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High resolution footprinting of EcoRI and distamycin with Rh(phi)_2(bpy)^(3+), a new photofootprinting reagent

Uchida, Kiyoshi and Pyle, Anna Marie and Morii, Takashi and Barton, Jacqueline K. (1989) High resolution footprinting of EcoRI and distamycin with Rh(phi)_2(bpy)^(3+), a new photofootprinting reagent. Nucleic Acids Research, 17 (24). pp. 10259-10279. ISSN 0305-1048. PMCID PMC335299. doi:10.1093/nar/17.24.10259.

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The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)_2(bpy)^(3+), cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootprinting reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5–7 base pair footprint for the drug at its A_6 binding site and a 10–12 base pair footprint for the enzyme centered at its recognition site (5′-GAATTC-3′). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg^(2+) or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.

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Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 1989 IRL Press. Received September 18, 1989; Revised and Accepted November 8, 1989. K.U. conducted this research as a Fulbright Fellow. We are grateful also to the National Institute of General Medical Science (GM33309 to J.K.B.; National Research Service Training Award for A.M.P., GM07216), to the National Foundation for Cancer Research, and to Bethesda Research Laboratories for their financial support.
Funding AgencyGrant Number
NIH Predoctoral FellowshipGM07216
National Foundation for Cancer ResearchUNSPECIFIED
Bethesda Research LaboratoriesUNSPECIFIED
Issue or Number:24
PubMed Central ID:PMC335299
Record Number:CaltechAUTHORS:20160513-121954121
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Official Citation:Kiyoshi Uchida, Anna Marie Pyle, Takashi Morii, and Jacqueline K. Barton High resolution footprinting of EcoRI and distamycin with Rh(phi)2(bpy)3+, a new photofootprinting reagent Nucl. Acids Res. (1989) 17 (24): 10259-10279 doi:10.1093/nar/17.24.10259
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67080
Deposited By: Ruth Sustaita
Deposited On:13 May 2016 19:37
Last Modified:11 Nov 2021 00:26

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