Biomethylation of deoxyribonucleic acid in cultured human tumour cells (HeLa). Methylated bases other than 5-methylcytosine not detected

Lawley, P.D.; Crathorn, A. R.; Shah, S. A.; Smith, Barbara A.

doi:10.1042/bj1280133

Cited by 44 publications

(29 citation statements)

References 16 publications

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“…In all the cell lines or strains investigated little or no MeAde was detected; the values of MeAde ranged from 0.01% in T637 cells to 0.02% in KB and HEK cells. This finding confirms previous reports in which the absence of MeAde was noted in eukaryotic cells (7,8). On the other hand, the DNA of all cell lines examined contained relatively high amounts of MeCyt.…”

Section: Resultssupporting

confidence: 81%

“…Viral DNA accounts for 30-50% of the total intracellular DNA at late times after infection (2, 3). Concomitantly with the onset of viral DNA replication, host DNA synthesis is drastically reduced (4).In previous DNA base analyses of mammalian cells 3-6% 5-methylcytosine (MeCyt) per cytosine (Cyt) was found (5, 6), while N6-methyladenine (MeAde) could not be detected (7,8).Little is known about the biological role of DNA methylation. There are reports on tissue specificities in the content of MeCyt in mammalian cells (5,6,9).…”

mentioning

confidence: 99%

“…In previous DNA base analyses of mammalian cells 3-6% 5-methylcytosine (MeCyt) per cytosine (Cyt) was found (5, 6), while N6-methyladenine (MeAde) could not be detected (7,8).…”

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confidence: 99%

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DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.

Günthert¹,

Schweiger²,

Stupp³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

137

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DNAs of adenovirus type 2 and type 12 contain low amounts of methylated bases (0.01 and 0.02% N6-methyladenine per adenine, if any, and 0.04 and 0.06% 5-methylcytosine per cytosine for type 2 and type 12, respectively), whereas the DNA of the mammalian host cells contains much more 5-methylcytosine (3.57% for human KB cells). The Methylation of DNA was determined by a sensitive method based on two consecutive steps of two-dimensional thin-layer chromatography of the radioactively labeled DNA bases. By this procedure the detection limits of 5-methylcytosine and N6-methyladenine could be lowered to 0.01% per main base. During replication of adenoviruses in permissive human cells, a large amount of viral DNA is synthesized in the nucleus of the host cell (1-3). Viral DNA accounts for 30-50% of the total intracellular DNA at late times after infection (2, 3). Concomitantly with the onset of viral DNA replication, host DNA synthesis is drastically reduced (4).In previous DNA base analyses of mammalian cells 3-6% 5-methylcytosine (MeCyt) per cytosine (Cyt) was found (5, 6), while N6-methyladenine (MeAde) could not be detected (7,8).Little is known about the biological role of DNA methylation. There are reports on tissue specificities in the content of MeCyt in mammalian cells (5,6,9). Certain developmental stages are associated with variations in the content of methylated DNA bases in pro-and eukaryotes (10, 11). The only established function of certain DNA methyltransferases is the modification of DNA in bacteria leading to resistance against restriction endonucleases of the same specificity (12, 13).Since it is presumed that synthesis of adenovirus DNA in human cells is mediated, at least in part, by the host replication system, one would expect viral DNA to show the methylation pattern characteristic of host DNA. However, in contrast to host -DNA, adenovirus DNA has a low level of methylation. Comparison of untransformed cells and cells transformed by adenovirus has shown a difference in the levels of DNA methyl- (Calbiochem). The method of further purification has been described (21).In some experiments the total cellular DNA and the nonencapsidated viral DNA were isolated from Ad2-infected KB cells 40-48 hr after infection. Cell extracts were prepared by ultrasonic treatment, and the virions were isolated by equilibrium sedimentation in CsCl density gradients with a mean density of 1.334 g/cm3 as described (3). All free intracellular DNA was recovered from the pellet of this gradient and recentrifuged to equilibrium in a CsCl density gradient with a mean density of 1.70 g/cm3.Purification of Viral and Cellular DNA. The DNA was precipitated with two volumes of ethanol and kept for 4 hr at -20°. Traces of RNA were removed by hydrolysis in 0.25 M KOH 16-18 hr at 37°. Subsequently, the DNA was again pre-3923

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 99%

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DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.

Günthert¹,

Schweiger²,

Stupp³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

137

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“…The composition of each nucleotide is shown in Table 1. 10 15 20 EL£/J/ON T/ME, Minutes ELUT/ON TIME, Minutfes It is noted that the content of 5-methylcytosine is 0.6%, which is comparable to a published value of 0.4% for human DNA (10). The treatment of human DNA with DMS resulted in the formation of modified bases, as is evident in Figure 5.…”

Section: Example Of Postlabeling Methods Dms Modification Of Dnasupporting

confidence: 50%

“…The occurrence of 5'-dUMP is found in DNA as a result of the deamination of cytosine. The occurrence of 5-methylcytosine results from methylation of cytosine and represents about 0.4% of the total bases in DNA of HeLa cells (10). The separation of5'-deoxynucleotides from digested human DNA is shown in Figure 3.…”

Section: Example Of Postlabeling Methods Dms Modification Of Dnamentioning

confidence: 99%

New methods for detection of low levels of DNA damage in human populations.

Haseltine¹,

Franklin²,

Lippke³

1983

Environ Health Perspect

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The use of a postlabeling method to characterize and to detect infrequent base modifications in DNA is outlined. This method has the advantage that low levels of DNA modifications, approximately 1 modified base per 105 nucleotides, can be detected. Moreover, a broad spectrum of modification can be identified by using this methodology. The basis for the method involves transfer of a radioactive phosphate from the y position of ATP to the 5'-hydroxyl terminus of 3'-phosphoryl nucleotides that are derived from modified DNA by appropriate nuclease digestion.The second method involves use of a defined DNA sequence within human cells. The a sequence is used as a probe for DNA damage to specific nucleotides. The a DNA sequence is reiterated approximately 300,000 times in the human genome and exists in tandem arrays. It comprises approximately 1% of the entire genome. The reiterated sequence is sufficiently homogeneous to permit its use as a probe for a site specific in DNA damage.Examples of the application of both of these methodologies to DNA damage inflicted in human cells by chemicals and ultraviolet light are provided. DMS reacts with purines in DNA (2), the principal adducts formed being 7-methylguanine, 3-methyladenine and 1-methyladenine (3). Following DMS treatment, the DNA was isolated, and digested to nucleotides. The nucleotides were then postlabeled and were resolved by HPLC. DMS-methylated bases, in nucleotide form, were readily resolved from the four normal DNA bases and were detected along with two other minor products, 5-methylcytosine and uracil.

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N6-methyladenine: the other methylated base of DNA

et al. 2006

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Contrary to mammalian DNA, which is thought to contain only 5-methylcytosine (m5C), bacterial DNA contains two additional methylated bases, namely N6-methyladenine (m6A), and N4-methylcytosine (m4C). However, if the main function of m5C and m4C in bacteria is protection against restriction enzymes, the roles of m6A are multiple and include, for example, the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA. Interestingly, even if adenine methylation is usually considered a bacterial DNA feature, the presence of m6A has been found in protist and plant DNAs. Furthermore, indirect evidence suggests the presence of m6A in mammal DNA, raising the possibility that this base has remained undetected due to the low sensitivity of the analytical methods used. This highlights the importance of considering m6A as the sixth element of DNA.

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Biomethylation of deoxyribonucleic acid in cultured human tumour cells (HeLa). Methylated bases other than 5-methylcytosine not detected

Cited by 44 publications

References 16 publications

DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.

DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.

New methods for detection of low levels of DNA damage in human populations.

N6-methyladenine: the other methylated base of DNA

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