An affinity of human replication protein A for ultraviolet-damaged DNA

Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication. Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg2+ in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.     

Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.     

Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages

We used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages.     

Isolation and characterization of a protein with high affinity for DNA: the glutamine synthetase of Thermus thermophilus 111

In a search of proteins from the thermophilic bacterium Thermus thermophilus 111 with a high affinity for DNA, the selected protein from this screening appears to be the glutamine synthetase (GS). The purified product gives one band in SDS-polyacrylamide gel electrophoresis (53,700 Da). The N-terminal 32 residues have been identified and present an homology of 80% with the glutamine synthetase of Bacillus subtilis and 76% with that of Thermotoga maritima. The protein displays the characteristic dodecameric structure of the eubacteria glutamine synthetase. From a detailed study of the interaction of this protein with DNA by dark-field electron microscopy and agarose gel electrophoresis, it is concluded that double-stranded DNA wraps the protein by a full turn of 150 bp length. An even number of GS molecules bound to a closed relaxed plasmid DNA does not alter its null topology. By using an inverted dimer DNA fragment, which contains twice a curved kinetoplast DNA insert in its central part, it is shown that DNA curvature rules the order in which GS binds to the DNA. DNA ends are also sites of high affinity for the GS. Supercoiling does not favor the binding of GS to the DNA with the exception of the apices that are by essence bent regions. By saturating a DNA molecule with GS one obtains a novel characteristic scalloped configuration in which the DNA undulates from one GS to the next. The DNA is condensed at least three times in these structures. By increasing the ratio of GS to DNA in solution the resulting material migrates as discrete bands relative to the free DNA in an agarose gel. By gel retardation and EM statistical distribution analysis of GS within the complexes, an average affinity constant of 10(7) M-1 was obtained. The potential implications of this novel interaction of the glutamine synthetase with DNA for the regulation of its own gene are briefly discussed.     

Cloning of a cDNA for DNA supercoiling factor reveals a distinctive Ca(2+)-binding protein

DNA supercoiling factor is a protein capable of generating negative supercoils into a relaxed DNA in conjunction with eukaryotic DNA topoisomerase II. A cDNA clone encoding the Bombyx mori supercoiling factor has been isolated and characterized. The cDNA sequence contains an open reading frame of 322 amino acids rich in acidic residues. A local region of the predicted polypeptide shares a weak homology with the central portion of bacterial DNA gyrase A subunit. The cDNA sequence also predicts two direct repeats within the factor. Each repeat unit consists of a potential EF-hand domain. The factor contains three other potential EF-hand domains and actually binds Ca2+. Moreover, the supercoiling reaction has been shown to be activated by Ca2+. These results implicate Ca2+ in the control of the superhelical state of DNA.