Protection analysis (or "footprinting") of specific protein-DNA complexes in crude nuclear extracts using methidiumpropyl-EDTA-iron (II).
Endonuclease protection or "footprinting" analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction. DNase I is the most often employed endonucleolytic agent; however, this endonuclease does not exhibit the true nonsequence-specific cleavage desired for this type of analysis. Methidiumpropyl-EDTA (MPE) is a synthetic DNA intercalator that cleaves DNA in the presence of ferrous ion and oxygen. Cleavage by MPE exhibits no sequence specificity, a characteristic that makes this reagent better suited for protection analysis. Here we report a generally applicable technique for MPE protection (or "footprinting") analysis of specific DNA-protein complexes from a crude nuclear extract. We have used this method to identify the nucleotides of the immunoglobulin (Ig) heavy chain promoter region that are involved in complex formation with a protein that binds the octameric sequence ATGCAAAT, and we compare our results to those obtained previously using DNase I.
Landolfi
N F
NF
University of Texas, Southwestern Medical Center.
Yin
X M
XM
Capra
J D
JD
Tucker
P W
PW
D001483:Base Sequence; D001709:Biotechnology; D002467:Cell Nucleus / Q000032:analysis; D004247:DNA / Q000302:isolation & purification*; D003850:Deoxyribonuclease I; D004492:Edetic Acid / Q000031:analogs & derivatives; D007502:Iron Chelating Agents; D008969:Molecular Sequence Data; D011506:Proteins / Q000302:isolation & purification*
D007502:Iron Chelating Agents; D011506:Proteins; C038200:methidiumpropyl-EDTA-iron(II); D004247:DNA; D004492:Edetic Acid; D003850:Deoxyribonuclease I