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1.
C Giamarchi M Solanas C Chailleux P Augereau F Vignon H Rochefort H Richard-Foy 《Canadian Metallurgical Quarterly》1999,18(2):533-541
We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5'-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located -10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor alpha, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor alpha, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions -2.3 Kb and -3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position -4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells. 相似文献
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L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome. 相似文献
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Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene
EA Berkowitz CP Hecht JC Azizkhan X Chen DC Lee 《Canadian Metallurgical Quarterly》1997,14(18):2229-2238
DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site. 相似文献
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ML Craig OV Tsodikov KL McQuade PE Schlax MW Capp RM Saecker MT Record 《Canadian Metallurgical Quarterly》1998,283(4):741-756
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Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases. 相似文献
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The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway. 相似文献
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We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome. 相似文献
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Y Matsuo A Yamada K Tsukamoto H Tamura H Ikezawa H Nakamura K Nishikawa 《Canadian Metallurgical Quarterly》1996,5(12):2459-2467
The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution. 相似文献
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DNase I footprinting of the apoA-II promoter using sterol regulatory element binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified four protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs. Potassium permanganate and dimethyl sulfate interference experiments using the AIIAB region as probe showed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and two 5' T residues participate in DNA-protein interactions. SREBP-2 transactivated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-fold in HepG2 cells. Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding site AIIK, was abolished by mutations in element AIIAB. An SREBP-2 mutant defective in DNA binding caused a dose-dependent repression of the apoA-II promoter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites. In contrast, a double SREBP-2 mutant which lacks both the DNA binding and the activation domains has no effect on the apoA-II promoter activity. Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites. Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of positive activator(s) which appear to recognize the activation domain of SREBP-2. 相似文献
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K Sorimachi MF Le Gal-Co?ffet G Williamson DB Archer MP Williamson 《Canadian Metallurgical Quarterly》1997,5(5):647-661
BACKGROUND: Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. RESULTS: The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. CONCLUSIONS: The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules. 相似文献
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Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence 总被引:1,自引:0,他引:1
The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids. In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA. In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I. In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction. Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA. This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed. 相似文献
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We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides [Chenna and Singer, Chem. Res. Toxicol., 8, 865-874], is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct. There are, however, significant differences in the rate of cleavage of this adduct by these enzymes. The two bacterial AP endonucleases are both much more efficient than the human repair enzyme. In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage. The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV. This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis. 相似文献
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The recognition and cleavage of tRNAPhe and the TAR RNA of HIV-1 by metallopeptides of the general form Ni(II).Xaa-Gly-His (where Xaa is Gly, Lys, or Arg) were investigated. The results of RNA cleavage analyses suggest that KHSO5- or magnesium monoperoxyphthalate-activated metallopeptides (1) induce nucleobase damage which requires aniline acetate for complete RNA strand scission and (2) selectively target the loops of stem-loop structures of the above-named substrates. In targeting RNA loop regions, the metallopeptides may be sensitive to intraloop structural features, including the overall structural environment of the loop itself and possibly the presence of intraloop hydrogen bonding. Overall, these results suggest that the metallopeptides interact selectively within a loop, in a fashion reminiscent of many RNA binding proteins, instead of targeting RNA single-stranded character alone. These observations further suggest a possible metallopeptide-based strategy for the molecular recognition of native RNA structures and insight with regard to the general features available for ligand binding site discrimination. 相似文献
17.
F Vonderviszt K Imada Y Furukawa H Uedaira H Taniguchi K Namba 《Canadian Metallurgical Quarterly》1998,284(5):1399-1416
HAP2 forms a capping structure, which binds very tightly to the distal end of flagellar filaments and still allows insertion of flagellin subunits below the cap by an unknown mechanism. Terminal regions of HAP2 from Salmonella typhimurium were found to be quickly degraded by various proteases, indicating that HAP2 also possesses disordered terminal regions like other axial proteins of bacterial flagellum. Removal of these portions by trypsin results in a fragment of 40 kDa (HP40), which lacks 42 NH2-terminal and 51 COOH-terminal residues. HAP2 in solution readily associates into a decameric structure without any significant population of intermediate oligomeric forms. The HP40 fragments, however, do not form decamers, while they can assemble into pentamers, as revealed by chemical cross-linking and analytical ultracentrifugation. Decameric HAP2 also dissociates into pentamers and smaller oligomers upon a heat induced conformational transition around 36 degreesC. While the highly mobile terminal regions are immobilized in decameric HAP2 complexes, they are still largely disordered in the pentameric state. These results demonstrate that the intersubunit interactions within the pentamers are mainly through the HP40 portions, whereas the terminal regions are responsible for association of pentamers into decameric complexes. Several observations indicate that HAP2 performs its capping function as a pentamer. We suggest that binding of the pentameric HAP2 cap to the filament is mediated by the highly flexible terminal regions. Indeed, HP40 fragments are unable to cap the end of filaments, while removal of about 30 residues from both terminal regions of HAP2 results in a highly reduced capping ability. A model is presented to explain the molecular mechanism of capping, in which conformational entropy in the disordered terminal regions moderates the otherwise too tight HAP2-filament interactions to allow insertion of flagellin subunits below the cap. 相似文献
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Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome. 相似文献