Correlation of doxorubicin footprints with deletion endpoints in lacO of E. coli.
This study explored the possibility that the sequence location of doxorubicin-induced deletion endpoints might relate to DNA structural alterations caused by doxorubicin binding to DNA. The 3'-OH endpoints of doxorubicin-induced deletions terminating in the 35-bp region of lacO appear to distribute differently from spontaneous deletion endpoints. Doxorubicin-induced deletions focus in the 26-bp palindrome which is separated by a 9-bp region with no reverse complementary, whereas spontaneous deletion 3'-OH endpoints are found distributed throughout the operator region. In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA labeled at the 5' end of each of the complementary DNA strands encompassed by lacO. Doxorubicin protected the 9-bp region between the palindromic sequences from DNase I cutting and caused enhanced DNase I cleavage at symmetrical sites in the palindrome, which were inherently resistant to the nuclease in the absence of the drug. These symmetrical sites also define regions in which the occurrence of deletion endpoints is enhanced 6-fold in the presence of doxorubicin. This enhanced cutting and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sites of the drug. Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adjacent to regions where doxorubicin appeared to inhibit cutting by DNase I. These results suggest that the binding of doxorubicin in the palindrome directs both the frequency and the specificity of deletion formation in this gene region.
Sedwick
W D
WD
Department of Medicine, Case Western Reserve University, Case Western Reserve Veterans Hospital, Cleveland, OH.
Anderson
R D
RD
Baxter
J
J
Donover
S
S
Schneiter
S
S
Veigl
M L
ML
D001483:Base Sequence; D004269:DNA, Bacterial / Q000187:drug effects; D003850:Deoxyribonuclease I; D004317:Doxorubicin / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics*; D007763:Lac Operon; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009875:Operator Regions, Genetic; D017384:Sequence Deletion
D004269:DNA, Bacterial; D004317:Doxorubicin; D003850:Deoxyribonuclease I