Jlu1j

Bgi n

5' gatccacaccg C cggcgcc ^

gtgtggcGO gccgcggctag

NgOMTV or gtgtggcGOgccgcggctag gatccacaccgCcggcgcc NgOMIV

ps pSA(plate)

Bgi n

Bgi n

5' gatccacaccg C cggcgcc ^

gtgtggcGO gccgcggctag

NgOMTV

transfection v cells

A : Mutagenesis assay bacteria

B : Repair assay

The shuttle vector used to construct the monomodified plasmids is derived from the pS189 shuttle vector and was given by M. Seidman (the National Institutes of Health, Bethesda, MD).

The replicative plasmids containing a unique 8-oxoG lesion paired with cyto-sine (GO: C) (Fig. 1) are constructed as described previously.4 Briefly, a single-stranded modified plasmid pS(C) carrying a cytosine in the position opposite the lesion is constructed by inserting a 19-mer oligonucleotide complementary to the modified oligonucleotide carrying the 8-oxoG into the original plasmid DNA. The active M13 replication origin contained in pS189 is used to produce single-stranded DNA (ssDNA), using M13 helper phage (Pharmacia, Piscataway, NJ). A gapped double-stranded plasmid is then constructed by hybridizing the single-stranded pS(C) in lx saline-sodium citrate (SSC) buffer with double-stranded DNA linearized at the BgUl site. The mixture is heated at 98° for 10 min, cooled slowly to 65°, allowed to renature for 1 hr, and then ethanol precipitated. Twenty micrograms of gapped duplex plasmid DNA thus formed is hybridized overnight with 5 jig of 5'-32P-phosphorylated 8-oxoG-containing oligonucleotide in the ligation buffer containing 1 mM ATP, and then ligated for 20 min with 400 units of T4 DNA ligase (Biolabs, Hertfordshire, UK) at 12°. Finally, covalently closed molecules are purified by isopycnic centrifugation on an ethidium bromide-cesium chloride gradient. Fractions collected from the gradient are analyzed on a 1 % (w/v) agarose gel and those corresponding to the closed circular double-stranded DNA vector as detected by the 32P label are pooled, dialyzed against TE [1 mM Tris (pH 7.8), 1 mM EDTA] and then ethanol precipitated (Fig. 2).

Plasmid containing an 8-oxoG in a nontranscribed sequence is constructed as described above, but beginning with a plasmid deleted for the late promoter sequences. The plasmid pSApiate is constructed by a two-step protocol, first by digestion of single-stranded plasmid with Sphl and FnuAYil enzymes after hybridization of oligonucleotides at the restriction sites. The oligonucleotides are removed by heating and filtration through a Spun size S-400 column (Pharmacia) and the longest fragment is gel purified. The resulting single-stranded plasmid deleted of 25 bp is then digested with BamHl and BstNl restriction enzymes, using

Fig. 1. Experimental scheme and genetic maps of the various plasmids used. (A) Mutagenesis assay: The plasmid pS contains an 8-oxoG.C in a transcribed sequence under the control of the SV40 early promoter. The pSA(piate) plasmid has been deleted in the SV40 late promoter sequence (hatched) and contains an 8-oxoG.C mispair in the opposite position in a nontranscribed sequence. After transfection and replication in human cells, plasmid DNA is recovered and amplified in recA~ bacteria. Digestion by NgoMIV of clones allows the selection of mutants. (B) Repair assay: The plasmid pSAoriSV has a deletion in the eukaryotic replication origin (hatched) and contains an 8-oxoG.C mispair in a transcribed sequence under the control of the SV40 early promoter. pSA(peariy) has a deletion of both the replication origin and the early promoter (hatched) that eliminates transcription of the 8-oxoG.C mispair. After transfection and repair in human cells, plasmid DNA was recovered and amplified in fpg"7mutY~ bacteria. Digestion by NgoMTV of plasmid DNA from individual colonies allows the selection of mutants.

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