A method for the purification of DNA/protein complexes applied to DNA topoisomerase II cleavage sites.
The object of this study was to devise a purification method for DNA/topoisomerase II complexes, with which to examine the enzyme's cleavage site specificity in cellular differentiation. Retinoic acid-induced differentiation involves topoisomerase II-mediated transient changes in DNA supercoiling, but it is not known whether this occurs at specific sites in the genome. Topoisomerase II forms a covalent DNA enzyme complex as it acts, which can be recovered by the sodium dodecyl sulfate (SDS)/KCl precipitation method, but this method fails to recover significantly more DNA from cells induced to differentiate. This may in part reflect the low numbers of retinoic acid-induced protein-linked breaks in DNA and also the method's relative inefficiency for DNA with few attached topoisomerase molecules. This suggested that an additional purification method would be required to enrich sufficiently for cleavage site DNA to address the issue of site specificity. The principle of our method is to couple poly(ethylene glycol) (PEG) to topoisomerase while it is covalently attached to DNA and then to use phase partitioning in an aqueous two-phase system of PEG and phosphate to separate free DNA from DNA bound to PEG-modified topoisomerases (which have high affinities for the phosphate-rich and PEG-rich phases, respectively). The method can be used in conjunction with DNase protection and, unlike the SDS/KCl method, can fractionate short fragments of DNA to which single protein molecules are attached. Using the SDS/KCl precipitation and new method in series, we have recovered protein-linked DNA from HL60 cells induced to differentiate to the granulocyte lineage (by retinoic acid) or to the monocyte/macrophage lineage (by phorbol myristate acetate) and have demonstrated that specific sequences become protein linked, probably to topoisomerase II, during induced differentiation.
Anderson
R J
RJ
Molecular Cell Pathology Laboratory, Royal Free Hospital School of Medicine, London, United Kingdom.
Delgado
C
C
Fisher
D
D
Cunningham
J M
JM
Francis
G E
GE
D005591:Chemical Fractionation; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry; D013010:Sonication; D013450:Sulfones / Q000737:chemistry; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D013450:Sulfones; C069114:2,2,2-trifluoroethane sulfonyl-monomethoxypolyethylene glycol; D011092:Polyethylene Glycols; D005047:Etoposide; D004247:DNA; D004250:DNA Topoisomerases, Type II