Through the specific binding of a negative calciumresponsive element to its binding protein in response to extracellular Ca (Ca by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.The Ku antigen (KuAg), 1 which consists of two subunits, p70 and p80 (p86), plays a crucial role in double-stranded break repair of DNA (1-6). In this process, its ability to bind to DNA ends nonspecifically is postulated to be related to subsequent actions such as DNA recombination or unwinding (1-3). Furthermore, such binding has been reported to be directly coupled with DNA-dependent protein kinase activity, which is elicited by the putative catalytic unit of KuAg, p350 (7,8). On the other hand, sequence-specific binding of KuAg has been demonstrated in some genes, such as the small nuclear RNA (9), T cell receptor (10), transferrin receptor (11), collagenase (12), ribosomal RNA (13, 14), and heat shock protein genes (15, 16). Although expression of most of these genes is stimulated by KuAg, transcription of the latter two is repressed by KuAg (14 -16). However, neither common KuAg-responsive DNA elements nor detailed domain structure of KuAg were identified in either type of gene regulation (9 -16). In this regard, it is of note that multimer formation of KuAg with another proteins was suggested to be involved in some of the above examples of sequence-specific gene regulation by KuAg (12,15,16) We previously reported that two DNA elements located far upstream of the human parathyroid hormone gene mediated negative gene regulation by extracellular Ca (Ca 2ϩ e ). These DNA elements (negative calcium-responsive elements (nCaREs)) bound to common nuclear proteins (nCaRE binding proteins (nCaREBs)) in a sequence-specific and Ca 2ϩ e -dependent manner (17,18). We further demonstrated that a redox factor protein, REF1, was one component of nCaREB by using the protein-DNA binding (Southwestern) assay (19). REF1 was first identified as a mammalian homologue of bacterial apurinic endonuclease/repair enzyme (20). Subsequently, it was reported to potentiate DNA binding activity of several transcription factors such as AP1 and NFB by modifying the redox state of these proteins (21). In addition to such activities of REF1, we first reported that it also possessed the sequencespecific transcriptional repressor function of nCaRE (19). However, REF1 alone could not explain all the characteristics of nCaREB activity, and we predicted the existence of another nuclear protein(s) that functions as nCaREB by cooperating with REF1 (19). By employing an oligonucleotide affinity column (22) and amino acid microsequencing (23), we demonstrate here that both subunits of KuAg interact with REF1 to bind t...
Bacillus brevis HPD31 contains a surface (S)-layer protein, termed the HWP, which forms a hexagonal array in the cell wall. The 5' region of the HWP gene was isolated from a DNA library constructed in bacteriophage vector EMBL3 from a partial BamHI digest of the chromosomal DNA. The 3' region contained in a 2.7-kilobase BgEl fragment of the DNA was cloned into Escherichia coli, using pUC118 as a vector. On the basis of the chemically determined N-terminal amino acid sequence, the HWP gene was found to encode a polypeptide consisting of 1,087 amino acid residues with a signal peptide of 53 or 23 amino acid residues. The deduced amino acid composition was similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic relative to basic amino acids and in the very low content of sulfur-containing amino acids. The deduced amino acid sequence showed high homology (78%) with that of the middle wall protein of B. brevis 47. Furthermore, the multiple 5' ends of the HWP gene transcripts detected on S1 nuclease analysis closely resembled those of the middle wall protein gene transcripts. This complex structure was also conserved (greater than 85%) in the regulatory regions of two other cell wall protein genes isolated from B. brevis HPD52 and HP033, suggesting that the synthesis of the cell wall proteins is intricately regulated through a similar mechanism in protein-producing B. brevis.A number of gram-positive and gram-negative bacteria possess a regular surface layer, the so-called S layer, which is now defined as a two-dimensional crystalline array of proteinaceous subunits forming a surface layer on procaryotic cells (28,29). The morphological properties of S layers have been extensively characterized for a wide range of microorganisms (25-27), whereas the genes for S-layerforming proteins have been isolated from only a few microorganisms, such as Bacillus brevis 47 (35, 37, 39, 41), Halobacterium halobium (11), Deinococcus radiodurans (18, 19), Caulobactor crescentus (30), and Aeromonas salmonicida (4). Too little is known at present about the regulation of these genes to elucidate the mechanisms involved in the biosynthesis, transport, and assembly of Slayer proteins. Isolation and characterization of the genes encoding S-layer-forming proteins from diverse origins are essential.Recently, we newly isolated many protein-producing B. brevis strains from soil from diverse origins (32) for comparison with B. brevis 47, the S layer of which has been extensively characterized from various aspects: morphology (40), chemical and immunological properties of the S-layerforming proteins, and characterization of genes for those proteins (35, 37). Protein-producing B. brevis strains all have S layers showing hexagonal symmetry, with a lattice constant of approximately 18 nm (8).The genes for two S-layer proteins, termed the outer wall protein and middle wall protein (MWP), of B. brevis 47 constitute a cotranscriptional unit (cwp operon) and are transcribed from several tandem promoters lo...
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