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At least 55 records · Page 3Linked to original sources
PubMed1981

The mat-1 gene in Chlamydomonas regulates DNA methylation during gametogenesis.

The inheritance of chloroplast genes in Chlamydomonas is regulated by methylation of chloroplast DNA during gametogenesis. The wild-type pattern of maternal inheritance results from the methylation of chloroplast DNA in female (mt+) but not in male (mt-) gametes, leading to preferential degradation of chloroplast DNA of male origin in zygotes. This paper describes the distribution of 5-methyl cytosine residues in restriction fragments of chloroplast DNA sampled during gametogenesis by two methods: ethidium bromide staining of agarose gels, and binding of antibody directed against 5-methyl cytosine onto restriction fragments blotted to nitro-cellulose paper. Methylated cytosines are located in most if not all Eco RI and Msp I fragments, but the extent of methylation is not proportional to fragment size. The mat-1 mutation carried by males converts maternal inheritance. Chloroplast DNA of male gametes carrying the mat-1 mutation becomes methylated during gametogenesis. This methylation protects against restriction enzyme-promoted degradation in zygotes, as shown by physical data demonstrating the transmission to progeny of chloroplast genes carried on chloroplast DNA of the mat-1 male parent. Thus the mat-1 gene, which is linked to the mating-type locus, determines whether or not methylation of chloroplast DNA will occur in males during gametogenesis.

Chlamydomonas↗
PubMed2005

Translational repression of C. elegans p53 by GLD-1 regulates DNA damage-induced apoptosis.

p53 is a tumor suppressor gene whose regulation is crucial to maintaining genome stability and for the apoptotic elimination of abnormal, potentially cancer-predisposing cells. C. elegans contains a primordial p53 gene, cep-1, that acts as a transcription factor necessary for DNA damage-induced apoptosis. In a genetic screen for negative regulators of CEP-1, we identified a mutation in GLD-1, a translational repressor implicated in multiple C. elegans germ cell fate decisions and related to mammalian Quaking proteins. CEP-1-dependent transcription of proapoptotic genes is upregulated in the gld-1(op236) mutant and an elevation of p53-mediated germ cell apoptosis in response to DNA damage is observed. Further, we demonstrate that GLD-1 mediates its repressive effect by directly binding to the 3'UTR of cep-1/p53 mRNA and repressing its translation. This study reveals that the regulation of cep-1/p53 translation influences DNA damage-induced apoptosis and demonstrates the physiological importance of this mechanism.

3' Untranslated Regions↗
PubMed1995

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.

Animals↗
PubMed2002

Retinoblastoma tumor suppressor targets dNTP metabolism to regulate DNA replication.

The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G(2) phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control S-phase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.

Adenoviridae↗
PubMed1997

Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex.

The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA. These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

Adenosine Triphosphatases↗
PubMed1999

L-arginine metabolites regulate DNA synthesis and nitric oxide synthase activity in cultured human dermal microvascular endothelial cells--potential positive and negative regulators of angiogenesis derived from L-arginine.

Human dermal microvascular endothelial cell (HDMVEC) growth and proliferation is important for skin angiogenesis. Our data support the hypothesis that a regulatory switch from an angiogenic to a nonangiogenic stimulus involves a change in l-arginine metabolism from polyamines (PAs) to nitric oxide (NO) and, conversely, a change to an angiogenic stimulus is expected to drive l-arginine metabolism in favor of PA synthesis. Current studies with cultured HDMVECs demonstrated that NO inhibited, whereas the PA putrescine stimulated DNA synthesis. The nitric oxide synthase (NOS) inhibitor NG-nitro l-arginine methyl ester stimulated DNA synthesis with a significant increase from control at 2.5 and 5 mM (p < 0.05); in contrast, the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethyl ornithine (DFMO) inhibited DNA synthesis with a significant decrease from control at 19.65, 29.48, and 39.48 microM (p < 0.05). NO donors sodium nitroprusside dihydrate, S-nitroso-N-penicillamine, and l-hydroxy-2-oxo-3(3-aminopropyl)-3-propyl-triazine all inhibited DNA synthesis. There was a significant increase in NOS activity with DFMO treatment (p < 0.05) and a less pronounced decrease in NOS activity with direct PA treatment. In summary, NO and PAs are important mediators of HDMVEC growth and PAs downregulate NOS activity in these cells. NOS and ODC enzyme products may not only be important components in the regulation of angiogenesis in the skin but they may also regulate their own synthesis via feedback mechanisms.

Arginine↗
PubMed1994

12(S)-hydroxyeicosatetraenoic acid regulates DNA synthesis and protooncogene expression induced by epidermal growth factor and insulin in rat lens epithelium.

Neonatal rat lens epithelium has a high 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] synthetic capacity, which decreases as epithelial cell proliferation decreases with age. To determine whether products of the 12-lipoxygenase pathway are involved in lens cell proliferation, we measured the effect of 12-lipoxygenase inhibitors on endogenous 12-HETE production, epidermal growth factor/insulin-stimulated DNA synthesis and protooncogene expression in cultured neonatal rat lens epithelial cells. Incubation of neonatal rat lenses in epidermal growth factor plus insulin, which stimulated endogenous 12-HETE production 8- to 10-fold, also produced a transient induction of c-fos and c-myc mRNAs after 2 to 3 h, followed by a round of DNA synthesis approximately 20 h later. The lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, strongly inhibited both the endogenous 12-HETE synthesis and growth factor-stimulated DNA synthesis with a half-maximal inhibition between 10 and 20 microM. Cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (10 microM) also inhibited the expression of c-fos and c-myc mRNA and, to a lesser extent, c-jun mRNA. The inhibitory effects of cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate on protooncogene expression and DNA synthesis were prevented by 0.3 microM 12(S)-HETE but not by equivalent concentrations of either 5(S)-HETE or 15(S)-HETE. These findings suggest that endogenously synthesized 12(S)-HETE may mediate epidermal growth factor/insulin-stimulated DNA synthesis in neonatal rat lens epithelial cells by regulating protooncogene expression.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2001

Inhibitory module of Ets-1 allosterically regulates DNA binding through a dipole-facilitated phosphate contact.

DNA binding of the transcription factor Ets-1 is negatively regulated by three inhibitory helices that lie near the ETS domain. The current model suggests that this negative regulation, termed autoinhibition, is caused by the energetic expense of a DNA-induced structural transition that includes the unfolding of one inhibitory helix. This report investigates the role of helix H1 of the ETS domain in the autoinhibition mechanism. Previous structural studies modeled the inhibitory helices packing together and connecting with helix H1, suggesting a role of this helix in the configuration of an inhibitory module. Recently, high-resolution structures of the ETS domain-DNA interface indicate that the N terminus of helix H1 directly contacts DNA. The contact, which is augmented by the macrodipole of helix H1, consists of a hydrogen bond between the amide NH of leucine 337 in helix H1 and the oxygen of a corresponding phosphate. We propose that this hydrogen bond positions helix H1 to be a link between autoinhibition and DNA binding. Four independent approaches tested this hypothesis. First, the hydrogen bond was disrupted by removal of the phosphate in a missing phosphate analysis. Second, base pairs that surround the helix H1-contacting phosphate and appear to dictate DNA backbone conformation were mutated. Next, a hydrophobic residue in helix H1 that is expected to position the N terminus of the helix was altered. Finally, a residue on the surface of helix H1 that may contact the inhibitory elements was changed. In each case DNA binding and autoinhibition was affected. Taken together, the results demonstrate the role of the dipole-facilitated phosphate contact in DNA binding. Furthermore, the findings support a model in which helix H1 links the inhibitory elements to the ETS domain. We speculate that this helix, which is conserved in all Ets proteins, provides a common route to regulation.

Allosteric Regulation↗
PubMed1998

Identification of redox-sensitive cysteines in GA-binding protein-alpha that regulate DNA binding and heterodimerization.

The transcription factor GA-binding protein (GABP) is composed of two subunits, GABPalpha and GABPbeta. The DNA-binding subunit, GABPalpha, is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABPbeta, which lacks a discernible DNA binding domain, through ankyrin repeats in the NH2 terminus of GABPbeta. We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABPalpha. To determine the roles of individual cysteines in GABP redox regulation, we generated a series of serine substitution mutants by site-directed mutagenesis and identified three redox-sensitive cysteine residues in GABPalpha (Cys388, Cys401, and Cys421). Sulfhydryl modification of Cys388 and Cys401 inhibits DNA binding by GABPalpha, whereas, modification of Cys421 has no effect on GABPalpha DNA binding but inhibits dimerization with GABPbeta. The positions of Cys388 and Cys401 within the known Ets-domain structure suggest two very different mechanisms for redox regulation of DNA binding. Sulfhydryl modification of Cys388 could directly interfere with DNA binding or might alter the positioning of the DNA-binding helix 3. Modification of Cys401 may inhibit DNA binding through stabilization of an inhibitory helix similar to that described in the Ets-1 protein. Thus, GABP is regulated through at least two redox-sensitive activities, DNA binding and heterodimerization.

Alkylation↗
PubMed2006

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication.

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.

Base Sequence↗
PubMed2025

Transposition element MERVL regulates DNA demethylation through TET3 in oxidative-damaged mouse preimplantation embryos.

Transposable elements (TEs) comprise approximately half of eukaryotic genomes and significantly contribute to genome plasticity. In this study, we focused on a specific TE, MERVL, which exhibits particular expression during the 2-cell stage and commonly serves as an indicator of embryonic totipotency. However, its precise role in embryo development remains mysterious. We utilized DRUG-seq to investigate the effects of oxidative damage on genes and TEs expression. Our findings revealed that exposure to hydrogen peroxide (H2O2) could induce DNA damage, apoptosis, and incomplete DNA demethylation in embryos, which were potentially associated with MERVL expression. To further explore its function, antisense nucleotides (ASO) targeting MERVL were constructed to knockdown the expression in early embryos. Notably, this knockdown led to the occurrence of DNA damage and apoptosis as early as the 2-cell stage, consequently reducing the number of embryos that could progress to the blastocyst stage. Moreover, we discovered that MERVL exerted an influence on the reprogramming of embryonic DNA methylation. In MERVL-deficient embryos, the activity of the DNA demethylase ten-eleven translocation 3 (TET3) was suppressed, resulting in impaired demethylation when compared to normal development. This impairment might underpin the mechanism that impacts embryonic development. Collectively, our study not only verified the crucial role of MERVL in embryonic development but also probed its regulatory function in DNA methylation reprogramming, thereby laying a solid foundation for further investigations into MERVL's role.

Animals↗
PubMed1995

Dexamethasone inversely regulates DNA synthesis and phosphoenolpyruvate carboxykinase mRNA levels in cultured rat hepatocytes: interactions with insulin, glucagon, and transforming growth factor beta 1.

In hepatocytes, glucocorticoids control the expression of several genes and exert significant, but complex, regulation of the proliferation. To shed more light on the growth responses to glucocorticoids in these cells, we treated adult rat hepatocytes in primary culture with dexamethasone, in various combinations with other hormones (insulin, glucagon, transforming growth factor beta 1 (TGF beta 1)), and examined the relationship between the effects on the DNA synthesis and the mRNA level of phosphoenolpyruvate carboxykinase, a gene typically expressed in differentiated hepatocytes. Insulin exhibited the previously observed suppressing effect on the glucocorticoid-induced phosphoenolpyruvate carboxykinase mRNA level, and also reversed growth-inhibitory effects of the glucocorticoid. Dexamethasone and glucagon (via cAMP) acted strongly synergistically both in enhancing the phosphoenolpyruvate carboxykinase expression and inhibiting the growth, the inhibitory effect of glucagon on DNA synthesis being totally dependent on dexamethasone. The effects of dexamethasone plus glucagon on both the phosphoenolpyruvate carboxykinase mRNA abundance and the DNA synthesis were partially counteracted by insulin. Dexamethasone is permissive for a promoting effect of TGF beta 1 on phosphoenolpyruvate carboxykinase expression, and was found to increase the maximal inhibitory effect of (but reduced the sensitivity to) TGF beta 1 on the DNA synthesis. The results indicate that there is an inverse glucocorticoid-induced regulation of the DNA synthesis and the expression of a liver-typical gene.

Animals↗
PubMed2004

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start.

Acetyltransferases↗
PubMed2006

Developmentally regulated DNA methylation in Dictyostelium discoideum.

Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.

Animals↗