- following prophage induction by a variety of treatments including U.V. irradiation (Dunn & Smith, 1958; Yudelevich & Gold, 1969).
(b) Fate of 3H-label from [methyl-3H]methionine When [methyZ-3H]methionine is used as a labeled methyl donor, the 3H-label can be incorporated into positions 2 and 8 of purine rings and into the methyl group of thymine through one-carbon metabolism as shown in Figure 2(a). The addition of sodium formate to the growth medium effectively eliminated the incorporation of label into the ring structure of adenine and guanine by diluting the radioactivity in the one-carbon pool (Kit, Beck, Graham & Gross, 1958) (see Fig. 2(b)). Label was still found in thymine. E. coli 15T- (555-7) is blocked in thymidylate synthetase and like most similar thymine-requiring strains, the block may not be complete (Barner & Cohen, 1954; Seno & Melechen, 1964). It was also possible that the label in thymine was due in part to an enzymic deamination of 5-MeCyt at the polymer level as reported by Grippo et al. (1970) in sea urchin embryos, or to a chemical deamination of 5-MeCyt during acid hydrolysis of DNA. An enzymic deamination of 5-MeCyt was eliminated by the following experiment. Methionine-requiring cells can synthesize unmethylated DNA by growth in the absence of methionine (Billen, 1968; Lark, 1968aJ). During amino acid starvation, the round of DNA replication in progress goes to completion and new rounds of replication are not initiated (Maalse & Hanawalt, 1961). When methionine is supplied,
DNA
METHYLATION
FOLLOWING
Distance FIG.
2. Incorporation
of 3H-label
from
IRRADIATION
367
from origin km1
[m&hyZ-3H]methionine
into
bases in DNA.
Plots are shown of the distribution of 3H-oounts on chromatograms of formic acid-hydrolyzed DNA from cells grown in the presence of [methyl-sH]methionine. Procedures are described in Materials and Methods. The samples chromatographed are: (a) hydrolyzed DNA from an exponential cell culture without sodium formate; and (b) with 20 mra-sodium formate. Abbreviations: guanine (Gus). 5-methylcytosine (5MeCyt), thymine (Thy), 6.methyladenine (6-MeAde) and adenine (Ade).
the DNA synthesized during the starvation period is methyl&ted. A log-phase culture of cells was transferred to medium lacking methionine, arginine s,nd tryptophan and incubated 90 minutes. The cells were then shifted to medium containing [mmethyL3H] methionine but lacking arginine, tryptophan and thymine and incubated 15 minutes. This permitted methylation of the unmethylated DNA while continuing the inhibition of DNA and protein synthesis. DNA from half the sample was analyzed as a control. Methyl&ion was 70% complete after 15 minutes, with 98% of the 3H-label in 6-MeAde and 5-MeCyt as shown in Figure 3(a). The remaining 2% of the label was in thymine. The other half-sample was harvested on a Millipore filter, washed with MS-7 salts and transferred to complete medium with ten times the normal concentration cd unlabeled methionine to dilute out any remaining labeled methionine in the pool. The cells were incubated one hour to permit deamination to occur. If an enzymic deamination of B-MeCyt occurred during this time, there should be an increase of 3H-label in thymine and a decrease in 5-MeCyt. There was no change in the distribu.tion of 3H-label its shown in Figure 3(b). It is, therefore, likely that the label appearing in thymine is a result of an incomplete block in thymidylate synthetase with a small amount of chemical deamination during hydrolysis. (c) Hype-methyl&ion of newly synthesized DNA following ultraviolet irradiation An exponential culture of E. coli at 3 x lo8 cells per ml. was harvested on a Millipore filter and resuspended in cold MS-7, divided and treated as in the previous experiment. Following 400 ergs/mm2 U.V. irradiation, each sample was added to complete medium containing [14C]thymine and [methyL3H]methionine with 20 mM-SOdhm formate and incubated 90 minutes. DNA was purified and hydrolyzed as described. As shown in Table 1, DNA synthesized after U.V. irradiation was under-methylated by 25% as compared to DNA from unirradiated cells. The ratio of 6-MeAde to 5-MeCyt remained unchanged at 1.4.
368
B.
L.
WHITFIELD
5
AND
IO
I5 20
Dstance
FIG.
3. Methyl&ion
of DNA
D.
BILLEN
25 30
from oqn
following
35
40
(cm)
methionine
starvation.
Following 90 min of methionine, arginine and tryptophan starvation the cells were shifted to medium containing [methyZ-3H]methionine, but lacking arginine, tryptophan and thymine, and incubated 15 min. (a) Is a plot of the distribution of 3H-counts on a chromcttogram of hydrolyzed DNA extracted immediately after the 15.min methyl&ion period. (b) The chromatogrem is of hydrolyzed DNA from cells that were incubated 60 min in complete medium with no labels after the 15-min methyl&ion period. Abbreviations are the s&me as in Fig. 2.
TABLE
Methylation
of DNA
synthesized Methyl&ion (%)
Sample
Control u.v. (400 ergs/mm2) X-rays (10,000 r)
1 after irradiation Ratio (6.MeAde/5-MeCyt)
100 75 115
1.4kO.l 1*4+0.1 1.5*0.1
The relative extent of methyl&ion w&s determined by dividing the amount of DNA synthesized (as determined by [l%]thymine counts) by the amount of methyl&ion (as determined by sH-counts in methylated bases). A comparison of the 14C/3H ratios in the irradiated samples to the ratio in the control gave the percentage methyl&ion. The ratio of 6-MeAde to 5-MeCyt was determined by the distribution of 3H-label on chrometograms of hydrolyzed DNA.
(d) Does pool dilution
contribute to hype-methyl&on following ultraviolet irradiation
of DNA
synthesized
?
This possibility was eliminated by first labeling cells with [methyL3H]methionine and determining if any label appeared in DNA subsequently synthesized. The cells were grown to an optical density of 0.4 in the presence of labeled methionine and [14C]thymine. They were harvested and irradiated with 400 ergs/mm2 as in previous experiments with post-irradiation incubation in fully supplemented MS-7 medium containing Ekbromouracil. Cells were lysed and DNA banded as before. Hybrid DNA was collected and denatured by heating for 10 minutes at 100°C followed by rapid cooling. The denatured DNA was rebanded in CsCl to separate the old and newly
DNA
METHYLATION
FOLLOWING
IRRADIATION
369
made DNA strands. No 3H-label was found in the newly made strand, and we concluded that pool dilution did not influence the observed methylation after U.V. irradiation. (e) Is hype-methyl&on
a result of recall of the chromosomul origin following ultraviolet irra4Sation ?
Following U.V. irradiation of bacteria, the DNA replication fork in progress either stops or is greatly slowed. When replication resumes, it starts from the genetic origin (Billen, 1969). If the first half of the chromosome normally contains fewer methylated bases than the terminal half, hypo-methylation could be expected in our experiments. To determine if this was the explanation, a log-phase culture of cells was deprived of arginine and tryptophan for 90 minutes to align the chromosome. One-half of the sample was added to complete medium containing [methyL3H]methionine and [14C]thymine and incubated 35 minutes, then transferred to complete medium with no radioisotopic label for 20 minutes more. The other half-sample was added to medium containing no radioisotopic label for the first 35 minutes, then transferred to medium containing [methyL3H]methionine and [14C]thymine for the last 20 minutes. This permitted us to examine DNA selectively labeled, on the average, in the first half and last half of the chromosome. There was no difference found in either extent of methylation or ratio of methylated bases in the two samples. Recall of the origin is unlikely to be responsible for the hype-methylation observed after U.V. irradiation. (f) Methyl&ion
of unmethylated DNA
after ultraviolet irradiation
Cells were starved for methionine and other required amino acids to permit the synthesis of unmethylated DNA as described in an earlier section. [14C]Thymine was present during starvation to measure DNA synthesis. The culture was divided and one half irradiated with 400 ergs/mm2 U.V. Each sample was allowed to methylate the unmethylated DNA by addition of [methyL3H]methionine in the absence of additional DNA or protein synthesis. There was no difference in the ability of irradiated and control cells subsequently to methylate the unmethylated DNA, indicating no detectable damage to the methylating system. When examined 15 minutes after re-addition of methionine, the ratio of 6-MeAde to 5-MeCyt in DNA was 1.8 & 0.2 in both control and irradiated cells. The ratio decreased to the log-phase value when methylation was allowed to continue one hour (see Table 2). The decrease primarily reflected an increase in 5-MeCyt content. TABLE
2
DNA methyl&ion followins methionine starvation Time after methionine addition (mb)
Relative methyl&ion (%)
10 30 60 The relative methylation described in Table 1.
percentages
Ratio (B-MeAde/5-MeCyt)
60 90 100 and
ratios
l+Jf0.2 1.5*0.1 1.4,tO.l of 6.MeAde
to 5-MeCyt
were
determined
aa
370
B.
L.
WHITFIELD
(g) Methyl&on
AND
D.
BILLEN
of DNA following X-irradiation
To determine the extent of methylation and ratio of methylated bases in DNA synthesized after X-irradiation, the experimental procedures were as described for the ultraviolet studies. After 10,000 r, the newly synthesized DNA was methylated approximately 15% more than control DNA, as shown in Table 1. The ratio of 6-MeAde t,o 5-MeCyt was slightly increased. Parental DNA did not undergo additional methylation after X-irradiation.
4. Discussion It is apparent that when methylation occurs on DNA being synthesized from an ultraviolet damaged template, the newly made DNA does not contain the full complement of methylated bases. Hypo-methylation is not the only abnormality of this DNA. It has been shown in E. coli K12 (Rupp & Howard-Flanders, 1968; Smith & Meun, 1970) and E. coli B/r (Billen & Carreira, 1971) that the DNA synthesized following U.V. irradiation exists in smaller pieces than normal for a time after synthesis. In Her+ cells the small pieces become larger with time; however, in Hercells the DNA remains in small pieces. Billen & Bruns (1970) have reported that the DNA synthesized after U.V. irradiation is less likely to be replicated in each succeeding replication cycle than normal DNA. Finally, the DNA replication fork present at the time of U.V. irradiation is known to be slowed or stopped, with most new synthesis occurring at a new replication fork starting at the replicative origin (Billen, 1969). The possibility that the DNA synthesized after U.V. irradiation is viral or episomal in origin rather than cellular, is eliminated by earlier published studies with E. coli 15T- (555-7). Hewitt & Billen (1965) using density-shift techniques showed that post-irradiation DNA synthesis after U.V. irradiation primarily reflects replication of the E. coli DNA. A similar study with X-rays leading to the same conclusion was reported by Billen, Hewitt, Lapthisophon & Achey (1967). In contrast to methylation of DNA synthesized after U.V. irradiation, the methylation of previously synthesized unmethylated DNA does not appear to be affected by ultraviolet light. Since DNA is normally methylated close to or at the time of synthesis, it is possible that the methylases and any required base sequence recognition proteins are part of a larger DNA replication complex. The different response to U.V. irradiation of methylation of DNA synthesized post-irradiation versus methylation of previously synthesized DNA may indicate some difference in the methylation process under the two conditions. Several investigators have reported that unmethylated DNA synthesized in t,he absence of methionine was normally methylated when methionine was supplied (Billen, 1968; Lark, 1968a$). Billen & Hewitt (1966) reported that this DNA subsequently replicated slower than normal. Data in the present paper show that methylation of the unmethylated DNA is not normal for at least one hour after methionine is added in either control or u.v.-irradiated cells. It is obvious that methionine starvation is an abnormal condition and can induce significant physiological changes. The changing 6-Megde to 5-MeCyt ratio with time after addition of methionine may be due either to selective turnover of methylases during starvation or to an inherent difference in rate of act)ion of the two met.hylases. However, the results may not
DNA
METHYLATION
FOLLOWING
IRRADIATION
371
have been affected by starvation but may simply reflect the normal operation of methylases not involved in semi-conservative replication at the replication fork. The presence of unmethylated DNA at sites other than the immediate vicinity of the replication fork is not a usual condition of a cell, except for small segments synthesized by the repair system. It doesnot necessarily follow that a methylase system free to methylate any region of the chromosome such as a repaired segment should be the samein all respects to the system used at the time of semi-conservative synthesis. The most likely explanation for hypo-methylation of the DNA synthesized postu.v.-irradiation is that the u.v.-damaged DNA cannot serve as a proper template, at least with respect to methylation. For example, the damaged template might lead to the failure to methylate an adenine or cytosine residue in someof the basesequences normally lesspreferred for methylation in the new strand. If damage to the DNA template is responsiblefor the hypo-methylation observed in our experiments, other types of damage might affect methylation differently. X-irradiation produces single- and double-strand breaks in DNA, interstrand crosslinks, depurination and other effects, as opposedto pyrimidine dimers as the primary effect of U.V. irradiation. DNA synthesized after 10,000 r X-rays, a dose giving survival comparable to the U.V. studies, was not hypo-methylated and was in fact methylated about 15% more than normal, with a slight increase in the 6-MeAde to 5MeCyt rat,io. No attempt was made to explore the X-ray data further, and no significance is being given to the data beyond the fact that hypo-methylation does not occur in DNA synthesized post-X-irradiation. This may indicate that the type of damage the DNA sustainsis important in subsequent methylation. This investigation AT-(4&l)-3596.
was supported
in part by the U.S. Atomic Energy Commission
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56,
1003.