Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions.

Abstract

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.