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

• Published in: Mutagenesis Vol. 16; no. 1; pp. 65 - 69
• Main Authors: Pontén, Ingrid, Waters, Laura S., Sayer, Jane M., Pilcher, Anthony S., Dipple, Anthony, Jerina, Donald M.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-01-2001
• Subjects: Bacteriophage M13 - genetics; Base Sequence; benzo(c)phenanthrene-diol epoxide; Biological and medical sciences; Blotting, Southern; Circular Dichroism; deoxyadenosine; Deoxyadenosines - chemistry; DNA Adducts - chemistry; DNA, Bacterial - chemistry; DNA, Circular - chemistry; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Mutagens - chemistry; Nucleic Acid Hybridization; Oligonucleotides - chemistry; Phenanthrenes - chemistry; Phenanthrene derivatives; Mutagenesis; Nucleotide sequence; Mutagenicity testing; Toxicity; Escherichia coli; Bacteria; Oligonucleotide; Lesion; Vector; Molecular adduct; Enterobacteriaceae
• ISSN: 0267-8357; 1464-3804; 1464-3804
• DOI: 10.1093/mutage/16.1.65

The adduct that would arise from cis opening of (+)-(1S,2R,3R,4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the underlined site into four oligonucleotides, 5′-CAGATTTAGAGTCTGC-3′, 5′-CAGTGCAGATTTAGAG-3′, 5′-GTGCAGATTTAGA-3′ and 5′-TGCAGATTTA-3′. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the underlined A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation 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 oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40–58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.