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PubMed1997

In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor.

In vitro experiments point to fatty acyl coenzymes A (acyl-CoAs) rather than unesterified fatty acids as the small-molecule ligands regulating DNA binding by the FadR protein of Escherichia coli. To provide an in vivo test of this specificity, unesterified fatty acids were generated within the cellular cytosol. These fatty acids were found to be efficient modulators of FadR action only when the acids could be converted to acyl-CoAs.

Acyl Coenzyme A↗

PubMed2005

SHP-2 phosphatase regulates DNA damage-induced apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively.

SHP-2, a tyrosine phosphatase implicated in diverse signaling pathways induced by growth factors and cytokines, is also involved in DNA damage-triggered signaling and cellular responses. We previously demonstrated that SHP-2 played an important role in DNA damage-induced apoptosis and G2/M cell cycle checkpoint. In the present studies, we have provided evidence that SHP-2 functions in DNA damage apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively. Mutant embryonic fibroblasts with the Exon 3 deletion mutation in SHP-2 showed decreased apoptosis and diminished G2/M arrest in response to cisplatin treatment. Wild type (WT), but not catalytically inactive mutant SHP-2 (SHP-2 C459S), rescued the apoptotic response of the mutant cells. Interestingly, both WT and SHP-2 C459S efficiently restored the G2/M arrest response. Furthermore, inhibition of the catalytic activity of endogenous SHP-2 in WT cells by overexpression of SHP-2 C459S greatly decreased cell death but not G2/M arrest induced by cisplatin. Biochemical analyses revealed that activation of c-Abl kinase was decreased in SHP-2 C459S-overexpressing cells. However, DNA damage-induced translocation of Cdc25C from the nucleus to the cytoplasm was fully restored in both WT and SHP-2 C459S "rescued" cells. Additionally, we demonstrated that the role of SHP-2 in DNA damage-induced cellular responses was independent of the tumor suppressor p53. Embryonic stem cells with the SHP-2 deletion mutation showed markedly decreased sensitivity to cisplatin-induced apoptosis, attributed to impaired induction of p73 but not p53. In agreement with these results, DNA damage-induced apoptosis and G2/M arrest were also decreased in SHP-2/p53 double mutant embryonic fibroblasts. Collectively, these studies have further defined the mechanisms by which SHP-2 phosphatase regulates DNA damage responses.

Animals↗

PubMed2005

Death inducer obliterator protein 1 in the context of DNA regulation. Sequence analyses of distant homologues point to a novel functional role.

Death inducer obliterator protein 1 [DIDO1; also termed DIO-1 and death-associated transcription factor 1 (DATF-1)] is encoded by a gene thus far described only in higher vertebrates. Current gene ontology descriptions for this gene assign its function to an apoptosis-related process. The protein presents distinct splice variants and is distributed ubiquitously. Exhaustive sequence analyses of all DIDO variants identify distant homologues in yeast and other organisms. These homologues have a role in DNA regulation and chromatin stability, and form part of higher complexes linked to active chromatin. Further domain composition analyses performed in the context of related homologues suggest that DIDO-induced apoptosis is a secondary effect. Gene-targeted mice show alterations that include lagging chromosomes, and overexpression of the gene generates asymmetric nuclear divisions. Here we describe the analysis of these eukaryote-restricted proteins and propose a novel, DNA regulatory function for the DIDO protein in mammals.

Anaphase↗

PubMed1997

The Jun kinase/stress-activated protein kinase pathway functions to regulate DNA repair and inhibition of the pathway sensitizes tumor cells to cisplatin.

We have studied the role of Jun/stress-activated protein kinase (JNK/SAPK) pathway in DNA repair and cisplatin resistance in T98G glioblastoma cells. JUN/SAPK is activated by DNA damage and phosphorylates serines 63 and 73 in the N-terminal domain of c-Jun, which is known to increase its transactivation properties. We show that treatment of T98G glioblastoma cells with cisplatin but not the transplatin isomer activates JNK/SAPK about 10-fold. T98G cells, which are highly resistent to cisplatin (IC50 = 140 +/- 13 microM), modified to express a nonphosphorylatable dominant negative c-Jun (termed dnJun) exhibit decreased viability following treatment with cisplatin, but not transplatin, in proportion (rPearson = 0.98) to the level of dnJun expressed leading to a 7-fold decreased IC50. Similar effects are observed in U87 cells, PC-3 cells, and MCF-7 cells, as well as in T98G cells modified to express TAM-67, a known inhibitor of c-Jun function. In contrast, no sensitization effect was observed in cells modified to express wild-type c-Jun. Furthermore, through quantitative polymerase chain reaction-stop assays, we show that dnJun expressing cells were inhibited in repair of cisplatin adducts (p = 0.55), whereas repair is readily detectable (p = 0.003) in parental cells. These observations indicate that the JNK/SAPK pathway is activated by cisplatin-induced DNA damage and that this response is required for DNA repair and viability following cisplatin treatment. Regulation of DNA repair following genotoxic stress may be a normal physiological role of the JNK/SAPK pathway.

Antineoplastic Agents↗

PubMed2003

Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation.

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

Amino Acid Sequence↗

PubMed1992

DNA synthesis control in yeast: an evolutionarily conserved mechanism for regulating DNA synthesis genes?

After yeast cells commit to the cell cycle in a process called START, genes required for DNA synthesis are expressed in late G1. Periodicity is mediated by a hexameric sequence, known as a MCB element, present in all DNA synthesis gene promoters. A complex that specifically binds MCBs has been identified. One polypeptide in the MCB complex is Swi6, a transcription factor that together with Swi4 also binds G1 cyclin promoters and participates in a positive feedback loop at START. The finding that Swi6 is directly involved in both START and DNA synthesis gene control suggest a model in which Swi6, activated through its participation in START, serves as the central transcription factor in coordinating late G1 gene expression. The mechanism may be conserved in all eukaryotic cells.

Animals↗

PubMed2002

Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei).

Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241). Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.

Amino Acid Sequence↗

PubMed2003

Structural polymorphism of telomeric DNA regulated by pH and divalent cation.

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.

Cations, Divalent↗

PubMed2004

Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylum.

The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria.

Bacterial Proteins↗

PubMed2000

Roles of zinc fingers and other regions of the transcription factor human MTF-1 in zinc-regulated DNA binding.

Mammalian metallothionein genes are transcriptionally regulated by heavy metals through cis-acting metal responsive elements (MREs). The MRE-binding transcription factor-1 (MTF-1), a protein containing six C(2)H(2)-type Zn fingers, is essential for MRE-mediated transcriptional activation. DNA binding of MTF-1 is known to be stimulated by Zn in vitro, but the binding was also largely influenced by redox conditions, suggesting that redox signals could modulate MTF-1 activity. To locate the functional domain required for Zn regulation, several deletion mutants of human MTF-1b, a newly cloned transcriptionally active MTF-1 variant, were characterized. This analysis showed that the N-terminal region and Zn-finger domain play roles in metal response. Functional roles of individual Zn fingers were estimated by co-transfection assays by using an MRE-driven reporter gene and vectors that express MTF-1b mutants each carrying one defective finger. Mutations in the N-terminal four fingers dramatically reduced the transcriptional activity, and at least for three of them the transcriptional defect was due to reduced DNA binding. These results indicate that the six Zn fingers are not functionally equivalent, probably sharing distinct roles such as direct DNA recognition and regulatory functions.

Binding Sites↗

PubMed2005

Oxidative stress-induced retinal damage up-regulates DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase in photoreceptor synaptic mitochondria.

Bright light triggers biphasic photoreceptor nuclear DNA fragmentation, suggesting a DNA-repair response (Invest Ophthalmol Vis Sci 43:3511; 2002; Adv Med Biol 533:229-240; Mol Neurobiol 28:111-122). Here, we demonstrate a remarkable increase in expression of the mitochondrial DNA-repair enzymes, DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase, following bright light treatment in rats. DNA polymerase gamma and 8-oxoguanine, the product of guanine oxidation, were selectively localized in photoreceptor synaptic terminals only within the superior central retinal region, where most light damage occurred. All induced DNA polymerase gamma was localized in photoreceptor synaptic terminals after 5 hr of light exposure, despite the fact that most photoreceptor cell mitochondria are confined to the inner segments. The neuroprotective platelet-activating factor-receptor antagonist LAU-0901 decreased mitochondrial DNA polymerase gamma up-regulation, suggesting that its neuroprotective effect is exerted upstream from this event. During aging, the ability to repair damaged photoreceptor DNA greatly declines. Thus, DNA-repair enzymes such as polymerase gamma and 8-oxoguanine-DNA-glycosylase may provide novel pharmacologic targets to promote DNA repair and rescue photoreceptors in retinal degenerative diseases.

Aging↗

PubMed1980

Isopentenyladenine as a mediator of mevalonate-regulated DNA replication.

The mechanism by which the cholesterol precursor, mevalonate, regulates S-phase DNA replication was examined in synchronized BHK cells. As previously demonstrated by this laboratory, blocking 3-hydroxy-3-methylglutaryl-CoA reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] with the competitive inhibitor compactin suppresses DNA synthesis specifically during the S phase of the cell cycle. In the present study, known mevalonate derivatives were examined as possible mediators by which mevalonate controls DNA replication. Of the compounds studied, only isopentenyladenine and its 4'-hydroxylated analogue, zeatin, could substitute for mevalonate in restoring DNA replication in compactin-blocked cells. Moreover, these two derivatives proved to be at least 100 times more active than mevalonate, and both restored DNA relication to normal within 15 min of their being added to the medium. In addition, isopentenyladenine, like mevalonate, stimulated DNA synthesis specifically during the S phase of the cell cycle. Isopentenyladenine also reversed the inhibition of DNA synthesis caused by nalidixic acid, an antibiotic that does not inhibit cholesterol synthesis. These findings indicate that isopentenyladenine or a closely related derivative may mediate the regulatory role of mevalonate in DNA replication and suggest that such isoprenes may act upon DNA replication at a site common to that inhibited by nalidixic acid.

Adenine↗

PubMed2005

Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8.

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.

Animals↗

PubMed1996

Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast.

Iron deprivation of Saccharomyces cerevisiae induces transcription of genes required for high-affinity iron uptake. AFT1 mediates this transcriptional control. In this report, the 5'-flanking region of FET3, which encodes a copper-dependent oxidase required for iron transport, was analyzed and found to contain a DNA sequence responsible for AFT1-regulated gene expression. AFT1 was capable of interacting specifically with this DNA sequence. A core element within this DNA sequence necessary for the binding of AFT1 was also determined. In vivo footprinting demonstrated occupancy of the AFT1 binding site in cells deprived of iron and not in cells grown in the presence of iron. Thus, the environmental signal resulting from iron deprivation was transduced through the regulated binding of AFT1 to the FET3 promoter, followed by the activation of transcription. A regulon of genes under the control of AFT1 could be defined. AFT1 was able to bind to a consensus binding site (PyPuCACCCPu) in the 5' region of FRE1, FRE2, FTR1, FTH1 and CCC2.

Base Sequence↗

PubMed2005

Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells.

BACKGROUND AND PURPOSE: Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions. METHODS AND MATERIALS: The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1alpha-inducing agent, CoCl2 (100 microM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression. RESULTS: Induction of hypoxia was confirmed using HIF1alpha and VEGF expression in gas- and CoCl2-treated cultures. Hypoxia (48-72 h of 0.2% O2) decreased RNA expression of a number of HR-related genes (e.g. Rad51, Rad52, Rad54, BRCA1, BRCA2) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70, DNA-PKcs, DNA Ligase IV, Xrcc4) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl2-treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution. CONCLUSIONS: Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.

Apoptosis↗

PubMed2006

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling.

Double-strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint-mediated cell-cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1- and Tel1-dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1-dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2-mediated inhibition of MRX signalling.

Blotting, Western↗

PubMed2001

Antitumor activity of 2',3'-dideoxycytidine nucleotide analog against tumors up-regulating DNA polymerase beta.

DNA polymerase beta (Pol beta), an error-prone DNA-synthesizing enzyme tightly down-regulated in healthy somatic cells, has been shown to be overexpressed in many human tumors. In this study, we show that treatment with the 2',3'-dideoxycytidine (ddC) nucleoside analog inhibited in vitro and in vivo the proliferation of Pol beta-transfected B16 melanoma cells, which up-regulate Pol beta compared with control isogenic cells. The administration of ddC also increased specifically the survival of mice bearing Pol beta-overexpressing B16 melanoma. When the phosphorylated form of ddC was electrotransfered into Pol beta-transfected melanoma, the cell growth inhibition was strengthened, strongly suggesting that the cytotoxic effect results from incorporation of the chain terminator into DNA. Using in vitro single- and double-stranded DNA synthesis assays, we demonstrated that excess Pol beta perturbs the replicative machinery, favors ddC-TP incorporation into DNA, and consequently promotes chain termination. Therefore, the use of chain terminator anticancer agents could be suitable for the treatment of tumors with a high level of Pol beta.

Animals↗

PubMed2002

CDK phosphorylation of Drc1 regulates DNA replication in fission yeast.

Cyclin-dependent kinases (CDKs) are absolutely required for DNA replication in eukaryotic cells. CDKs are thought to activate one or more replication factors, but the identities of these proteins are unknown. Here we describe fission yeast Drc1, a protein required for DNA replication that is phosphorylated by Cdc2. Drc1 depletion leads to catastrophic mitotic divisions with incompletely replicated DNA, indicating that Drc1 is required for DNA synthesis and S-M replication checkpoint control. Drc1 associates with Cdc2 and is phosphorylated at the onset of S phase when Cdc2 is activated. Mutant Drc1 that lacks CDK phosphorylation sites is nonfunctional and fails to interact with Cut5 replication factor. These data suggest that Cdc2 promotes DNA replication by phosphorylating Drc1 and regulating its association with Cut5.

CDC2 Protein Kinase↗