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At least 19 recordsLinked to original sources
PubMed1998

The DNA binding characteristics of the trimeric EcoKI methyltransferase and its partially assembled dimeric form determined by fluorescence polarisation and DNA footprinting.

The type I DNA restriction and modification systems of enteric bacteria display several enzymatic activities due to their oligomeric structure. Partially assembled forms of the EcoKI enzyme from E. coli K12 can display specific DNA binding properties and modification methyltransferase activity. The heterodimer of one specificity (S) subunit and one modification (M) subunit can only bind DNA whereas the addition of a second modification subunit to form M2S1 also confers methyltransferase activity. We have examined the DNA binding specificity of M1S1 and M2S1 using the change in fluorescence anisotropy which occurs on binding of a DNA probe labelled with a hexachlorofluorescein fluorophore. The dimer has much weaker affinity for the EcoKI target sequence than the trimer and slightly less ability to discriminate against other DNA sequences. Binding of both proteins is strongly dependent on salt concentration. The fluorescence results compare favourably with those obtained with the gel retardation method. DNA footprinting using exonucleaseIII and DNaseI, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer. This indicates that the dimer is a mixture of the two possible forms, M1S1 and S1M1. The dimer has a footprint on the DNA substrate of the same length as the trimer implying that the modification subunits are located on either side of the DNA helical axis rather than lying along the helical axis.

Base Sequence↗
PubMed1989

Interaction of bisantrene anti-cancer agents with DNA: footprinting, structural requirements for DNA unwinding, kinetics and mechanism of binding and correlation of structural and kinetic parameters with anti-cancer activity.

A topoisomerase I assay in conjunction with covalently closed circular DNA afforded unwinding angles for a series of eight analogs of the anti-cancer agent bisantrene. Values ranging from 33 degrees to zero comprised three classes wherein the extent of DNA interaction correlates with anti-cancer activity. The binding of bisantrene compounds to T4 DNA suggests at least partial interaction via the minor groove. MPE footprinting evaluation of bisantrene showed no evidence for selective protection of a Hind III-EcoRI fragment of pBR322 suggesting a sequence neutral binding mode. Stopped flow spectrophotometry studies indicate an association rate constant for bisantrene with DNA of greater than 1 x 10(7) M-1 S-1 at 20 degrees at ionic strength of 0.1 and is considerably slower than ethidium or propidium at all ionic strengths. At least two exponential components are required to match the kinetic curves indicating a biphasic mechanism and estimates of k1 and k2 were obtained for seven drugs. The ionic strength dependence of the surfactant driven dissociation of bisantrene is in accord with a competitive mechanism for intercalation of the drugs at alternative sites. The residence time and t1/2 (app) for bisantrene are slightly larger for poly d(G.C)2 than poly d(A.T)2 and calf thymus DNA is at an intermediate value. Considerations of structural factors in the drug dissociation kinetics permit a correlation with observed anti-cancer properties of these bisantrene analogs.

Animals↗
PubMed1991

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction.

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.

Autoradiography↗
PubMed2001

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference.

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

Adenine↗
PubMed2001

Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein-DNA footprinting.

Nucleic acid fragmentation (footprinting) by *OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100-300 ms. Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity. In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein-DNA protection portraits at sub-second timescales.

Bacterial Proteins↗
PubMed1991

DNA footprint enhancement using tandem binding sites.

A concatenated DNA fragment containing a five-repeat binding site was used for DNase I footprinting. Under the same conditions, the tandem repeat assay greatly enhanced the DNA footprint as compared with a native DNA sequence with only one binding site. This technique provides an approach for improving poor DNA footprints.

Base Sequence↗
PubMed2002

Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein-DNA interactions in vivo.

A variety of methods are available to analyze protein-DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein-protein interactions. Furthermore, the results of in vivo footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine beta-globin promoter in MEL cells. DMS footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the beta-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein-DNA interactions in vivo.

Animals↗
PubMed1989

Identification of the DNA-binding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting.

Expression of the ompF and ompC genes of Escherichia coli requires the OmpR protein for transcriptional activation. In vivo binding of the OmpR protein to the ompF and ompC promoter regions was observed using an in vivo dimethyl sulfate DNA footprinting technique. Two different sequence motifs were found to be protected by OmpR in both the ompF and ompC promoter regions. This technique was further used to localize the DNA-binding domain of OmpR to be within the C-terminal 117 amino acid residues. Binding of the C-terminal portion OmpR to the ompF and ompC promoter regions, however, did not result in activation of transcription. Our results, together with sequence homologies between OmpR and other regulatory proteins, suggests that OmpR has separable domain structures: the C-terminal portion for binding-specific DNA sequences and the N-terminal portion for interacting with RNA polymerase and/or other transcription factors.

Bacterial Outer Membrane Proteins↗
PubMed1997

Quantitating oligonucleotide affinities for duplex DNA: footprinting vs electrophoretic mobility shift assays.

Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNa-seI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods.

DNA↗
PubMed2001

DNA footprints of European hedgehogs, Erinaceus europaeus and E. concolor: Pleistocene refugia, postglacial expansion and colonization routes.

European hedgehogs, Erinaceus europaeus and E. concolor, are among the many European plant and animal taxa that have been subjected to cyclical restriction to glacial refugia and interglacial expansion. An analysis of 95 mitotypes, comprising partial cytochrome b and control region sequences, shows deep divergence between the two hedgehog species. Three europaeus and two concolor clades are clearly identified and are consistent with previously identified refugia for Europe: the Iberian peninsula, Italy, and the Balkans. The degree of mitochondrial divergence among these clades suggests pre-Pleistocene separation of the refugial populations. In contrast, analysis of two nuclear introns clearly separates the two concolor clades, as in the mitochondrial data, but cannot discriminate the three europaeus clades. This discrepancy between nuclear and mitochondrial data is attributed to historical differences in the refugial population size of europaeus and concolor. The geographical distribution of mitotypes is analysed using nested clade analysis. This method, by including unobserved ('missing') mitotypes, can identify mitotype groupings that remain undetected in conventional analyses. However, the application of nested clade analysis to the study of refugial populations may be hampered by such factors as the loss of haplotypes from the refugial areas by repeated contractions of the population and the recent time scale of colonization relative to mutation rate.

Animals↗
PubMed1997

DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism.

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the alpha-subunit truncation mutant (alpha-235) of RNA polymerase was sharply reduced, indicating that the alpha-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

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↗