Enzymatic methylation of cytosine in DNA is prevented by adjacent O6-methylguanine residues

The effect of O6-alkylation of guanine residues on the enzymic methylation of cytosine has been studied using synthetic oligonucleotides in which all guanines in cytosine-guanine sequences at potentially methylatable sites are replaced by O6-methylguanine. In contrast with the unmodified forms, which showed high acceptance activity for methyl-3H-labeled groups from S-adenosyl-L-[methyl-3H]methionine in the presence of DNA methylase, the modified oligonucleotides were not substrates for the enzyme either in the single-stranded or annealed forms. In view of the importance of cytosine methylation in the down-regulation of certain genes, the potential to affect gene expression by this mechanism may be a contributory factor in the toxic and carcinogenic effects of chemical methylating agents.     

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.     

Mechanism of CpG DNA methyltransferases M.SssI and Dnmt3a studied by DNA containing 2-aminopurine

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase-DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme-substrate complexes were observed.     

Mechanism of CpG DNA Methyltransferases M.SssI and Dnmt3a Studied by DNA Containing 2-Aminopurine

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase–DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme–substrate complexes were observed.     

Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype

Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases. Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine. Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type. Using a Ung1p–green fluorescent protein (GFP) fusion construct, we demonstrate that yeast yUng1–GFP protein localizes to both mitochondria and the nucleus, indicating that Ung1p must contain both a mitochondrial localization signal (MLS) and a nuclear localization signal. Our study reveals that the first 16 amino acids at the N-terminus contain the yUng1p MLS. Deletion of 16 amino acids resulted in the yUng1p–GFP fusion protein being transported to the nucleus. We also investigated the intracellular localization of human hUng1p–GFP in yeast. Our data indicate that hUng1p–GFP predominately localizes to the mitochondria. Further analysis identified the N-terminal 16 amino acids as important for localization of hUng1 protein into the mitochondria. Expression of both yeast and human UNG1 cDNA suppressed the frequency of mitochondrial mutation in UNG1-deficient cells. However, expression of yUNG1 in wild-type cells increased the frequency of mutations in mtDNA, suggesting that elevated expression of Ung1p is mutagenic. An increase in the frequency of mitochondrial mutants was also observed when hUNG1 site-directed mutants (Y147C and Y147S) were expressed in mitochondria. Our study suggests that deamination of cytosine is a frequent event in S.cerevisiae mitochondria and both yeast and human Ung1p repairs deaminated cytosine in mitochondria.     

Normal somatic hypermutation of Ig genes in the absence of 8-hydroxyguanine-DNA glycosylase

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and kappa L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1(-/-) and Ogg1(+/+) clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1(-/-) clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.     

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

Germline ablation of SMUG1 DNA glycosylase causes loss of 5-hydroxymethyluracil- and UNG-backup uracil-excision activities and increases cancer predisposition of Ung-/-Msh2-/- mice

Deamination of cytosine (C), 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) occurs spontaneously in mammalian DNA with several hundred deaminations occurring in each cell every day. The resulting potentially mutagenic mispairs of uracil (U), thymine (T) or 5-hydroxymethyluracil (hmU) with guanine (G) are substrates for repair by various DNA glycosylases. Here, we show that targeted inactivation of the mouse Smug1 DNA glycosylase gene is sufficient to ablate nearly all hmU-DNA excision activity as judged by assay of tissue extracts from knockout mice as well as by the resistance of their embryo fibroblasts to 5-hydroxymethyldeoxyuridine toxicity. Inactivation of Smug1 when combined with inactivation of the Ung uracil-DNA glycosylase gene leads to a loss of nearly all detectable uracil excision activity. Thus, SMUG1 is the dominant glycosylase responsible for hmU-excision in mice as well as the major UNG-backup for U-excision. Both Smug1-knockout and Smug1/Ung-double knockout mice breed normally and remain apparently healthy beyond 1 year of age. However, combined deficiency in SMUG1 and UNG exacerbates the cancer predisposition of Msh2(-/-) mice suggesting that when both base excision and mismatch repair pathways are defective, the mutagenic effects of spontaneous cytosine deamination are sufficient to increase cancer incidence but do not preclude mouse development.     

Comparison of dose-response relationships for ethyl methanesulfonate and 1-ethyl-1-nitrosourea in Drosophila melanogaster spermatozoa

The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents. 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine. Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site. To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa. For both mutagens the dose response curve was linear and extrapolated to the origin. The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3). Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS. In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G. If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS. In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS. It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis.     

DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues.

Derivatives of phage M13 were constructed and used for the in vitro preparation of heteroduplex DNA molecules containing base/base mismatches that mimick DNA lesions caused by hydrolytic deamination of 5-meC residues in Escherichia coli DNA (i.e. they carry a T/G mismatch in the special sequence context provided by the recognition site -CCA/TGG-of the Dcm-methyltransferase). Upon introduction of these heteroduplex DNAs into CaCl2-treated E. coli cells, the mismatches are efficiently repaired with high bias in favour of the DNA strand containing the mismatched guanine residue. This special DNA mismatch-repair operates on fully dam-methylated DNA and is independent of gene mutH. It thus fulfills the salient requirements of a repair pathway responsible for counteracting the spontaneous hydrolytic deamination of 5-meC in vivo. The repair efficiency is boosted by a 5-methyl group present on the cytosine residue at the next-nearest position to the 5' side of the mismatched guanine. The repair is severely impaired in host strains carrying a mutation in any of the three loci dcm, mutL and mutS.     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.     

Nickel-guanine interactions in DNA: crystal structure of nickel-d[CGTGTACACG]2

The aim of this study was to clarify whether Ni2+ ions could bind to guanine bases in a standard B-DNA duplex and eventually induce a B-->Z transition. We have determined by X-ray crystallography at 3.1 A resolution the structure of the alternating deoxynucleotide d(CGTGTACACG), which contains both internal and terminal guanines. The duplex is in the B form. It is shown that nickel ions bind selectively to the N7 atom of guanine 10, which is in an extra-helical position, and guanine 2, which is in the terminal position of the duplex. It does not bind to guanine 4, which lies within a standard B-DNA tract. This simple but unambiguous result proves that nickel ions select between different guanines via steric accessibility. Guanine-Ni2+-guanine bridges among symmetry-related duplexes have also been found. These bridges may explain why Ni2+ ions may act either as a precipitant or a renaturing agent for DNA under certain conditions. The biochemical interaction of nickel with DNA can thus be related to its capacity to specifically bind to B-DNA regions with exposed guanines. Also, from the structural point of view, we have found a terminal cytosine, which forms a C.G:C reverse-Hoogsteen triple structure with a base pair of a neighbor duplex. This type of triplet is seldom found and is here described for the first time for a DNA structure.     

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.     

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.     

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.     

Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alkA umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada'-lacZ' and umuC'-lacZ' plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine.     

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]₂. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.     

Mechanism of translocation of uracil-DNA glycosylase from Escherichia coli between distributed lesions

Uracil–DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg²⁺ ions suppressed the search in double-stranded DNA to a higher extent than K⁺ likely due to tight binding of Mg²⁺ to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ∼100 nucleotides and translocation rate constant of ∼2 × 10⁶ s⁻¹.     

A review of the genetic effects of ethyl methanesulfonate

Ethyl methanesulfonate (EMS) is a monofunctional ethylating agent that has been found to be mutagenic in a wide variety of genetic test systems from viruses to mammals. It has also been shown to be carcinogenic in mammals. Alkylation of cellular, nucleophilic sites by EMS occurs via a mixed SN1/SN2 reaction mechanism. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups. Genetic data obtained using microorganisms suggest that EMS may produce both GC to AT and AT to GC transition mutations. There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions. In higher organisms, there is clear-cut evidence that EMS is able to break chromosomes, although the mechanisms involved are not well understood. An often cited hypothesis is that DNA bases ethylated by EMS (mostly the N-7 position of guanine) gradually hydrolyze from the deoxyribose on the DNA backbone leaving behind an apurinic (or possibly an apyrimidinic) site that is unstable and can lead to single-strand breakage of the DNA. Data also exist that suggest that ethylation of some chromosomal proteins in mouse spermatids by EMS may be an important factor in causing chromosome breakage.