High-resolution footprinting of sequence-specific protein–DNA contacts

Abstract

Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the regulatory sites in the genome and to identify the proteins that bind them. Because members of transcription factor families often exhibit similar sequence preferences, methods for determining intermolecular contacts in protein-DNA interfaces must be sensitive to even subtle structural differences. The most detailed structural views of protein-DNA interfaces have been obtained through X-ray crystallography and NMR spectroscopy, and these methods have revolutionized the understanding of the structural determinants of sequence-specific recognition. Neither crystallography nor NMR, however, is particularly well-suited to high-throughput applications such as pan-genomic elucidation of regulatory sequences; in addition, these methods yield no information on the energetic contribution of particular contacts. Here we report a straightforward, high-resolution biochemical method for mapping, at single-nucleotide resolution, DNA bases that are subject to sequence-specific contacts by regulatory proteins.

Detection of plasmid DNA vectors following gene transfer to the murine airways

“…The relationship between the delivery of vector DNA and gene expression is an important one in models Detection of plasmid DNA in the mouse lung IA Pringle et al of gene therapy as the detection of vector DNA has been used a positive outcome marker in gene therapy clinical trials. 9 There are methods available that allow the fine mapping of transcriptionally active regions of DNA, 37 but these methods do not have the required sensitivity to quantify the actively transcribed component of plasmid DNA in a potentially excessive background of untranscribed plasmid DNA. Therefore if these relationships are to be made meaningful, more sensitive assays perhaps coupled with methods to purify specific cell populations (and even subcellular compartments) will have to be developed.…”

Analysis of RNA polymerase-promoter complex formation

“…Interference studies have also been carried out with RNAP using promoter fragments with randomly-positioned bases that contain modifications (e.g., dUTP substitutions; [81]; see also [83] for review of template-directed interference footprinting procedures) or fragments with randomly-positioned missing bases or missing nucleoside (e.g., [84]). These experiments can be used to scan the promoter for positions that are critical for complex formation.…”

A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides

“…However, TDI footprinting involves only low-level statistical incorporation of an analog, whereas ICCC method requires complete substitution of a normal base by the analog, a much more demanding operation. Therefore, two TDI analogs, 5-hydroxy-dUTP and 7-deaza-7-nitro-dATP (15,19), as well as 5-hydroxy-dCTP and 7-deaza-7-nitro-dGTP (20), which have similar structural and electronic characteristics, were developed for use in the ICCC genotyping platform (Fig. 1).…”