'footprinting' proteins on DNA with peroxonitrous acid

Peter A. King; Elizabeth Jamison; Daniel Strahs; Vernon E. Anderson; Michael Brenowitz

doi:10.1093/nar/21.10.2473

'footprinting' proteins on DNA with peroxonitrous acid

Peter A. King, Elizabeth Jamison, Daniel Strahs, Vernon E. Anderson, Michael Brenowitz

Biochemistry

Research output: Contribution to journal › Article › peer-review

99 Scopus citations

Abstract

The peroxonltrite anlon (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 nonspeclfically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the d-repressor (cl) of phage λ with the right operator, O_R. 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 proteln-DNA interactions.

Original language	English (US)
Pages (from-to)	2473-2478
Number of pages	6
Journal	Nucleic acids research
Volume	21
Issue number	10
DOIs	https://doi.org/10.1093/nar/21.10.2473
State	Published - May 25 1993

ASJC Scopus subject areas

Genetics

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10.1093/nar/21.10.2473

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title = "'footprinting' proteins on DNA with peroxonitrous acid",

abstract = "The peroxonltrite anlon (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 nonspeclfically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the d-repressor (cl) of phage λ 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 proteln-DNA interactions.",

author = "King, {Peter A.} and Elizabeth Jamison and Daniel Strahs and Anderson, {Vernon E.} and Michael Brenowitz",

note = "Funding Information: This work was supported by NIH grants GM39929 (M.B.) and GM36562 (V.E.A.), a Sloan Foundation Fellowship (V.E.A.), a Junior Faculty Research Award from die American Cancer Society (M.B.), a Hirschl/Weill-Caulier Career Scientist Award (M.B.) and NTH training grant 5T32-GM07128 (D.S.). We wish to diank Dr John O.Edwards for his many helpful suggestions and insightful discussions.",

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doi = "10.1093/nar/21.10.2473",

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AU - King, Peter A.

AU - Jamison, Elizabeth

AU - Strahs, Daniel

AU - Anderson, Vernon E.

AU - Brenowitz, Michael

N1 - Funding Information: This work was supported by NIH grants GM39929 (M.B.) and GM36562 (V.E.A.), a Sloan Foundation Fellowship (V.E.A.), a Junior Faculty Research Award from die American Cancer Society (M.B.), a Hirschl/Weill-Caulier Career Scientist Award (M.B.) and NTH training grant 5T32-GM07128 (D.S.). We wish to diank Dr John O.Edwards for his many helpful suggestions and insightful discussions.

PY - 1993/5/25

Y1 - 1993/5/25

N2 - The peroxonltrite anlon (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 nonspeclfically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the d-repressor (cl) of phage λ 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 proteln-DNA interactions.

AB - The peroxonltrite anlon (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 nonspeclfically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the d-repressor (cl) of phage λ 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 proteln-DNA interactions.

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'footprinting' proteins on DNA with peroxonitrous acid

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