scholarly journals Interaction of bleomycin with a bent DNA fragment

1992 ◽  
Vol 284 (3) ◽  
pp. 929-934 ◽  
Author(s):  
K P Nightingale ◽  
K R Fox
Keyword(s):  
Binding Sites ◽  
Dna Bending ◽  
Cobalt Complex ◽  
Dnase I ◽  
Cleavage Sites ◽  
Kinetoplast Dna ◽  
Bent Dna ◽  
Diethyl Pyrocarbonate ◽  
Dnase I Footprinting ◽  
Dna Fragment

The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3′ side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.

scholarly journals Footprinting studies on the effect of echinomycin on the structure of a bent DNA fragment

1990 ◽  
Vol 269 (1) ◽  
pp. 217-221 ◽  
Author(s):  
K R Fox ◽  
E Kentebe
Keyword(s):  
Binding Sites ◽  
Dna Structure ◽  
Drug Binding ◽  
Micrococcal Nuclease ◽  
Kinetoplast Dna ◽  
Bent Dna ◽  
Diethyl Pyrocarbonate ◽  
Drug Binding Sites ◽  
Dna Fragment

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

scholarly journals Dissociation of the AT-specific bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM from TpA sites in DNA

1995 ◽  
Vol 306 (1) ◽  
pp. 15-19 ◽  
Author(s):  
M C Fletcher ◽  
R K Olsen ◽  
K R Fox
Keyword(s):  
Dissociation Rate ◽  
Dnase I ◽  
Time Dependent ◽  
Base Pairs ◽  
Unusual Structure ◽  
Dnase I Footprinting ◽  
Calf Thymus ◽  
Stability Of Complexes ◽  
Dna Fragment ◽  
The Stability

We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

DNase I Footprinting to Identify Protein Binding Sites

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (14) ◽  
Author(s):  
Isabelle Gaugué ◽  
Dominique Bréchemier-Baey ◽  
Jacqueline Plumbridge
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

Mapping Protein-binding Sites on DNA by DNase I Footprinting

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3947
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes

1989 ◽  
Vol 9 (11) ◽  
pp. 5022-5033
Author(s):  
L A Gossett ◽  
D J Kelvin ◽  
E A Sternberg ◽  
E N Olson
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Dnase I ◽  
Strong Activity ◽  
Enhancer Element ◽  
Diethyl Pyrocarbonate ◽  
Dnase I Footprinting ◽  
Binding Factor ◽  
Deficient Medium

Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909, 1988). To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specific enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A + T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor, designated MEF-2, was undetectable in nuclear extracts from C2 or BC3H1 myoblasts or several nonmyogenic cell lines. MEF-2 was first detectable within 2 h after exposure of myoblasts to mitogen-deficient medium and increased in abundance for 24 to 48 h thereafter. The appearance of MEF-2 required ongoing protein synthesis and was prevented by fibroblast growth factor and type beta transforming growth factor, which block the induction of muscle-specific genes. A myoblast-specific factor that is down regulated within 4 h after removal of growth factors was also found to bind to the MEF-2 recognition site. A 10-bp sequence, which was shown by DNase I footprinting and diethyl pyrocarbonate interference to interact directly with MEF-2, was identified within the rat and human mck enhancers, the rat myosin light-chain (mlc)-1/3 enhancer, and the chicken cardiac mlc-2A promoter. Oligomers corresponding to the region of the mlc-1/3 enhancer, which encompasses this conserved sequence, bound MEF-2 and competed for its binding to the mck enhancer. These results thus provide evidence for a novel myocyte-specific enhancer-binding factor, MEF-2, that is expressed early in the differentiation program and is suppressed by specific polypeptide growth factors. The ability of MEF-2 to recognize conserved activating elements associated with multiple-specific genes suggests that this factor may participate in the coordinate regulation of genes during myogenesis.

A photochemical method to map ethidium bromide binding sites on DNA: application to a bent DNA fragment

Biochemistry ◽  
1990 ◽  
Vol 29 (4) ◽  
pp. 981-988 ◽  
Author(s):  
Girija Krishnamurthy ◽  
Thomas Polte ◽  
Thomas Rooney ◽  
Michael E. Hogan
Keyword(s):  
Ethidium Bromide ◽  
Binding Sites ◽  
Bent Dna ◽  
Photochemical Method ◽  
Dna Fragment

Transcriptional Regulation of the SHP-1, a Candidate Tumor Suppressor Gene in Hematological Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4346-4346
Author(s):  
Koichi Nakase ◽  
Jihua Cheng ◽  
Quan Zhu ◽  
Wayne A. Marasco
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Binding Sites ◽  
Hematological Malignancies ◽  
Core Promoter ◽  
Tyrosine Phosphatase ◽  
Dnase I ◽  
Jurkat Cells ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
P2 Promoter

Abstract The SH2-Containing Protein-Tyrosine Phosphatase-1 (SHP-1) is a hematopoietic cell specific protein-tyrosine phosphatase and a candidate tumor suppressor. It has been shown that expression of SHP-1 is repressed in HTLV-1 transformed lymphocyte cell lines, Adult T-cell Leukemia and other hematological malignancies. However, the mechanisms that regulate SHP-1 expression remain unclear. We previously identified that the −120 to +157 of the SHP-1 P2 promoter (core promoter) exhibits similar levels of activity as the wild type promoter. Here we report the involvement of NF-kB and Oct-1 in the transcriptional regulation of the SHP-1 P2 promoter. To elucidate the mechanisms by which SHP-1 promoter is regulated, in vitro DNase I footprinting experiments were performed. Incubation of nuclear extracts from Jurkat cells with the SHP-1 P2 promoter probes revealed two DNase I protected regions between −100 and −90 and between −80 and −55 in the SHP-1 P2 promoter. Elk-1, Ik-1, NF-kB and Oct-1 were identified through database search as likely candidate factors binding to these regions. Electrophoretic Mobility Shift Assay (EMSA) was carried out using a double strand DNA probe that covers the previous DNase I footprinting area (−120 to −19) and Jurkat nuclear extract. Transcription factors NF-kB and Oct-1 were confirmed as binding to the SHP-1 P2 promoter through antibody supershift, probe competition, and/or site directed mutagenesis. Results from in vivo Chromatin Immunoprecipitation (ChIP) assay with anti-NF-kB and anti-Oct-1 antibody also strongly suggested that Oct-1 and NF-kB bound the SHP-1 P2 promoter in Jurkat cells. In transient transfection experiments using luciferase as a reporter, mutations at Oct-1 binding sites and NF-kB binding sites showed significant reduction in SHP-1 promoter activity compared to wild type core promoter, further indicating the involvement of these factors in the SHP-1 P2 promoter transcription regulation. In summary, this is the first report demonstrating that Oct-1 and NF-kB are involved in the regulation of SHP-1 P2 promoter. Modulation of these transcription factors could provide new targets for treatment of hematological malignancies.

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