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  • The DNA intercalators ethidium bromide and propidium iodide also bind to core histones. 24649406

    Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. 25879486

    The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3034
    Product Catalog Name:
    Anti-DNA Antibody, single stranded, clone 16-19

  • Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding. 17209048

    We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

  • A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer. 21946329

    DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The DNA damage checkpoint regulates a transition between yeast and hyphal growth in schizosaccharomyces japonicus. 20368354

    Dimorphic yeasts change between unicellular growth and filamentous growth. Many dimorphic yeasts species are pathogenic for humans and plants, being infectious as invasive hypha. We have studied the determinants of the dimorphic switch in the nonpathogenic fission yeast Schizosaccharomyces japonicus, which is evolutionarily close to the well-characterized fission yeast S. pombe. We report that camptothecin, an inhibitor of topoisomerase I, reversibly induced the unicellular to hyphal transition in S. japonicus at low concentrations of camptothecin that did not induce checkpoint arrest and the transition required the DNA checkpoint kinase Chk1. Furthermore, a mutation of chk1 induced hyphal transition without camptothecin. Thus, we identify a second function for Chk1 distinct from its role in checkpoint arrest. Activation of the switch from single cell bipolar growth to monopolar filamentous growth may assist cells to evade the source of DNA damage.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Active DNA demethylation in human postmitotic cells correlates with activating histone modifications, but not transcription levels. 20565882

    In mammals, the dynamics of DNA methylation, in particular the regulated, active removal of cytosine methylation, has remained a mystery, partly due to the lack of appropriate model systems to study DNA demethylation. Previous work has largely focused on proliferating cell types that are mitotically arrested using pharmacological inhibitors to distinguish between active and passive mechanisms of DNA demethylation.We explored this epigenetic phenomenon in a natural setting of post-mitotic cells: the differentiation of human peripheral blood monocytes into macrophages or dendritic cells, which proceeds without cell division. Using a global, comparative CpG methylation profiling approach, we identified many novel examples of active DNA demethylation and characterized accompanying transcriptional and epigenetic events at these sites during monocytic differentiation. We show that active DNA demethylation is not restricted to proximal promoters and that the time-course of demethylation varies for individual CpGs. Irrespective of their location, the removal of methylated cytosines always coincided with the appearance of activating histone marks.Demethylation events are highly reproducible in monocyte-derived dendritic cells from different individuals. Our data suggest that active DNA demethylation is a precisely targeted event that parallels or follows the modification of histones, but is not necessarily coupled to alterations in transcriptional activity.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse. 23788429

    In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. 20948999

    Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein-RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4