DNA-methyltransferase SsoII interaction with own promoter region binding site.

The investigation of Sso II DNA-methyltransferase (M.Sso II) interaction with the intergenic region of Sso II restriction-modification system was carried out. Seven guanine residues protected by M. Sso II from methylation with dimethylsulfate and thus probably involved in enzyme-DNA recognition were identified. Six of them are located symmetrically within the 15 bp inverted repeat inside the Sso II promoter region. The crosslinking of Sso II methyltransferase with DNA duplexes containing 5-bromo-2'-deoxyuridine (br5dU) instead of thymidine was performed. The crosslinked products were obtained in all cases, thus proving that tested thymines were in proximity with enzyme. The ability to produce the crosslinked products in one case was 2-5-fold higher than in other ones. This allowed us to imply that thymine residue in this position of the inverted repeat could be in contact with M. Sso II. Based on the experimental data, two symmetrical 4 bp clusters (GGAC), which could be involved in the interaction with M. Sso II in the DNA-protein complex, were identified. The model of M. Sso II interaction with its own promoter region was proposed.     

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.     

Regulation of EcoRII methyltransferase: effect of mutations on gene expression and in vitro binding to the promoter region.

EcoRII Methyltransferase (M.EcoRII) which methylates the second C in the sequence CCWGG (W = A/T) is autogenously regulated by binding to the 5' regulatory region of its gene. DNase I footprinting experiments demonstrated that purified M.EcoRII protected a 47-49 bp region of DNA immediately upstream of the ecoRIIM coding region. We have studied this interaction with mutants of the enzyme, in vitro by DNA binding and in vivo by investigating the repression in trans of expression of beta-galactosidase from an ecoRIIM-lacZ operon fusion. Two catalytically active mutants failed to repress expression of the fusion whereas catalytically inactive mutants had repressor activity. However, with one of the catalytically inactive mutants, C186S, in which the catalytic Cys was replaced with Ser, and which bound unmethylated CCWGG sequences, repression could only be demonstrated when those sequences in cellular DNA were methylated by supplying a cloned dcm gene in trans. In vitro binding of the DNA fragment containing the ecoRIIM regulatory region was detected only with the mutants that showed repressor activity, including C186S. Results indicate that down-regulation of the gene in vivo and binding to the promoter in vitro are not dependent on the catalytic properties of M.EcoRII. Mobility shift experiments with C186S also revealed that it could bind either the promoter or unmethylated CCWGG sites, but not both. We conclude that the concentration of unmethylated CCWGG sites controls expression from the ecoRIIM promoter.     

DNA topoisomerase II cleaves at specific sites in the 5'' flanking region of c-fos proto-oncogenes in vitro.

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.     

Mycobacterium tuberculosis FurA autoregulates its own expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.     

Self-association and DNA binding properties of the human topoisomerase IIA alpha2HTH module

The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant alpha(2)HTH module of human topoisomerase II alpha and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha(2)HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 A. Circular dichroism spectra showed that the helical content of human alpha(2)HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha(2)HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha(2)HTH, HTH and alpha(4), but not alpha(3), bind efficiently to DNA (dissociation constants of 3.10(-7) M for alpha(2)HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3)). Correlatively, alpha(2)HTH, alpha(4) and HTH, but not alpha(3), were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha(2)HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.