FREQUENCY AND FIDELITY OF TRANSLESION SYNTHESIS OF SITE-SPECIFIC N-2-ACETYLAMINOFLUORENE ADDUCTS DURING DNA-REPLICATION IN A HUMAN CELL EXTRACT

We have previously analyzed the effects of site-specific N-2-acetylami nofluorene (AAF) adducts on the efficiency and frameshift fidelity of SV40-based DNA replication in a human cell extract (Thomas, D. C., Vea ute, X,, Kunkel, T. A., and Fuchs, R. P. P. (1994) Proc. Natl. Acad. S ci. U.S.A. 91, 7752-7756). Here we use two sets of substrates to exami ne the probability of replication termination and error-free and error -prone bypass of AAF adducts. The substrates contained site-specific a dducts at one of three guanines in a NarI sequence (5'-GGCGCC-3') plac ed within the lacZ alpha reporter gene and located on the template for either leading or lagging strand replication. The presence of the add uct at any position strongly reduces the efficiency of a single round of replication in a HeLa cell extract, Product analysis reveals prefer ential replication of the undamaged strand and termination of replicat ion of the damaged strand occurring one nucleotide before incorporatio n opposite either a leading or lagging strand adduct, Products resista nt to restriction endonuclease cleavage at the adducted site were gene rated in amounts consistent with 16-48% lesion bypass during replicati on, Most of this bypass was error-free, However, two-nucleotide deleti on errors were detected in the replication products of DNA containing an AAF adduct in either the leading or lagging strand, but only when p resent at the third guanine position. Collectively, the data suggest t hat the replication apparatus in a HeLa cell extract generates a templ ate-primer slippage error at an AAF adduct once for every 30-100 bypas s events.