CORRELATION OF DOXORUBICIN FOOTPRINTS WITH DELETION END-POINTS IN LACO OF ESCHERICHIA-COLI

This study explored the possibility that the sequence location of doxo rubicin-induced deletion endpoints might relate to DNA structural alte rations caused by doxorubicin binding to DNA. The 3'-OH endpoints of d oxorubicin-induced deletions terminating in the 35-bp region of lacO a ppear to distribute differently from spontaneous deletion endpoints. D oxorubicin-induced deletions focus in the 26-bp palindrome which is se parated by a 9-bp region with no reverse complementary, whereas sponta neous deletion 3'-OH endpoints are found distributed throughout the op erator region. In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA l abeled at the 5' end of each of the complementary DNA strands encompas sed by lacO. Doxorubicin protected the 9-bp region between the palindr omic sequences from DNase I cutting and caused enhanced DNase I cleava ge at symmetrical sites in the palindrome, which were inherently resis tant to the nuclease in the absence of the drug. These symmetrical sit es also define regions in which the occurrence of deletion endpoints i s enhanced 6-fold in the presence of doxorubicin. This enhanced cuttin g and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sit es of the drug. Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adja cent to regions where doxorubicin appeared to inhibit cutting by DNase I. These results suggest that the binding of doxorubicin in the palin drome directs both the frequency and the specificity of deletion forma tion in this gene region.