Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2

The adduct that would arise from cis opening of (+)-(1S,2R,3R,4S)-3,4-dihyd roxy-1,2-epoxy-benzo[c]phenanthrene (benzo[c]phenanthrene diol epoxide-2, w here the benzylic hydroxyl group and the epoxide oxygen are traits) by the exocyclic N-6-amino group of deoxyadenosine was incorporated at the underli ned site into four oligonucleotides, 5'-CAGATTTAGAGTCTGC-3', 5'-CAGTGCAGATT TAGAG-3', 5'-GTGCAGATTTAGA-3' and 5'-TGCAGATTTA-3'. The oligonucleotides we re inserted into M13mp7L2 acid the vector transfected into SOS-induced Esch erichia coli SMH77 which were then plated on agar plates, The experiments r eported here were designed to test the effect of the lesion position (the u nderlined A in the sequences above) on the ligation efficiency of the inser t and the frequency of failed constructs, as well as any possible effects o n the mutagenic consequences of the lesion, The construct survival was esti mated from the number of plaques formed following transformation, and mutat ion frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucl eotide, and changing the insert length or the adduct location did not marke dly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the addu ct) remains constant, the size of the oligonucleotide insert and the positi on of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.