‘Footprinting’ proteins on DNA with peroxonitrous acid

Abstract

The peroxonitrite anion (ONOO-) is a stable species in alkaline solution that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on the procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly generates a strong oxidant capable of nonspecifically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the cl-repressor (cl) of phage lambda with the right operator, OR. In addition, we show that the individual-site binding isotherms determined by quantitative DNase I, Fe-EDTA and ONOOK footprinting are identical within experimental error. The identical isotherms obtained with the three different reagents with greatly differing sampling times indicates that the sampling time of the footprinting probe need not be short relative to the kinetic dissociation constants that govern protein-DNA interactions.

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

“…The model in its general form, as demonstrated by our data, does not make any specific distinctions between processive polymerases and other enzymes, such as HIV-1 RT that dissociate more frequently from their nucleic acid substrates. The site-specific footprints can be seen as snapshots taken within a few seconds that compose the short lifetime of HOONO of ϳ4 s at neutral pH (39). The relative distribution of the two bands at position Ϫ7 and Ϫ8 reflect the relative distribution of pre-and post-translocational stages within this time interval.…”

A method for probing the topography and interactions of proteins: footprinting of myoglobin.

“…The E helix, which forms a ridge in the figure might be the main target of the antibody, since the sites of differential reactivity observed surround this helix. The size of the enzymes used as probes precludes defining the antibody epitope on the protein to high resolution (31,32); smaller probes (10,11) should improve the resolution. Figs.…”

Catching RNA polymerase in the act of binding: Intermediates in transcription illuminated by synchrotron footprinting

“…Reactivity patterns can be generated and analyzed with resolution as fine as a single nucleotide. Chemical and radiolytic methods have been used to generate • OH for static, equilibrium, and time-resolved nucleic acid footprinting (6,(8)(9)(10)(11). A singular advantage to the use of a synchrotron x-ray beam [such as that used by Sclavi et al (1)] for radiolytic generation of • OH is that millisecond exposure yields sufficient concentrations of radicals for footprinting.…”