DNA sequence specificity of doxorubicin-induced mutational damage in uvrB- Escherichia coli

In the absence of excision repair, doxorubicin caused a striking (41-fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac repressor gene (lacI) of Escherichia coli. In contrast, there was only a 2-fold increase in the frequency of small deletions despite a 3-fold increase in overall mutation frequency. The 5'-endpoints of doxorubicin-induced lacO and lacI/lacO deletions occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent to 5'-dGdC or 5'-dCdG doublets (34%). Ninety % (27 of 30) of the doxorubicin-induced deletions involving the region of the lacO palindrome had 3'-endpoints within the palindrome sequence as compared with 40% (4 of 10) spontaneous deletions in an untreated set. Doxorubicin-induced single base substitutions were highly focused at one site (4 of 6) in the i-d region of lacI, in contrast to the spontaneous distribution of point mutations, where 16 mutants were recovered at 12 different sites. An increased frequency (3-fold) of highly focused base substitutions was also observed at 2 sites in the lac operator region (at lacO +6, which is a transition "hotspot" in the spontaneous spectra of both wild type and uvrB- organisms and at the adjacent +5 site). Notably, the frequency of 1- and 2-base frameshifts did not increase in the doxorubicin-induced spectrum, relative to the spontaneous mutation spectrum. These in vivo observations in E. coli suggest that in the absence of excision repair, doxorubicin causes highly focused deletions and base substitutions. These mutations occur adjacent to DNA sequences identified in previous in vitro studies as preferential sites of doxorubicin binding.