Stable DNA-protein complexes in yeast Saccharomyces cerevisiae. Protein composition analysis

Proteins tightly bound to DNA (TBP) are a protein group that remains attached to DNA by covalent or non-covalent bonds after its deproteinisation Key words: DNA-protein interaction, stable DNA-protein complexes Abbreviations: TBP -eukaryotic non-histone proteins tightly bound to DNA

Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53

Evaluation of some S/MAR sequence properties on DNA sequences involved in tightly bound DNA... 1 Evaluation of some S/MAR sequence properties on DNA sequences involved in tightly bound DNA protein complexes of murine cells

Abbreviations: MEL -Friend erythroleukemia cells; TBD -eukaryotic non-histone proteins tightly bound to DNA; S/MARs -conserved DNA sequences associated with matrix and/or scaffold Here we report the DNA sequence-based characterization of DNA fragments involved in stable complexes with proteins in mouse chromatin. Computer analysis of nine independently isolated and sequenced clones containing such sequences revealed some similar properties known to comprise S/MAR sequences. These characteristics (ori signals, TA, CA, TG richness, curved DNA, topoisomerase II binding sites) suggest that DNAs presented in tightly DNA-bound complexes have a certain characteristic sequence and conformation properties typical of S/MARs

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei