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PubMed1978

Modification of DNA by aflatoxin B1 creates alkali-labile lesions in DNA at positions of guanine and adenine.

The damage to DNA by the hepatocarcinogen aflatoxin B1 was investigated. A DNA fragment of known sequence of the lactose promoter-operator region was used as a substrate for modification by aflatoxin B1. The DNA was incubated with aflatoxin B1 in crude mammalian liver extracts or with purified microsomes. Treatment of the DNA incubated in the complete system with either 1 M piperidine or 0.1 M NaOH at 90 degrees revealed alkali-labile lesions in the DNA. The exact location of the cleavage site was determined by comparison of the length of the cleavage products with the known sequence on polyacrylamide gels. The lengths of the cleavage products were the same as those produced by alkali-induced breakage of the same sequence of DNA that had been modified with dimethyl sulfate. The major cleavage products of the aflatoxin B1-modified DNA were at positions of guanine and the minor cleavage products were at positions of adenine. These studies show that modification of DNA by aflatoxin B1 creates alkali-labile sites at positions of guanine and, to a lesser extent, adenine.

Aflatoxins↗
PubMed1997

Laser-induced modification of DNA and Poly[A,G] at guanine moiety using acetone as photosensitizer.

The interactions of triplet acetone with polyadenylic acid (Poly[A]), polyguanylic acid (Poly[G]), polyadenylic-guanylic acid (Poly[A,G]) and single-stranded DNA (ssDNA) were investigated in neutral aqueous solution using KrF (248 nm) laser flash photolysis. The transient absorption spectra and kinetics of DNA and polynucleotides obtained under acetone sensitization demonstrated that the predominant transient species was guanine radical. These novel findings have offered time-resolved evidence for photochemical modification of DNA and Poly[A,G] at guanine moiety.

Acetone↗
PubMed1993

Bile acid-induced modifications in DNA synthesis by the regenerating perfused rat liver.

Liver cell proliferation is a complex process that can be affected by a large number of factors such as bile acids, which have been reported to be associated to the pathogenesis of liver cancer. In this work, bile acid-induced modifications in DNA synthesis by regenerating perfused rat liver were investigated. Two-thirds hepatectomy was carried out 24 hr before perfusion of liver with recirculating, erythrocyte-free Krebs-Henseleit solution. The viability of the preparations was maintained under all experimental conditions, as indicated by bile flow, oxygen uptake, perfusion pressure, perfusion flow and release of lactate dehydrogenase and potassium into the perfusate. Livers received (min 10 to min 60) bile acid infusion at a rate of 25 nmol/min/gm liver (i.e., maximal secretion rate/2) in regenerating livers as calculated for taurocholate in separate experiments). Trace amounts of [methyl-14C]thymidine were added to the perfusate at min 30. At the end of the experiments (min 60) the livers were washed, removed, weighed and homogenized to determine radioactivity in whole tissue, in DNA and in non-DNA-related fractions. Taurocholate and, to a lesser extent, taurodeoxycholate and dehydrocholate (but not ursodeoxycholate) were found to reduce 14C incorporation into DNA. This was not due to changes in the content of 14C in whole, regenerating liver tissue. Taurocholate, taurodeoxycholate, dehydrocholate and ursodeoxycholate had no effect on thymidine uptake; moreover, the proportion of 14C found in bile was negligible. However, bile acid-induced modification in the fate of intracellular thymidine was observed. In regenerating livers receiving no bile acid, the 14C carried by thymidine metabolites accounted for about 60% of 14C in whole liver tissue. Taurocholate markedly increased this proportion to about 80%. Reverse-phase high-pressure liquid chromatography revealed that most of this 14C (about 80%) was recovered at the elution time, corresponding to thymidine catabolites rather than to DNA precursors. These results suggest that bile acids induce enhancement of thymidine catabolism that reduces its incorporation into DNA; inhibition in the process of DNA synthesis itself, leading to a subsequent increase in the metabolism of DNA precursors; or both. Moreover, from the diversity in this property for bile acid species it might be inferred that changes in the composition and size of the bile acid pool during liver carcinogenesis or regeneration play a role in the modulation of the proliferative process.

Animals↗
PubMed1993

[Porphyrin derivatives of oligonucleotides. I. Synthesis of oligonucleotide derivatives bearing 2,4-di(alpha-(2-hydroxyethoxy)ethyl)- deuteroporphyrin IX or a metal complex of it and study of the oxidative modification of DNA by these derivatives].

A method for coupling 2,4-di[alpha-(2-hydroxyethoxy)ethyl] deuteroporphyrin dimethyl ether (IX), DDPOH and its complexes with metals to the 5'- or 3'-end of oligonucleotides was elaborated. In the presence of an oxidizing agent (H2O2), Fe(III)DDP-derivatives of oligonucleotides modified single-stranded DNA. The reaction was strictly site-specific and occurred at two neighbouring guanosine residues. A few types of modification were observed: cross-linking, modification leading to DNA cleavage upon piperidine treatment, and direct chain scission. The total modification yield reached 90%. Covalent attachment of Fe(III)DDP-group to oligonucleotides increased the efficiency of their uptake and the melting temperature of their complementary complexes.

Base Sequence↗
PubMed1978

Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: relation to chromatin structure and reconstitution.

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S1 than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the 'open' regions of chromatin, and 35% to 'closed' regions. These results indicate that although convalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.

Animals↗
PubMed1994

DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate.

Ferric nitrilotriacetate (Fe-NTA) causes renal proximal tubular necrosis, a consequence of iron ion-mediated free-radical-associated damage, that finally leads to a high incidence of renal adenocarcinoma in male rats and mice. We have investigated the levels of typical hydroxyl radical-induced DNA base modifications in renal chromatin of male Wistar rats treated with a single or repeated administrations of Fe-NTA. Five pyrimidine-derived and 5 purine-derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected-ion monitoring. The modified bases were 5-hydroxy-5-methylhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, thymine glycol, 5,6-dihydroxyuracil, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine and 8-hydroxyguanine. The amounts of most of these compounds were significantly increased over control levels in renal chromatin of Fe-NTA-treated rats as measured 3 and 24 hr after treatment. Elevated levels of modified bases were accompanied by proximal tubular necrosis. On the 19th day, however, accumulation of modified DNA bases was not observed. Morphologically, scattered karyomegalic cells were seen in the proximal tubules, but necrosis was rarely found. Some of the identified DNA base lesions are known to be promutagenic, although others have not been investigated. Presence of modified DNA bases concomitant with necrosis and regeneration of the renal proximal tubules may be a critical step in Fe-NTA-induced carcinogenesis.

Animals↗
PubMedDate not supplied

DNA methylation and epigenetic mechanisms.

Genes are essential for the transmission of genetic information from generation to generation, and this mechanism of inheritance is fully understood. Genes are also essential for unfolding the genetic program for development, but the rules governing this process are obscure. Epigenetics comprises the study of the switching on and off of genes during development, the segregation of gene activities following somatic cell division, and the stable inheritance of a given spectrum of gene activities in specific cells. Some of these processes may be explained by DNA modification, particularly changes in the pattern of DNA methylation and the heritability of that pattern. There is strong evidence that DNA methylation plays an important role in the control of gene activity in cultured mammalian cells, and the properties of a CHO mutant strain affected in DNA methylation are described. Human diploid cells progressively lose cytosine methylation during serial subculture, and this may be related to their in vitro senescence. There is also evidence that DNA modifications can be inherited through the germ line. Classical genetics is based on the study of all types of change in DNA base sequence, but the rules governing the activity of genes by epigenetic mechanisms are necessarily different. Their elucidation will depend both on a theoretical framework for development and on experimental studies at the molecular, chromosomal, and cellular levels.

Animals↗
PubMed1997

[Interaction of short nucleotide derivatives with nucleic acids. IV. Modification of DNA by an alkylating tetranucleotide reagents in the presence of effectors in perfect and imperfect complexes].

It was demonstrated that any mismatches in a complex formed by an ssDNA target and a tetranucleotide at 25 or 37 degrees C can be discriminated by alkylating the DNA with a tetranucleotide carrying a 4-[N-methyl-N-(2-chloroethyl)]aminobenzylethylamine residue at the 5'-terminal phosphate in the presence of a pair of flanking effectors, octanucleotide di-N-(2-hydroxyethyl)-phenazinium derivatives. The discrimination factor (ratio of the extent of the target modification in the perfect and mismatch-containing complexes) for a single mismatch in the tetranucleotide binding site at 25 degrees C varied between 4 and 500 depending on the type of mismatch and its location in the complex and exceeded 400 at 37 degrees C for all the investigated mismatches. The DNA target modification by the alkylating derivative of the 3'-estrone ester of tetranucleotide pCAGX (mean = C, T, A or G) was selective in the presence of a pair of hydrophobic effectors, octanucleotide 5'-cholesteryl-3'-phenazinium derivatives. The discrimination factors for 3'-terminal mismatches T.G, A.G, and G.G were 1,8,400, and 400, respectively.

Alkylating Agents↗
PubMed1989

Hydroxyl radical mediated DNA base modification by manmade mineral fibres.

Manmade mineral fibres (MMMFs) were examined for their ability to hydroxylate 2-deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OH-dG), a reaction that is mediated by hydroxyl radicals. It appeared that (1) catalase and the hydroxyl radical scavengers, dimethylsulphoxide and sodium benzoate, inhibited the hydroxylation, whereas Fe2+ and H2O2 potentiated it; (2) pretreatment of MMMFs with the iron chelator, deferoxamine, or with extensive heat (200-400 degrees C), attenuated the hydroxylation; (3) the hydroxylation obtained by various MMMFs varied considerably; (4) there was no apparent correlation between the hydroxylation and the surface area of different MMMFs, although increasing the surface area of a fibre by crushing it increased its hydroxylating capacity; and (5) there was good correlation between the hydroxylation of dG residues in DNA and the hydroxylation of pure dG in solution for the 16 different MMMFs investigated. These findings indicate that MMMFs cause a hydroxyl radical mediated DNA base modification in vitro and that there is considerable variation in the reactivity of different fibre species. The DNA modifying ability seems to depend on physical or chemical characteristics, or both, of the fibre.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1989

Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities.

I-compounds are non-polar covalent DNA modifications of as yet undetermined structure that tend to accumulate in an age-dependent manner in tissues of untreated animals. They are detectable by 32P-postlabeling assay because of their adduct-like properties and chromatographically resemble DNA nucleotides containing bulky/hydrophobic moieties. To determine which factors may be involved in their formation, I-compounds were examined by 32P-postlabeling in liver and kidney DNA of female and male Sprague-Dawley rats and Syrian hamsters of different ages (1, 4 and 10 months and 1, 2.5 and 9.5 months, respectively). The following results were obtained: (i) Every tissue DNA studied contained characteristic I-compounds. (ii) Patterns and amounts of I-compounds were reproducible among animals of the same kind. (iii) There were pronounced organ and species differences. (iv) I-compound patterns were sex-dependent. (v) I-compound levels increased with age in all tissues studied, except in male hamster kidney, a target organ of estrogen-induced carcinogenesis. The highest levels were observed in liver and kidney of 10-month-old female rats. (vi) The rise of I-compound levels was less steep during the later part of the observation period for female but not male animals. (vii) Gonadectomy decreased I-compound levels in female hamster kidney DNA, while causing a slight increase in male animals later in life. These I-compounds were identical to previously reported DNA modifications that increased in male hamster kidneys after prolonged estrogen treatment. Points, iv, vi and vii strongly implicated sex hormones in I-compound formation. The qualitative effects of species, tissue differentiation, gender and sex hormones on these DNA modifications support the hypothesis that I-compounds are formed by the binding of endogenous electrophiles to DNA. As persistent DNA alterations, they are likely to affect DNA replication and to play a role in spontaneous and chemically induced carcinogenesis and in aging.

Aging↗
PubMed1996

DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata.

Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.

Alkylation↗
PubMed1980

Virus adaptation to host cells: the non-classical modification of phage T3.

Bacterial virus T3 undergoes host-controlled modification which is not based on "classical" processes of DNA modification and restriction. The adsorption and thus the growth of T3 on Escherichia coli W cells (E. coli K12 derivative) decisively depends on the host strain on which the virus was previously propagated. Depending on the modification conferred to the virus by its last host, its efficiency of plating (e.o.p.) on E. coli W varies by six orders of magnitude between 10(-7) and 10(-1). This does not reflect the appearance of T3 host-range mutants, but a fully reversible modification of genotypically unchanged T3 wild-type phage. The behaviour of T3 in the described host system constitutes a second case of so-called non-classical modification and restriction (KRUGER et al. 1977, Molec. gen. Genet. 153, 107-110) of bacteriophages. Non-classical modification (protein modification) is additive to and independent of DNA modification and restriction as demonstrated with the ocr- phage T3/R7. - Furthermore, our results suggest that the adsorption specificity of T3 is determined by at least two independent genetic factors; in both of these factors T3 differs from T7.

Adaptation, Physiological↗
PubMed1998

Sulfotransferase-mediated genotoxicity of propane 2-nitronate in cultured ovine seminal vesicle cells.

2-Nitropropane (2-NP) is a well-known genotoxin and carcinogen in rat liver. Several metabolic pathways, particularly cytochrome P450-, peroxidase- and sulfotransferase-dependent ones, have been suggested to lead to the formation of DNA-reactive species from 2-NP. Because rat liver cells express most types of xenobiotic-metabolizing enzymes, the role of specific pathways in the metabolic activation of 2-NP is difficult to assess in these cells. We have therefore investigated the genotoxicity of 2-NP and its anionic form, propane 2-nitronate (P2N), in cultured ovine seminal vesicle (OSV) cells. OSV cells lack cytochrome P450-dependent monooxygenase activity, but express prostaglandin-H-synthase (PHS) and, as we found out, phenol sulfotransferase. The induction of DNA repair synthesis and specific DNA modifications served as indicators for the genotoxicity of 2-NP and P2N. Both forms strongly induced repair, P2N being more active than 2-NP. The secondary nitroalkanes nitrocyclopentane and nitrocyclohexane also induced repair, whereas 1-nitropropane and the reduction product of 2-NP, acetone oxime, did not. P2N also elicited the formation of the characteristic DNA modifications 'DX1' and 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine residues in the DNA. Pretreatment of OSV cells with indomethacin, an inhibitor of PHS, affected neither the induction of repair nor the formation of the DNA modifications, and P2N was not a reducing substrate for the PHS-peroxidase activity. In contrast, the sulfotransferase inhibitor pentachlorophenol strongly reduced genotoxicity. The results show that cytochrome P450-dependent monooxygenases are not required for the metabolic conversion of secondary nitroalkanes or their nitronates into DNA-damaging products, nor is PHS involved in the metabolic activation. Instead, the data corroborate an essential role of sulfotransferase(s) in the genotoxicity and carcinogenicity of secondary nitroalkanes. Moreover, it is demonstrated for the first time that these compounds can be genotoxic in cells other than hepatocytes or hepatoma cells. This implies that in species other than the rat, organs other than the liver can be targets for the genotoxicity, and possibly carcinogenicity, of secondary nitroalkanes.

Animals↗
PubMed2002

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1999

Acute elevation by short-term dietary restriction or food deprivation of type I I-compound levels in rat liver DNA.

Type I I-compounds are bulky endogenous DNA modifications detectable by 32P postlabeling that exhibit age, species, tissue, genotype, gender, and diet dependence. Their formation appears unrelated to oxidative stress. In fact, several lines of indirect evidence suggest that many type I I-compounds may represent normal functional DNA modifications. For example, long-term dietary restriction (DR), which retards the development of age-related diseases including cancer and extends median and maximum life spans, unexpectedly elicits significant increases rather than decreases in the levels of many I-compounds in different rodent tissues. Positive linear correlations have been observed between such levels and median life spans of the animals. In the present work we have investigated 1) whether elevation of I-compound levels does not depend on chronic DR, i.e., occurs after a short period of DR or fasting, and 2) whether I-compound levels return to control values after the animals are returned to unrestricted feeding after food deprivation. Female Fischer 344 rats (approx 140 g each) were randomized into three groups. Group I was fed a natural ingredient (Purina 5001) diet ad libitum (AL) throughout the study, Group 2 was switched to 60% of the AL amount (40% DR) at 0 hour, and Group 3 was given no food for up to 72 hours and then returned to AL feeding until the end of the experiment. Liver DNA of individual rats (n = 4) was isolated for I-compound analysis at 24, 72, and 240 hours. Restricted and food-deprived rats showed elevated levels of hepatic I-compounds, with fasting eliciting the highest levels. These effects were seen as early as the 24-hour time point. Refeeding after 72 hours of food deprivation restored the levels to control values, measured at 240 hours. Our observations are discussed in relation to carcinogenesis and tumor promotion. The almost instantaneous changes of endogenous DNA modifications showed their exquisite sensitivity to nutritional factors and provided strong new evidence for precise regulation of their formation and removal.

Animals↗
PubMed2000

Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells.

Okadaic acid (OA) is a marine toxin, a tumour promoter and an inducer of apoptosis. It mainly inhibits protein-phosphatases, protein synthesis and enhances lipid peroxidation. Cadmium (Cd) is known to be carcinogenic in animals and humans (group 1 according to the International Agency for Research on Cancer (IARC) classification). Cd also induces oxidative stress in living organisms. Since they are sometimes found simultaneously in mussels, we have evaluated in the present investigation, the lipid peroxidation, as malondialdehyde (MDA) production, in the variation of the ratios of 8-(OH)-dG/10(5)dG and m5dC/(dC + m5dC) induced by OA and/or Cd in Caco-2 cells. When cells were treated exclusively by OA (15 ng/ml) or Cd (0.625 and 5 microg/ml) for 24 h, protein synthesis was inhibited (by 42 +/- 5%, 18 +/- 13%, and 90 +/- 4% respectively) while MDA production was 2,235 +/- 129, 1710 +/- 20, and 11,496 +/-1,624 pmol/mg protein respectively. In addition, each toxicant induced modified bases in DNA; increases in oxidised bases and methylated dC. The combination of OA and cadmium was more cytotoxic and caused more DNA base modifications; the ratio m(5)dC/(m(5)dC + dC) was increased from 3 +/- 0.15 to 9 +/- 0.15 and the ratio 8-(OH)-dG/10(5) dG also (from 36 +/- 2 to 76 +/- 6). The combination of OA and Cd also increased the level of MDA (1,6874 +/- 2,189 pmole/mg protein). The present results strongly suggest that DNA damage resulting from the oxidative stress induced by these two toxicants may significantly contribute to increasing their carcinogenicity via epigenetic processes.

5-Methylcytosine↗
PubMed2026

TRIM28 regulates the G2/M transition via histone modification and DNA damage repair during mouse oocyte meiosis.

TRIM28, a member of the tripartite motif (TRIM) family, functions as a transcriptional coregulator involved in maintaining genome stability during mitosis. In this study, we explored the role of TRIM28 in mouse oocyte meiotic maturation, where transcriptional activity is barely detectable. We found that TRIM28 was constitutively expressed during the early stages of oocyte meiotic maturation, with predominant nuclear localization in germinal vesicle (GV)-stage oocytes. TRIM28 depletion caused defective germinal vesicle breakdown (GVBD), but oocytes that successfully underwent GVBD displayed unimpaired first polar body (PB1) extrusion. TRIM28 depletion impaired CDK1 activity and reduced cyclin B1 levels, leading to a delay in the G2/M transition. This delay might be caused by altered levels of HDAC2-mediated H4K12ac and H3K4me2-modulated H3K9me2 in nonsurrounded nucleolus (NSN)-type GV oocytes, which decreased transcription activity. Additionally, TRIM28-depleted oocytes exhibited elevated γ-H2A.X expression, accompanied by aberrant expression of CHK1 and CHK2, as well as dysregulated expression of RAD51, which collectively contributed to GVBD failure in mouse oocytes. In conclusion, our findings indicate that TRIM28 participates in the regulation of the G2/M transition during mouse oocyte meiotic maturation, acting through the modulation of histone modifications and DNA damage repair.

Animals↗
PubMed1989

Base and conformational specificity of an amine modification of DNA.

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A-form, Z-form, or the single-strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH2O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of adduct formation under standard reaction conditions (27 degrees C, 0.63M CH2O, 0.007M n-butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI-dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG-dC) show enhanced 2'-endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA-dT) in the "X"-form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23, 1-4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B-form of polynucleotides as demonstrated by the failure of poly(dG-m5dC) in the stable Z-form to exhibit substantial reaction. The B-form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗