NEW STRATEGY FOR THE CONSTRUCTION OF SINGLE-STRANDED PLASMIDS WITH SINGLE MUTAGENIC LESIONS

Single-stranded DNA vectors containing single adducts offer a unique o pportunity to study the biochemistry and genetics of trans lesion synt hesis, a process during which a DNA polymerase synthesizes across a le sion. We describe a new and general strategy to produce high-quality s ingle-stranded plasmids containing a single adduct within a predetermi ned sequence context starting with a short oligonucleotide containing the lesion of interest. These vectors are isolated from the correspond ing double-stranded constructs by selective enzymatic degradation in v itro of the nonadducted uracil-containing strand. Efficient and comple te removal of this strand was achieved using uracil DNA glycosilase to generate AP sites followed by the action of the AP endonuclease assoc iated with exonuclease III and the robust 3' --> 5' exonuclease activi ty associated with T7 DNA polymerase. We show the utility of these con structs for the study of trans lesion synthesis in vitro and in vivo i n the case of the highly carcinogenic N-2-acetylaminofluorene adducts located within frameshift mutation hot spots. The possibility to const ruct both single-stranded and double-stranded plasmids, with the same origin of replication (i.e., ColE1), will allow a direct comparison be tween single-stranded and double-stranded DNA replication in site-spec ific mutagenesis studies.