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Results for “DNA Footprinting”

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At least 37 records · Page 2Linked to original sources
PubMed2005

Sequence-selective interaction of the minor-groove interstrand cross-linking agent SJG-136 with naked and cellular DNA: footprinting and enzyme inhibition studies.

SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5'-Pu-GATC-Py-3' (Pu = purine; Py = pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5'-G-GATC-C-3' site and is the first report of DNA footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage rather than C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5'-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5'-GAAC-3'/5'-GTTC-3' (preferred) or 5'-GAGC-3'/5'-GCTC-3' sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.

Bacterial Proteins↗
PubMed1999

Protein-DNA footprinting of the human epsilon-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents.

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein-DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy epsilon-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences - both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein-DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5'- and 3'-ends of the CCAAT box in K562 cells - this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the epsilonF1 DNA binding motif in K562 cells.

Bleomycin↗
PubMed1994

DNA binding mode of class-IIS restriction endonuclease FokI revealed by DNA footprinting analysis.

We investigate the interaction of FokI with its DNA recognition sequence by several footprinting techniques. Methylation of three guanine bases in the recognition sequence 5'-GGATG-3' is strongly protected by FokI binding, whereas other guanine bases are not masked from the modification. In footprinting using the methidiumpropyl-EDTA-Fe(II) complex, binding of FokI strongly inhibits cleavage by the footprinting reagent at and near the recognition sequence. In high-resolution footprinting techniques using hydroxyl radical and the bleomycin-Fe(II) complex, all footprints in each binding site clearly face one side of the DNA helix. Interference analysis with FokI digestion by preethylation of phosphate groups suggests that essential phosphates for FokI digestion are located at and near the recognition sequence and the cleavage site. Evidently, the results indicate that (i) the sequence-recognition of FokI occurs in the major groove and that (ii) the enzyme interacts with its target DNA from one side of the DNA helix.

Alkylation↗
PubMed1995

Perturbations in DNA structure upon interaction with porphyrins revealed by chemical probes, DNA footprinting and molecular modelling.

The interactions of several porphyrins with a 74 base-pair DNA sequence have been examined by footprinting and chemical protection methods. Tetra-(4-N-methyl-(pyridyl)) porphyrin (TMPy), two of its metal complexes and tetra-(4-trimethylanilinium) porphyrin (TMAP) bind to closely similar AT-rich sequences. The three TMPy ligands produce modest changes in DNA structure and base accessibility on binding, in contrast to the large-scale conformational changes observed with TMAP. Molecular modelling studies have been performed on TMPy and TMAP bound in the AT-rich minor groove of an oligonucleotide. These have shown that significant structural change is needed to accommodate the bulky trimethyl substituent groups of TMAP, in contrast to the facile minor groove fit of TMPy.

Base Sequence↗
PubMed1998

DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA.

Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Esigma70 RNA polymerase (R) and the lambdaPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0 degreesC, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends approximately 30 bases downstream and at least approximately 40 bases upstream from the start site. I1 has a short lifetime (</=15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degreesC to 0 degreesC, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degreesC. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Esigma70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0 degreesC, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k-2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures.

Bacteriophage lambda↗
PubMed1984

Interaction of tight binding repressors with lac operators. An analysis by DNA-footprinting.

To increase our understanding of protein-DNA interaction in general, and in particular that of lac repressor with lac operator, we have investigated the interaction of tight binding (Itb) repressors with wild type (WT) operator and Oc operators. Nine Oc and a WT operator were cloned and sequenced. Three different Oc and an O+ were then chosen for the footprint analysis of six Itb repressors and WT repressor. Distinct protection patterns for the various repressor-operator pairs were observed at low repressor concentrations whereas, at high repressor concentrations, a stretch of 24 bases of the lower strand of the four different operators was protected in most cases. This protection pattern at high repressor concentration was almost completely redundant for all repressor-operator pairs, in spite of the fact that the affinities of the various pairs differed by more than three orders of magnitude. Two exceptions to this general observation were the two tight binding repressors R67 and R78a. These had been mapped in a region that codes for amino acid residues involved in subunit interaction. The two repressors showed reduced protection of O+ and of some Oc operators at the 3' (right) end of the lower strand. Dimethylsulfoxide, which is known to increase the affinity of O+ for repressor, did not increase the number of bases protected by WT repressor on the lower strand of O+. The footprinting results presented here clearly demonstrate that lac repressor can maximally protect about 24 bases of the lower strand of the operator and that the number and kind of interactions occurring in this region determine the strength of the repressor-operator interaction.

Base Sequence↗
PubMed2000

Sequence-selectivity of 5,11-dimethyl-5H-indolo[2,3-b]quinoline binding to DNA. Footprinting and molecular modeling studies.

Indolo[2,3-b]quinolines are a new family of the DNA intercalators showing significant cytotoxic activity. The mechanism of their action is based on the inhibition of DNA topoisomerase II activity. It depends on their ability to induce and stabilize drug-topII-DNA cleavable complexes. Site-specific intercalation of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ) was analyzed in vitro by DNaseI footprinting and by molecular modeling. To model the DNA-intercalator complex, use was made of the CVFF and ESFF force fields implemented in Insight 97.0 software. Experimental results were verified using a simple statistical model. The DiMIQ molecule was found to bind preferentially to the pBR322 DNA plasmid in the 5'-TGCTAACGC-3' region between adjacent adenine bases.

Base Sequence↗
PubMed1988

DNA footprinting with hydroxyl radical.

High resolution images of proteins bound to DNA can be achieved through the use of a simple inorganic chemical system that produces the hydroxyl radical.

Binding Sites↗
PubMed1991

Elicitor-inducible and constitutive in vivo DNA footprints indicate novel cis-acting elements in the promoter of a parsley gene encoding pathogenesis-related protein 1.

The presence of three genes encoding pathogenesis-related protein 1 (PR1) in cultured parsley cells and the activation of all three genes by fungal elicitor are demonstrated. In vivo dimethyl sulfate footprinting was used to identify two putative sites of protein-DNA interaction in the promoter of one PR1 gene, located around positions -240 and -130 relative to the transcription start site. The TATA-distal footprint was elicitor dependent and appeared within 30 minutes of elicitor treatment, concomitant with the onset of PR1 transcription. The second footprint was observed irrespective of whether elicitor was present or absent. The two footprinted regions contain, in opposite orientation, nearly identical 11-base pair motifs that are unrelated to any known cis-acting element in elicitor-activated or pathogen-activated plant genes. The results demonstrate the usefulness of in vivo footprinting for the identification of cis-acting elements within promoters not accessible to other types of analysis.

Base Sequence↗
PubMed1989

Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene.

We began characterization of the protein--DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression experiments in parsley protoplasts. Two of the three footprinted sequence 'boxes' reside within the minimal promoter. Replacement of 10 bp within either of these 'boxes' leads to complete loss of light responsiveness. We conclude that these sequences define the necessary cis elements of the minimal CHS promoter's light responsive element. One of the functionally defined 'boxes' is homologous to an element implicated in regulation of genes involved in photosynthesis. These data represent the first example in a plant defense gene of an induced change in protein--DNA contacts necessary for transcriptional activation. Also, our data argue strongly that divergent light induced biosynthetic pathways share common regulatory units.

Acyltransferases↗
PubMed1989

DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters.

The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.

Bacterial Proteins↗
PubMed1990

Thermodynamic data from drug-DNA footprinting experiments.

Sequence-dependent thermodynamic quantities for the antiviral agent netropsin and a related bis(N-methylimidazole) dipeptide, lexitropsin, have been determined by DNase I footprinting techniques. The primary data are autoradiographic spot intensities derived from 10 footprinting experiments carried out in the temperature range 0-45 degrees C. After exclusion effects due to overlapped drug sites on DNA and redistribution phenomena associated with the enzyme were accounted for, sequence-dependent binding constants for the two ligands were calculated. Our approach does not require an independent determination of the free drug concentration, which is calculated, with individual site binding constants, by using only footprinting data. The temperature dependence of the binding constants for netropsin implied that the binding enthalpies for all the sites but one on a 139 base pair restriction fragment of pBR 322 DNA are exothermic. Their values roughly correlate with the free energies of binding, which are smaller for sites including a 5'-TA-3' sequence. The binding enthalpies for the lexitropsin to all its sites were exothermic and more negative than those of netropsin. This may be due to the greater ability of the lexitropsin, when compared to netropsin, to form hydrogen bonds with sites on DNA. The binding constants of the lexitropsin toward its GC interaction sequences were much lower than those of netropsin, as can be explained by the reduced charge of the former ligand. Although it is difficult to determine the specific origin of the thermodynamic effects measured, comparison between netropsin and the lexitropsin suggests that the degree of solvation in the minor groove of DNA may be a factor influencing the entropy of the binding process.

Base Sequence↗
PubMed1991

DNA footprinting with the hydroxyl radical.

The Fenton reaction of iron(II) EDTA with hydrogen peroxide, performed in the presence of ascorbate ion, has proven to be useful as a probe of structure in DNA systems. Two aspects of this chemistry are discussed: the identity of the active DNA cleaving agent produced by this reagent, and the application of the Fenton reaction to the determination of the structure of the Holliday junction, the four-stranded DNA molecule that is a key intermediate in recombination. The cleavage pattern of the Holliday junction has pseudo-twofold symmetry, putting important constraints on possible structures.

Bacteriophage lambda↗
PubMed1997

Chloroplast DNA footprints of postglacial recolonization by oaks.

Recolonization of Europe by forest tree species after the last glaciation is well documented in the fossil pollen record. This spread may have been achieved at low densities by rare events of long-distance dispersal, rather than by a compact wave of advance, generating a patchy genetic structure through founder effects. In long-lived oak species, this structure could still be discernible by using maternally transmitted genetic markers. To test this hypothesis, a fine-scale study of chloroplast DNA (cpDNA) variability of two sympatric oak species was carried out in western France. The distributions of six cpDNA length variants were analyzed at 188 localities over a 200 x 300 km area. A cpDNA map was obtained by applying geostatistics methods to the complete data set. Patches of several hundred square kilometers exist which are virtually fixed for a single haplotype for both oak species. This local systematic interspecific sharing of the maternal genome strongly suggests that long-distance seed dispersal events followed by interspecific exchanges were involved at the time of colonization, about 10,000 years ago.

Journal Article↗
PubMed1995

Functional roles of in vivo footprinted DNA motifs within an alpha-globin enhancer. Erythroid lineage and developmental stage specificities.

Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.

Adult↗