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DNA methylation and mammalian epigenetics.

Epigenetic modifications of DNA such as methylation are important for genome function during development and in adults. DNA methylation has central importance for genomic imprinting and other aspects of epigenetic control of gene expression, and during development methylation patterns are largely maintained in somatic lineages. The mammalian genome undergoes major reprogramming of methylation patterns in the germ cells and in the early embryo. Some of the factors that are involved both in maintenance and in reprogramming, such as methyltransferases, are being identified. Epigenetic changes are likely to be important in animal cloning, and influence the occurrence of epimutations and of epigenetic inheritance. Environmental factors can alter epigenetic modifications and may thus have long lasting effects on phenotype. Epigenetic engineering is likely to play an important role in medicine in the future.

Animals↗

Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53.

Nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) entails two principal mechanisms: modification of DNA ends prior to ligation (error-prone rejoining) or precise ligation without modification if the DNA ends are complementary (error-free repair). Error-prone rejoining is mutagenic, because it can lead to destruction of coding sequence or to chromosomal aberrations, and therefore must be tightly regulated. Previous studies on the role of the p53 tumor suppressor in the regulation of NHEJ have yielded conflicting results, but a rigorous analysis of NHEJ proficiency and fidelity in a purely chromosomal context has not been carried out. To this end, we created novel repair plasmid substrates that integrate into the genome. DSBs generated by the I-SceI endonuclease within these substrates were repaired by either error-prone rejoining or precise ligation. We found that the expression of wild-type p53 inhibited any repair-associated DNA sequence deletion, including a more than 250-fold inhibition of error-prone rejoining events compared to p53-null cells, while any promoting effect of p53 on precise ligation could not be directly evaluated. The role of p53 in NHEJ appeared to involve a direct transactivation-independent mechanism, possibly restricting DNA end-modification by blocking the annealing of single strands along flanking stretches of microhomology. The inhibition of error-prone rejoining by p53 did not apply to the rejoining of DSBs induced by ionizing radiation. In conclusion, our data suggest that p53 restricts the mutagenic effects of NHEJ without compromising repair proficiency or cell survival, thereby maintaining genomic stability.

Animals↗

Site-specific modification of Escherichia coli DNA polymerase I large fragment with pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.

Binding Sites↗

DNA methylation: biology and significance.

The modification of DNA by cytosine methylation is crucial for normal development. DNA methylation patterns are distinctive between tissues and are maintained with high fidelity during cell division. DNA methylation probably exerts its effects through alterations in chromatin structure, with a resultant effect on genetic transcription. 5-methylcytosine is also prone to spontaneous hydrolytic deamination to thymine. Whilst most G:T mismatches so produced are repaired, failure of mismatch repair leads to established mutation. Indeed, mutations that are the result of 5-methylcytosine transitions account for a disproportionate number of genetic mutations described in malignant and non-malignant disease. There is also evidence for substantial deregulation of DNA methylation in malignancy. Whether this deregulation is crucial for the transformation process, or simply an epiphenomenon associated with it, is still not established.

Animals↗

DNA base and deoxyribose modification by the carbon-centered radical generated from 4-(hydroxymethyl)benzenediazonium salt, a carcinogen in mushroom.

Modification of the base and the sugar moieties of DNA with 4-(hydroxymethyl)benzenediazonium salt (HMBD), a carcinogen in the mushroom Agaricus bisporus, was investigated. When deoxyribonucleosides dGuo, dAdo, dThd, and dCyd were incubated with HMBD at pH 7.4 and 37 degrees C, the levels of all the nucleosides were decreased. The decrease was inhibited by ethanol or Cys. When deoxyribose was incubated with HMBD, malonaldehyde was released as assessed by the thiobarbituric acid reactivity. The release was inhibited by ethanol. Major products of the reaction of dGuo and dAdo with HMBD were isolated, and their structures were established to be 8-[4-(hydroxymethyl)phenyl]dGuo (8-HMP-dGuo) and 8-[4-(hydroxymethyl)phenyl]dAdo), respectively. Calf thymus DNA treated with HMBD was enzymatically digested into nucleosides, in which 8-HMP-dGuo and 8-HMP-dAdo were detected. Formation of the modified nucleosides in DNA was inhibited by ethanol or 2-mercaptoethanol. Malonaldehyde was released from DNA treated with HMBD, which indicated that the deoxyribose moiety of DNA had been damaged. The results indicate that the 4-(hydroxymethyl)phenyl radical generated from HMBD can directly modify the base and the sugar moieties of DNA under the mild conditions. Inhibitory effect of ethanol was ascribable to its scavenging activity for the carbon-centered radical. The inhibitory effect of Cys and 2-mercaptoethanol was found to be due to the formation of the reversible adducts between HMBD and the SH compounds.

Adenosine↗

Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis.

DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.

Adenine↗

Poly (ADP-ribosylation) and DNA topoisomerase I in different cell lines.

The modification of DNA topoisomerase I activity by poly (ADP-ribosylation) is a potentially important control mechanism for this ubiquitous enzyme which is widely believed to play a role in replication (2, 3). Our studies of the phenomenon so far suggest that DNA topoisomerase I activity may be regulated in response to the state of the DNA in the nucleus since the modification enzyme, poly (ADP-ribose) synthetase, is sensitive to interruptions in the normal double helical structure of DNA (10). Although this phenomenon has been most extensively characterized for the purified enzymes from calf thymus (5), preliminary evidence for poly (ADP-ribosylation) of topoisomerase I is presented in a nonthymic system, the mammalian cell line CHO EM9. Attempts to detect poly (ADP-ribosylation) in yeast cells have so far proven to be unsuccessful.

Animals↗

Inhibition of cleavage by restriction endonucleases due to modifications induced in SV40 DNA by methyl methanesulfonate.

Methyl methanesulfonate (MMS), a direct mutagen, methylates DNA bases and causes distortions in DNA structure. Supercoiled SV40 DNA was treated in vitro with varying concentrations of MMS from 0.001 mM to 10 mM MMS either for 30 min or 3 h and analysed by electrophoresis in 1% neutral and alkaline agarose gels. The electrophoretic mobility (EPM) of native DNA did not change after treatment with the mutagen, while alkaline gels revealed low MW DNA fragments due to single strand breaks at alkali-sensitive sites generated by the action of MMS. By two-dimensional electrophoresis, we find that all three native DNA forms contain alkali-sensitive sites after treatment with MMS. To examine the effect of base modification by MMS on DNA-protein interactions, we have used as probes, restriction endonucleases. These cleave DNA in a sequence-specific manner, and their activity is dependent upon the methylation status of the substrate DNA. We find that cleavage by these restriction endonucleases is inhibited due to methylation by MMS.

DNA Damage↗

[Interaction of DNA with N-bromosuccinimide. G+C-specific reaction potentially useful for structural and functional studies].

Incomplete modification of DNA fragments labeled at one terminus with N-bromosuccinimide is shown to be useful for localization of G and C residues along the polynucleotide chain. After N-bromosuccinimide treatment the fragments can be split at modified residues with piperidine. The rate of the reaction of the single-stranded fragments is extremely fast and much higher than that of double-stranded DNA. The modification can be used for structure and function studies on DNA.

Base Sequence↗

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity.

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.

Antibiotics, Antineoplastic↗

Dissociation of single-stranded DNA from nucleosomes following modification with acetic anhydride.

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic strength.

Acetates↗

Unusual modification of bacteriophage Mu DNA.

Bacteriophage Mu DNA was labeled after induction in the presence of [2-(3)H]adenine or [8-(3)H]adenine. Both Mu mom(+).dam(+) DNA and Mu mom(-).dam(+) DNA have similar N(6)-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R.DpnI but resistant to cleavage by R.DpnII. These results indicate that the mom(+) protein does not alter the sequence specificity of the host dam(+) methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A(x), in Mu mom(+).dam(+) DNA; approximately 15% of the adenine residues are modified to A(x). Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N(6)-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A(x) is not present in Mu mom(+).dam(+) or Mu mom(-).dam(+) phage DNA or in cellular DNA from uninduced Mu mom(+).dam(+) lysogens. These results suggest that expression of the dam(+) and mom(+) genes are required for the A(x) modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom(+).dam(-) DNA and Mu mom(-).dam(-) DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5'... C-A(*)-C... 3' and that this methylation is mediated by the EcoK modification enzyme.

Adenine↗

[Genetic and immunologic studies after space flight].

The effect of spaceflight on lymphocytes of the peripheral blood was investigated in the Austrian cosmonaut flying the Austro-Soviet mission Austromir. Blood was sampled before and after flight, and the following parameters studied: quantities of lymphocytic subsets (T cells, helper cells, suppressor cells, B cells and natural killer cells); mitogenic stimulation of lymphocytes--DNA (= deoxyribonucleic acid) synthesis and expression of Interleukin-2-receptor; structural modification of DNA, unscheduled DNA synthesis and sister chromatid exchanges. Besides reducing the number of natural killer cells, spaceflight caused on impairment of lymphocyte activity and a slight modification of DNA structure. 4 weeks after flight, control values were reestablished, indicating efficient repair mechanisms.

Chromosome Aberrations↗

[DNA: what does it do, and what do we do with it?].

We present a bird's-eye view of the fundamentals and applications of the DNA technology. DNA is the carrier of genetic information. Separate pieces of DNA can be isolated in two different ways: cloning or via the polymerase chain reaction. Analysis of DNA has already found several applications in medical diagnosis and criminal investigations. Modification of DNA from lower organisms in order to produce proteins is now generally accepted. Genetic modification of plants and animals is promising, but is also the subject to a public and political debate. Gene therapy is a promise of the future. Analysis as well as modification of DNA has ethical consequences and both require an effective legislation.

Animals↗

Eucaryotic DNA replication complex: study of structure and function using the affinity modification technique.

Eucaryotic DNA replication complex is now one of the most intensively studied subjects of molecular biology and biochemistry. In addition to detailed studies on the structures and functions of individual DNA polymerases involved in this process, other enzymes and protein factors are also given much attention. The structures and functions of proteins in the replication complexes are studied by various approaches, including X-ray diffraction analysis. At present, this approach provides sufficient information about the structures and functions of individual biopolymers and their complexes with ligands. However, this approach is unsuitable for studies on proteins, which cannot be cloned and isolated in amounts sufficient for X-ray diffraction analysis. Moreover, this approach is inapplicable for studies on multicomponent systems, such as DNA replication and repair complexes. Furthermore, data of X-ray diffraction analysis virtually never characterize the variety of dynamic interactions in enzymatic systems. Affinity modification is an alternative and rather successful approach for studies on structure-functional organization of supramolecular structures. This approach can be used for studies on individual enzymes and their complexes with substrates and also on systems consisting of numerous interacting proteins and nucleic acids. The purpose of this review is to analyze the available data obtained by affinity modification studies on the eucaryotic replication complex.

Animals↗

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

Adenine↗

The epigenetic breakdown of cancer cells: from DNA methylation to histone modifications.

The recognition of epigenetic defects in all types of cancer has represented a revolutionary achievement in cancer research in recent years. DNA methylation aberrant changes (global hypomethylation and CpG island hypermethylation) were among the first events to be recognized. The overall scenario comprises a network of factors in which deregulation of DNA methyltransferases leads to a cancer-type specific profile of tumor suppressor genes that become epigenetically silenced. Over recent years, a better understanding of the machinery that connects DNA methylation, chromatin and transcriptional activity, in which histone modifications stand in a key position, has been achieved. The identification of these connections has contributed not only to understanding how epigenetic deregulation occurs in cancer but also to developing novel therapies that can reverse epigenetic defects in cancer cells.

DNA (Cytosine-5-)-Methyltransferase 1↗