DNAse footprinting

Earlier studies using thermal denaturation analysis and spectrophotomet-ric titration with TxA T and CxC-C" containing DNA triplexes showed that coralyne binds strongly to these triplexes by intercalation and does not exhibit a significant sequence-selectivity [222]. In a later study by Morau Allen et al. [217], employing DNase footprinting, thermal denaturation analysis, UV-visible spectrophotometric titrations, circular dichroism and NMR spectroscopy, showed that coralyne is fully intercalated into TxA T triplex DNA whereas in C GxC triplex, it is partially intercalated due to electrostatic repulsion between the cationic alkaloid and the protonated cytosine [217]. Kepler et al. [223] demonstrated that coralyne intercalated to parallel triplex DNA but did not intercalate to antiparallel triplex DNA. Recently Hud and coworkers [219,224] demonstrated that duplex poly(dA) poly(dT) is trans-... 

The Fur protein from E. coli was isolated in one step due to its high affinity for metal-chelate columns loaded with zinc. In DNase footprinting experiments, the Fur protein was shown to bind DNA in the promoter region of several iron-regulated genes. The consensus sequence, called the Fur box, is GATAATGATAATCATT ATC. In vitro binding is dependent on the divalent cations Co2+ Mn2+ /s Cd2+ Cu2+ at 150 iM, while Fe2+ seemed to be less active at this concentration, probably due to oxidation to Fe3+ (De Lorenzo et al., 1987). The unspecificity for divalent metals observed in vitro shows that the cells have to select the ions transported carefully and have to balance their active concentrations. In addition, it is a caveat for the experimenter to test a hypothesis on metal-ion specificity not only in vitro, but also in vivo. 

Identification of the HupR binding site at the hupS promoter by DNase footprinting analysis... 

Sequence selectivity was investigated by DNase/footprinting. The ( + )-isomer of 16b failed to inhibit DNase I cleavage, even at high concentrations. However, the (-)-isomer strongly protected certain sequences against cleavage by this nuclease... 

Galas, D., Schmitz, A. (1978) DNase Footprinting A Simple Method for the Detection of Protein-DNA Binding Specifidty, Nudeic Acids Res. 5, 3157-3170. 

TANDEM, Triostin A N-Demethylated, a synthetic analogue of triostin A. TANDEM is a bisintercalator with the capability to bind to 5 -TpA sequences on DNA by DNase footprinting [T. L. CiardeUi et al.,... 

Footprinting DNA with protein bound is resistant to digestion by DNase enzymes. When a sequencing reaction is performed using such DNA, a protected area, representing the footprint of the bound protein, will be detected. 

The 5 LTR has been extensively characterized in vitro, and binding sites for several cellular transcription factors have been identified using DNase I in vitro footprinting and gel retardation assays (Fig. Ic) (see for reviews Roebuck and Saifuddin, 1999 Pereira et ai, 2000 Rohr et al, 2003). 

Any technique designed to characterize binding interactions by determining the accessibility of the backbone of macromolecules to cleavage or modification reactions. For nucleic acid interactions, footprinting was originally accomplished by changes in phosphodiester accessibility to DNase 1, but numerous chemical and enzymatic methods continue to be elaborated. 

As in the case of the cisplatin interstrand cross-links, several techniques have been used to characterize the distortions induced in the DNA double helix by the transplatin interstrand cross-links. From gel electrophoresis [68] it has been deduced that the DNA double helix is unwound (12°) and its axis is bent (26°) toward the major groove. Chemical probes and DNase I footprinting indicate that the distortion of the double helix spreads over four-... 

Figure 28.3. Footprinting. One end of a DNA chain is labeled with 32p (shown as a red circle). This labeled DNA is then treated with DNAse I such that each fragment is cut only once. The same cleavage is carried out after a protein that binds to specific sites on the DNA has been added. The bound protein protects a segment on the DNA from the action of DNAse 1. Hence, certain fragments present in the reaction without protein will be missing. These missing bands in the gel pattern identify the binding site on DNA.

Detailed biochemical studies revealed how the Pol II preinitiation complex, comprising a Pol II molecule and general transcription factors bound to a promoter region of DNA, is assembled. In these studies DNase I footprinting and electrophoretic mobility shift assays were used to determine the order in which Pol II and general transcription factors bound to TATA-box promoters. Because the complete, multisubunit TFIID is difficult to purify, researchers used only the isolated TBP component of this general transcription factor in these experiments. Pol II can initiate transcription in vitro in the absence of the other TFIID subunits. 

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