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PubMed2025

100+ years of phase variation: the premier bacterial bet-hedging phenomenon.

Stochastic, reversible switches in the expression of Salmonella flagella variants were first described by Andrewes in 1922. Termed phase variation (PV), subsequent research found that this phenomenon was widespread among bacterial species and controlled expression of major determinants of bacterial-host interactions. Underlying mechanisms were not discovered until the 1970s/1980s but were found to encompass intrinsic aspects of DNA processes (i.e. DNA slippage and recombination) and DNA modifications (i.e. DNA methylation). Despite this long history, discoveries are ongoing with expansions of the phase-variable repertoire into new organisms and novel insights into the functions of known loci and switching mechanisms. Some of these discoveries are somewhat controversial as the term 'PV' is being applied without addressing key aspects of the phenomenon such as whether mutations or epigenetic changes are reversible and generated prior to selection. Another 'missing' aspect of PV research is the impact of these adaptive switches in real-world situations. This review provides a perspective on the historical timeline of the discovery of PV, the current state-of-the-art, controversial aspects of classifying phase-variable loci and possible 'missing' real-world effects of this phenomenon.

Gene Expression Regulation, Bacterial↗
PubMed1992

DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin.

The antineoplastic benzanthroquinone drug doxorubicin can undergo flavoenzyme-catalyzed one-electron reduction which, in an aerobic environment, leads to the generation of oxygen-derived species. We therefore sought to determine whether doxorubicin in the presence of NADH dehydrogenase and the transition metal ions Fe(III) or Cu(II) induces DNA base modifications in isolated human chromatin. NADH dehydrogenase-catalyzed reduction of doxorubicin (25-100 microM) caused hydroxyl radical production detected as methane generated from dimethyl sulfoxide; addition of isolated human chromatin to the system produced a concentration-dependent quenching of detectable hydroxyl radical formation. Doxorubicin (5-50 microM)-stimulated enzyme-catalyzed oxidation of NADH was also diminished, but still detectable, in the presence of chromatin. Doxorubicin-induced DNA base modifications in chromatin were measured by gas chromatography/mass spectrometry with selected-ion monitoring. Production of modified bases required the addition of transition metal ion and was enhanced by the addition of active flavoenzyme. The non-redox cycling analogue 5-iminodaunorubicin induced significantly less base modification than did doxorubicin. In the presence of Fe(III), NADH dehydrogenase-catalyzed reduction of doxorubicin caused enhancement in the content of all modified bases over control levels. Substitution of Cu(II) for Fe(III) altered both the degree and the pattern of doxorubicin/NADH dehydrogenase-induced base modifications. The scavengers of hydroxyl radical mannitol and dimethyl sulfoxide or catalase did not significantly affect doxorubicin/NADH/NADH dehydrogenase/transition metal ion-induced base modifications. Superoxide dismutase further enhanced production of all base modifications. The data demonstrate that flavoenzyme-catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, suggesting a possible mechanism for the mutagenicity of doxorubicin in vivo.

Chromatin↗
PubMed1992

DNA base modifications in chromatin of human cancerous tissues.

Free radical-induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady-state level of free radical-modified bases in cellular DNA. We have investigated endogenous levels of typical free radical-induced DNA base modifications in chromatin of various human cancerous tissues and their cancer-free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine-derived and six purine-derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected-ion monitoring. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack on DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts of modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified bases over the control levels. The results clearly indicate higher steady-state levels of modified DNA bases in cancerous tissues than in their cancer-free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.

Chromatin↗
PubMed1978

Bacteriophage Mu-induced modification of DNA is dependent upon a host function.

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.

Coliphages↗
PubMed1975

Host specificity of DNA in haemophilus influenzae: The physiological and genetical bases of instability of restriction and modification of DNA in strain Rd.

Further investigations of the instability of restriction and modification properties of H. influenzae Rd strain were carried out. It has been shown that the instable properties of hsd Hind1 system are maintained even after transfer of this system to another H. influenzae strain. The expression of hsd Hind1 system is very sensitive to various physiological changes which do not influence the other hsd systems present in the same Rd strain. The instability of hsd Hind1 system is postulated to be connected with some regulator gene(s).

DNA Restriction Enzymes↗
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↗
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↗
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↗
PubMed1976

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.

Adenine↗
PubMed1978

Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The structural modification of double-stranded circular DNA of simian virus 40 and plasmid ColE1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. Stepwise hydrolysis with endonuclease S1 and DNase followed by DNA base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of DNA, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. Of the two synthetic double-stranded polymers, poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), bound to the diol-epoxide, only the former showed a marked hydrolysis after endonuclease S1 treatment, whereas binding occurred 24-fold more on the latter.

Benzopyrenes↗
PubMed1979

Modification of DNA in chromatin with methyltransferase from Haemophilus influenzae Rd.

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein-free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA. Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers. Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.

Adenine↗
PubMed1977

Inhibition of transcription of supercoiled PM2 DNA by carbodiimide modification.

PM2 superhelican DNA (form I), which as been reacted with the single strand specific reagent, N-cyclohexyl-N'-beta-(methylmorpholinium)ethyl carbodiimide (CMC) is more than 95% inhibited in its ability to support transcription with E. coli B RNA polymerase in vitro. Almost complete inhibition of transcription was achieved after 2 hours of reaction with FI when only 1% of the bases were modified. A large increase in S20,* (from 26.8 S to 33.6 S) of FI DNA was observed during the course of reaction. Rifampicin resistant transcription is more susceptible to inhibition by CMC than total transcription, suggesting that the CMC is preferentially binding at promoter sites. These results clearly are in accord with the observation that supercoiled DNA contains localized regions of unpaired bases. The promotor sites for E. coli RNA polymerase in FI PM2 DNA appear to be located at or near these unpaired sites.

Carbodiimides↗
PubMed2026

SET domain bifurcated histone lysine methyltransferase 1 regulates histone modification and DNA damage response during zygotic genome activation in pigs.

SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) is a key epigenetic regulator that catalyzes histone H3 lysine 9 trimethylation (H3K9me3), a mark essential for transcriptional repression and heterochromatin formation. Here, we investigated the role of SETDB1 during zygotic genome activation (ZGA) in porcine embryos. SETDB1 knockdown (KD) was induced by microinjecting double-stranded RNA (dsRNA), and its impact on early embryonic development was evaluated. SETDB1 KD decreased H3K9me3 levels, markedly increased H3K9ac, and downregulated ZGA-associated genes. These epigenetic alterations were accompanied by impaired cleavage, reduced blastocyst formation, and a lower total cell number. Upon etoposide-induced DNA double-strand breaks, SETDB1 KD embryos showed reduced expression of key DNA repair proteins, failed to efficiently restore DNA integrity, and exhibited increased apoptosis, indicating a compromised DNA damage response and repair process. SETDB1 KD also reduced HDAC3 expression, suggesting that SETDB1 may regulate HDAC3 to maintain histone acetylation balance. Consistently, HDAC3 inhibition increased H3K9ac, decreased H3K9me3, and reduced SETDB1 protein levels, supporting a reciprocal regulatory relationship. Together, these findings indicate that SETDB1 is important for porcine embryonic development by coordinating histone modifications and safeguarding genomic integrity during ZGA, and they suggest that the interplay between SETDB1 and HDAC3 constitutes a potentially important epigenetic axis for proper histone modification dynamics and developmental competence.

Animals↗
PubMed1977

The photodynamic modification of DNA by hematoporphyrin.

Illumination (white light: 300-750 nm) of DNA in the presence of hematoporphyrin (less than or equal to 5-10 (-4) M) results in selective degradation of the guanine moiety. DNA so illuminated exhibits physical chemical properties (lowered sedimentation coefficients, lower temperatures of helix-coil transitions, increased buoyant density values) consistent with single-chain scissions (and the generation of single-stranded regions) which presumably are secondary to the photodegradation of the guanine residue. Illumination of DNA in the presence of low levels of hematoporphyrin (greater than or equal to 2.5 - 10 (-4) M) results in a biopolymer exhibiting all of the physical properties described above with the exception of a lowered sedimentation coefficient; on the contrary such DNA is aggregated. Of the four usual deoxynucleosides irradiated in the presence of hematoporphyrin, only deoxyguanosine is destroyed.

Binding Sites↗
PubMed1977

Chemical modification of DNA polymerase phosphoprotein from avian myeloblastosis virus.

Fractionation of purified avian myeloblastosis virus DNA polymerase, after phosphorylation in vitro, revealed the presence of a small acidic proten, a phosphate acceptor polypeptide with high specific activity. Its presence in the phosphorylated form with the polymerase resulted in as much as a 10-fold increase in the rate of DNA synthesis. Its presence in the dephosphorylated form with the polymerase had no effect in the rate of DNA synthesis.

Avian Leukosis Virus↗