New strategy for the construction of single-stranded plasmids with single mutagenic lesions

Single-stranded DNA vectors containing single adducts offer a unique opportunity to study the biochemistry and genetics of trans lesion synthesis, a process during which a DNA polymerase synthesizes across a lesion. We describe a new and general strategy to produce high-quality single-stranded plasmids containing a single adduct within a predetermined sequence context starting with a short oligonucleotide containing the lesion of interest. These vectors are isolated from the corresponding double-stranded constructs by selective enzymatic degradation in vitro of the nonadducted uracil-containing strand. Efficient and complete removal of this strand was achieved using uracil DNA glycosilase to generate AP sites followed by the action of the AP endonuclease associated with exonuclease III and the robust 3'-->5' exonuclease activity associated with T7 DNA polymerase. We show the utility of these constructs for the study of trans lesion synthesis in vitro and in vivo in the case of the highly carcinogenic N-2-acetylaminofluorene adducts located within frameshift mutation hot spots. The possibility to construct both single-stranded and double-stranded plasmids, with the same origin of replication (i.e., ColE1), will allow a direct comparison between single-stranded and double-stranded DNA replication in site-specific mutagenesis studies.