Site-directed cleavage of DNA by protein-Fe(II) EDTA conjugates within model chromatin complexes.
The nucleosome and other chromatin complexes are examples of complicated protein-DNA assemblies that are not easily studied by traditional structural methods. Site-directed cleavage of DNA is a method for mapping the location of interaction of a specific site in a protein such as a linker histone within a large complex such as the nucleosome. In this chapter we describe the application of the site-directed cleavage method, employing linker histones site-specifically modified with the chemical cleavage reagent Fe(II)(EDTA-2-aminoethyl) 2-pyridyl disulfide (ebr). Addition of hydrogen peroxide and a reducing agent to the complex containing the modified protein leads to the production of hydroxyl radicals from the iron center, resulting in cleavage of DNA backbones in the vicinity of the modified residue. The cleavages can then be mapped and ascribed to a particular location within the nucleosome, allowing the binding site of the protein within this structure to be determined.
Chafin
David R
DR
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Hayes
Jeffrey J
JJ
D019943:Amino Acid Substitution; D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003956:Dialysis; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D017665:Hydroxyl Radical / Q000378:metabolism; D007553:Isotope Labeling; D008967:Molecular Biology / Q000379:methods*; D009707:Nucleosomes / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011868:Radioisotopes; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology
D002843:Chromatin; C084719:EDTA-(2-aminoethyl 2-pyridyl disulfide)-Fe; D009707:Nucleosomes; D011506:Proteins; D011868:Radioisotopes; D017665:Hydroxyl Radical; D012965:Sodium Chloride; D004247:DNA; D004492:Edetic Acid; D004262:DNA Restriction Enzymes; D003545:Cysteine