PROTEIN-DNA INTERACTIONS IN THE HUMAN BETA-GLOBIN - LOCUS-CONTROL REGION HYPERSENSITIVE SITE-2 AS REVEALED BY 4 NITROGEN MUSTARDS

Four nitrogen mustards have been used in this study to examine protein -DNA interactions in intact human cells, specifically at the locus con trol region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus, Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the four th (chlorambucil) is a non-targeted mustard, The ligation-mediated PCR technique was used to determine the sites of damage at base pair reso lution on DNA sequencing gels. A densitometric comparison was made bet ween DNA damaged in intact erythroid K562 cells and in purified DNA. T he intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA, At the NF-E2 /AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor-erythroid 2 (NF-EP) protei n factor is thought to bind at this NF-E2/AP-1 motif in K562 cells, Ot her sites of protection and enhancement that corresponded to known tra nscription factor binding sites were also detected. These nitrogen mus tards are therefore very effective compounds for detection of transcri ption factor binding to DNA in intact cells and are superior to other commonly used agents, The sequence selectivity of the compounds was de termined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming addu cts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.