Stereospecific differences in repair by human cell extracts of synthesizedoligonucleotides containing trans-opened 7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol-9,10-epoxide-N-2-dG adduct stereoisomers located within the humanK-ras codon 12 sequence

The potent environmental carcinogen benzo[a]pyrene (BaP), following enzymat ic activation to enantiomeric pairs of bay-region 7,8-diol 9,10-epoxides (t he benzylic 7-hydroxyl group and epoxide oxygen are cis for DE-1 diastereom ers and trans for DE-2 diastereomers), reacts with DNA to form covalent add ucts predominately at the exocyclic amino groups of purines. Specific adduc ts, corresponding to the trans opening of each of the four optically active BaP DE isomers at C-10 by the N-2-amino group of dG, were synthesized as a ppropriately blocked phosphoramidites and were incorporated at either the f irst or second G of codon 12 within the G-rich sequence of human K-ms codon s 11-13: GCT G(1)G(2)T GGC. The adducted oligonucleotides were incorporated into plasmids by primer extension, followed by purification of the covalen tly closed circular constructs. Adducts derived from either (+)- or (-)-DE- 2, placed at either G(1) or G(2), presented strong blocks to in vitro trans cription elongation by bacteriophage T3 RNA polymerase, but only moderately blocked transcription elongation by human RNA polymerase II in nuclear ext racts. Adducts derived from all four DEs, placed on either G(1) or G(2), we re used as substrates in a DNA repair synthesis assay using human whole cel l extracts. Adducts derived from three of the DE stereoisomers exhibited si gnificant amounts of repair synthesis, but the (-)-DE-2 adduct experienced no repair synthesis above that of the control. Constructs containing a pre- existing nick at the sixth phosphodiester bond 3' to either (+)-DE-2 or (-) -DE-2 adducts exhibited increased repair synthesis.