Replication of a site-specific trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B-1 adduct by the exonuclease deficient Klenow fragment of DNA polymerase I

A 19-mer oligodeoxynucleotide containing a site-specific trans-8,9-dihydro- 8-(N7-guanyl)-9-hydroxyaflatoxin B-1 adduct was prepared and purified. This was used as a template for replication with DNA polymerase I exo(-) (Kleno w exo(-)) in vitro. The chemical stability of the modified template strand containing the cationic aflatoxin B1 adduct was monitored by mass spectrome try. Under the conditions used in these assays, the cationic aflatoxin B1 a dduct remained intact; quantitative conversion to the corresponding formami dopyrimidine adduct was not observed. The results revealed that the cationi c guanine AFB(1) N7 adduct blocked translesional DNA synthesis at the adduc ted site and one nucleotide 3' to the adducted site. Correct incorporation of cytosine opposite the lesion led to blockage, while incorrect incorporat ion of adenine allowed full-length extension. The in vitro experiments with polymerase I yielded base pair substitutions at the lesion site but not th e 5'-neighbor substitutions observed in vivo [Bailey, E. A., Iyer, R. S., S tone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Proc. Natl. Acad. S ci. U.S.A. 93, 1535-1539].